Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

Science.gov (United States)

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

DEFF Research Database (Denmark)

Goldfinger, L E; Hopkinson, S B; deHart, G W

1999-01-01

Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion

DEFF Research Database (Denmark)

Piwko-Czuchra, Aleksandra; Koegel, Heidi; Meyer, Hannelore

2009-01-01

BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in thei...... of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis....... that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon...... was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations...

Genetic analysis of beta1 integrin "activation motifs" in mice

DEFF Research Database (Denmark)

Czuchra, Aleksandra; Meyer, Hannelore; Legate, Kyle R

2006-01-01

-null phenotype in vivo. Surprisingly, neither the substitution of the tyrosines with phenylalanine nor the aspartic acid with alanine resulted in an obvious defect. These data suggest that the NPXY motifs of the beta1 integrin tail are essential for beta1 integrin function, whereas tyrosine phosphorylation...

Beta1 integrin-mediated adhesion signalling is essential for epidermal progenitor cell expansion.

Directory of Open Access Journals (Sweden)

Aleksandra Piwko-Czuchra

Full Text Available BACKGROUND: There is a major discrepancy between the in vitro and in vivo results regarding the role of beta1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of beta1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate this discrepancy we generated hypomorphic mice expressing reduced beta1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with beta1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of beta1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the beta1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of beta1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that expression of beta1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

DEFF Research Database (Denmark)

Iba, K; Albrechtsen, R; Gilpin, B

2000-01-01

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments......-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading......, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did...

The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin alpha4beta7.

Science.gov (United States)

Chen, JianFeng; Takagi, Junichi; Xie, Can; Xiao, Tsan; Luo, Bing-Hao; Springer, Timothy A

2004-12-31

We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.

Role of the beta1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

DEFF Research Database (Denmark)

Gustavsson, Anna; Armulik, Annika; Brakebusch, Cord

2002-01-01

Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta......1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine...... noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex...

Beta1 integrin promotes but is not essential for metastasis of ras-myc transformed fibroblasts

DEFF Research Database (Denmark)

Brakebusch, C; Wennerberg, K; Krell, H W

1999-01-01

To investigate the role of beta1 integrin during tumor metastasis, we established a ras-myc transformed fibroblastoid cell line with a disrupted beta1 integrin gene on both alleles (GERM 11). Stable transfection of this cell line with an expression vector encoding beta1A integrin resulted in beta1A......, tumors induced by the high expressing clones 1A10 and 2F2 were markedly smaller, suggesting an inverse correlation of tumor growth and beta1 integrin expression. The metastasis potential of all three beta1 integrin-expressing GERM 11 sublines tested was significantly higher than that of the beta1......-deficient GERM 11 cells. GERM 116 tumors led in all animals to severe metastasis in lung and liver, while GERM 11 tumors induced only a few metastatic foci in the lung. Stroma of both tumors contained nidogen and high amounts of tenascin C, but only a few very low levels of fibronectin, laminin-1...

Alpha5beta1 integrin-fibronectin interactions specify liquid to solid phase transition of 3D cellular aggregates.

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Carlos E Caicedo-Carvajal

2010-07-01

Full Text Available Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM connections, regulated by integrins. Integrin alpha5beta1 and soluble fibronectin (sFN are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin alpha5beta1 and sFN and its influence on tissue mechanical properties and cell sorting behavior.We generated a series of cell lines varying in alpha5beta1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin alpha5beta1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as alpha5beta1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high alpha5beta1 levels. We also show that differential expression of alpha5beta1 integrin can promote phase-separation between cells.The interplay between alpha5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level

Targeting of beta 1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells

NARCIS (Netherlands)

Dickreuter, E.; Eke, I.; Krause, M.; Borgmann, K.; van Vugt, M. A.; Cordes, N.

2016-01-01

beta 1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of beta 1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of beta 1 integrin targeting on the repair of radiation-induced

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

International Nuclear Information System (INIS)

Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A

2005-01-01

Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype

Beta1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesis

DEFF Research Database (Denmark)

Aszodi, Attila; Hunziker, Ernst B; Brakebusch, Cord

2003-01-01

Beta1 integrins are highly expressed on chondrocytes, where they mediate adhesion to cartilage matrix proteins. To assess the functions of beta1 integrin during skeletogenesis, we inactivated the beta1 integrin gene in chondrocytes. We show here that these mutant mice develop a chondrodysplasia...... of various severity. beta1-deficient chondrocytes had an abnormal shape and failed to arrange into columns in the growth plate. This is caused by a lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F......-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. The G1/S defect is, at least partially, caused by overexpression of Fgfr3, nuclear translocation of Stat1/Stat5a, and up-regulation of the cell cycle inhibitors p16 and p21...

Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes

DEFF Research Database (Denmark)

Brakebusch, C; Grose, R; Quondamatteo, F

2000-01-01

developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression......, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance...

beta 1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts

Energy Technology Data Exchange (ETDEWEB)

Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.

2008-06-02

{beta}1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of {beta}1 integrin signaling. We showed previously that {beta}1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and {beta}1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

Endothelial-monocyte activating polypeptide II alters fibronectin based endothelial cell adhesion and matrix assembly via alpha5 beta1 integrin

International Nuclear Information System (INIS)

Schwarz, Margaret A.; Zheng, Hiahua; Liu, Jie; Corbett, Siobhan; Schwarz, Roderich E.

2005-01-01

Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN-alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

Directory of Open Access Journals (Sweden)

Beecken Wolf-Dietrich

2005-01-01

Full Text Available Abstract Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a, alpha2beta1 (CD49b, alpha3beta1 (CD49c, alpha4beta1 (CD49d, alpha5beta1 (CD49e, and alpha6beta1 (CD49f receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.

Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.

Science.gov (United States)

Hermosilla, Tamara; Muñoz, Daniel; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Muñoz, Nicolás; Nham, Sang-Uk; Schneider, Pascal; Burridge, Keith; Quest, Andrew F G; Leyton, Lisette

2008-06-01

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, I

DEFF Research Database (Denmark)

Evans, Evan; Kinoshita, Koji; Simon, Scott

2010-01-01

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with inte......Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels...... with integrin activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling in leukocytes. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off......-based assays of soluble ICAM-1 dissociation from immobilized LFA-1, i.e., approximately 10(-2)/s in Mg2+ or Mn2+ and approximately 1/s in Ca2+. At the same time, as expected for adhesive function, we find that the beta2 integrin bonds activated in Mn2+ or Mg2+ possess significant and persistent mechanical...

Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization

DEFF Research Database (Denmark)

Henry, M D; Satz, J S; Brakebusch, C

2001-01-01

Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface...... integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells......, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential...

A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2.

Science.gov (United States)

Vararattanavech, Ardcharaporn; Chng, Choon-Peng; Parthasarathy, Krupakar; Tang, Xiao-Yan; Torres, Jaume; Tan, Suet-Mien

2010-05-14

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar

Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow

DEFF Research Database (Denmark)

Potocnik, A J; Brakebusch, C; Fässler, R

2000-01-01

hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult beta1 integrin null HSCs isolated from mice carrying loxP-tagged beta1 integrin alleles and ablated for beta1 integrin expression by retroviral cre transduction......Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that beta1 integrin-deficient HSCs from the para-aortic splanchnopleura and the fetal blood had...

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

Science.gov (United States)

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

Integrin activation dynamics between the RGD-binding site and the headpiece hinge.

Science.gov (United States)

Puklin-Faucher, Eileen; Vogel, Viola

2009-12-25

Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding

NARCIS (Netherlands)

Geerts, D.; Fontao, L.; Nievers, M. G.; Schaapveld, R. Q.; Purkis, P. E.; Wheeler, G. N.; Lane, E. B.; Leigh, I. M.; Sonnenberg, A.

1999-01-01

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

Science.gov (United States)

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Molecular cloning and phylogenetic analysis of integrins alpha v beta 1 and alpha v beta 6 of one-humped camel (Camelus dromedarius)

DEFF Research Database (Denmark)

Du, Junzheng; Larska, Magdalena Larska; Chang, Huiyun

2010-01-01

Bactrian camels can relatively easily be infected with FMDV, but dromedary camels remain resistant even to high doses of the virus. To understand the different susceptibility between the two camel species from the standpoint of viral receptors, this work reports the sequences of the dromedary camel...... into the Artiodactyla group, together with those of Bactrian camel, pig, sheep, and cattle that are susceptible to FMDV infection. Compared with the Bactrian camel integrins, 4, 10, and 8 amino acid changes were found in the dromedary camel alpha v, beta 1, and beta 6 subunits, respectively. This study...... will be of importance in understanding the differences of integrins as FMDV receptors among dromedary camel and other species. Crown...

Discovery, SAR, and Radiolabeling of Halogenated Benzimidazole Carboxamide Antagonists as Useful Tools for (alpha)4(beta)1 Integrin Expressed on T- and B-cell Lymphomas

Energy Technology Data Exchange (ETDEWEB)

Carpenter, R D; Natarajan, A; Lau, E Y; Andrei, M; Solano, D M; Lightstone, F C; DeNardo, S J; Lam, K S; Kurth, M J

2010-02-08

The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin is an attractive yet poorly understood target for selective diagnosis and treatment of T- and B-cell lymphomas. This report focuses on the rapid microwave preparation of medicinally pertinent benzimidazole heterocycles, structure-activity relationships (SAR) of novel halobenzimidazole carboxamide antagonists 3-6, and preliminary biological evaluation of radioiodinated agents 7, 8, and 18. The I-125 derivative 18 had good tumor uptake (12 {+-} 1% ID/g at 24 h; 4.5 {+-} 1% ID/g at 48 h) and tumor:kidney ratio ({approx}4:1 at 24 h; 2.5:1 at 48 h) in xenograft murine models of B-cell lymphoma. Molecular homology models of {alpha}{sub 4}{beta}{sub 1} integrin have predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. These high affinity ({approx} pM binding) halogenated ligands are attractive tools for medicinal and biological use; the fluoro and iodo derivatives are potential radiodiagnostic ({sup 18}F) or radiotherapeutic ({sup 131}I) agents, whereas the chloro and bromo analogues could provide structural insight into integrin-ligand interactions through photoaffinity cross-linking/mass spectroscopy experiments, as well as co-crystallization X-ray studies.

Tumor targeting with radiolabeled alpha(v)beta(3) integrin binding peptides in a nude mouse model.

NARCIS (Netherlands)

Janssen, M.L.H.; Oyen, W.J.G.; Dijkgraaf, I.; Massuger, L.F.A.G.; Frielink, C.; Edwards, D.S.; Rajopadhye, M.; Boonstra, H.; Corstens, F.H.M.; Boerman, O.C.

2002-01-01

The alpha(v)beta(3) integrin is expressed on proliferating endothelial cells such as those present in growing tumors, as well as on tumor cells of various origin. Tumor-induced angiogenesis can be blocked in vivo by antagonizing the alpha(v)beta(3) integrin with small peptides containing the

Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, II

DEFF Research Database (Denmark)

Kinoshita, Koji; Leung, Andrew; Simon, Scott

2010-01-01

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with acti......Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels......-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to beta2 integrin on the cell surface. Even though ICAM-1 bonds to recombinant alphaLbeta2 on microspheres in Mg2+ or Mn2+ can live for long periods of time under slow pulling, here we show that stimulation...... of neutrophils in Mg2+ plus the chemokine IL-8 (i.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg2+ or Mn2+ alone. Similar changes are observed with outside-in signaling, i...

Quantitative gene-expression of the tumor angiogenesis markers vascular endothelial growth factor, integrin alphaV and integrin beta3 in human neuroendocrine tumors

DEFF Research Database (Denmark)

Oxboel, Jytte; Binderup, Tina; Knigge, Ulrich

2009-01-01

, in neuroendocrine tumors. We used quantitative real-time PCR for measuring mRNA gene-expression of vascular endothelial growth factor (VEGF), integrin alphaV, and integrin beta3, and CD34 for a group of patients with neuroendocrine tumors (n=13). Tissue from patients with colorectal cancer liver metastases (n=14...... compared to both colorectal liver metastases (p=0.10) and normal liver tissue (p=0.06). In neuroendocrine tumors, gene-expression was highly variable of VEGF (530-fold), integrin alphaV (23-fold) and integrin beta3 (106-fold). Quantitative gene-expression levels of the key angiogenesis molecules VEGF......Anti-angiogenesis treatment is a promising new therapy for cancer that recently has also been suggested for patients with neuroendocrine tumors. The aim of the present study was therefore to investigate the level of tumor angiogenesis, and thereby the molecular basis for anti-angiogenesis treatment...

MicroPET/CT imaging of {alpha}{sub v}{beta}{sub 3} integrin via a novel {sup 68}Ga-NOTA-RGD peptidomimetic conjugate in rat myocardial infarction

Energy Technology Data Exchange (ETDEWEB)

Menichetti, Luca; Kusmic, Claudia; Panetta, Daniele; Petroni, Debora; Salvadori, Piero A. [CNR-Institute of Clinical Physiology (IFC), Pisa (Italy); Arosio, Daniela; Manzoni, Leonardo [CNR-Institute of Molecular Science and Technologies (ISTM), Milan (Italy); Matteucci, Marco [Scuola Superiore Sant' Anna, Pisa (Italy); Casagrande, Cesare [University of Milan, Department of Chemistry, Milan (Italy); L' Abbate, Antonio [CNR-Institute of Clinical Physiology (IFC), Pisa (Italy); Scuola Superiore Sant' Anna, Pisa (Italy)

2013-08-15

The {alpha}{sub v}{beta}{sub 3} integrin is expressed in angiogenic vessels and is a potential target for molecular imaging of evolving pathological processes. Its expression is upregulated in cancer lesions and metastases as well as in acute myocardial infarction (MI) as part of the infarct healing process. The purpose of our study was to determine the feasibility of a new imaging approach with a novel {sup 68}Ga-2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA)-arginine-glycine-aspartic acid (RGD) construct to assess integrin expression in the evolving MI. A straightforward labelling chemistry to attach the radionuclide {sup 68}Ga to a NOTA-based chelating agent conjugated with a cyclic RGD peptidomimetic is described. Affinity for {alpha}{sub v}{beta}{sub 3} integrin was assessed by in vitro receptor binding assay. The proof-of-concept in vivo studies combined the {sup 68}Ga-NOTA-RGD with the flow tracer {sup 13}N-NH{sub 3} imaging in order to obtain positron emission tomography (PET)/CT imaging of both integrin expression and perfusion defect at 4 weeks after infarction. Hearts were then processed for immunostaining of integrin {beta}{sub 3}. NOTA-RGD conjugate displayed a binding affinity for {alpha}{sub v}{beta}{sub 3} integrin of 27.9 {+-} 6.8 nM. {sup 68}Ga-NOTA-RGD showed stability without detectable degradation or formation of by-products in urine up to 2 h following injection in the rat. MI hearts exhibited {sup 68}Ga-NOTA-RGD uptake in correspondence to infarcted and border zone regions. The tracer signal drew a parallel with vascular remodelling due to ischaemia-induced angiogenesis as assessed by immunohistochemistry. As compared to similar imaging approaches using the {sup 18}F-galacto-derivative, we documented for the first time with microPET/CT imaging the {sup 68}Ga-NOTA-RGD derivative that appears eligible for PET imaging in animal models of vascular remodelling during evolving MI. The simple chemistry employed to

An integrin alpha4beta7•IgG heterodimeric chimera binds to MAdCAM-1 on high endothelial venules in gut-associated lymphoid tissue.

Science.gov (United States)

Hoshino, Hitomi; Kobayashi, Motohiro; Mitoma, Junya; Sato, Yoshiko; Fukuda, Minoru; Nakayama, Jun

2011-06-01

Lymphocyte homing is regulated by a multistep process mediated by sequential adhesive interactions between circulating lymphocytes and high endothelial venules (HEVs). In gut-associated lymphoid tissue (GALT), the initial interactive step, "tethering and rolling," is partly mediated by integrin α4β7 expressed on GALT-homing lymphocytes and its ligand MAdCAM-1, which is exclusively expressed on HEVs in GALT. To probe functional MAdCAM-1 in tissue sections, we developed a soluble integrin α4β7 heterodimeric IgG chimera by joining the extracellular region of mouse integrin α4 and β7 subunits to a human IgG Fc domain. Western blot analysis revealed that co-transfection of HEK 293T cells with expression vectors encoding integrin α4•IgG and β7•IgG results in the formation of α4β7•IgG heterodimeric chimeras. This complex preferentially binds to CHO cells expressing MAdCAM-1 and, to a lesser extent, to cells expressing VCAM-1, but not to cells expressing ICAM-1. Moreover, α4β7•IgG specifically binds to HEVs in GALT in situ in a divalent cation-dependent fashion and inhibits lymphocyte binding to HEVs in GALT. These findings indicate that α4β7•IgG can be used as a probe for functional MAdCAM-1 expressed on HEVs in GALT and could potentially serve as an anti-inflammatory drug inhibiting GALT-specific lymphocyte migration.

Noninvasive visualization and quantification of tumor {alpha}{sub V{beta}3} integrin expression using a novel positron emission tomography probe, {sup 64}Cu-cyclam-RAFT-c(-RGDfK-){sub 4}

Energy Technology Data Exchange (ETDEWEB)

Jin, Zhao-Hui [Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Furukawa, Takako, E-mail: tfuru@nirs.go.j [Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan); Galibert, Mathieu; Boturyn, Didier [Departement de Chimie Moleculaire, UMR 5250, CNRS-Universite Joseph Fourier, 38041 Grenoble Cedex 9 (France); Coll, Jean-Luc [INSERM U823, Institut Albert Bonniot and Universite Joseph Fourier, 38706 La Tronche Cedex, Grenoble (France); Fukumura, Toshimitsu; Saga, Tsuneo [Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Dumy, Pascal [Departement de Chimie Moleculaire, UMR 5250, CNRS-Universite Joseph Fourier, 38041 Grenoble Cedex 9 (France); Fujibayashi, Yasuhisa [Molecular Imaging Center, National Institute of Radiological Sciences, Chiba 263-8555 (Japan); Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193 (Japan)

2011-05-15

Introduction: The {alpha}{sub V{beta}3} integrin is a well-known transmembrane receptor involved in tumor invasion, angiogenesis and metastasis. Our aim was to evaluate a novel positron emission tomography (PET) probe, {sup 64}Cu-cyclam-RAFT-c(-RGDfK-){sub 4}, for noninvasive visualization and quantification of {alpha}{sub V{beta}3} integrin expression. Methods: RAFT-c(-RGDfK-){sub 4}, a tetrameric cyclic Arg-Gly-Asp (RGD)-based peptide, was conjugated with a bifunctional chelator, 1,4,8,11-tetraazacyclotetradecane (cyclam), radiolabeled with the positron emitter {sup 64}Cu and evaluated in vitro by cell binding and competitive inhibition assays and in vivo by biodistribution and receptor blocking studies, and PET imaging. The following cell lines, human embryonic kidney HEK293({beta}{sub 1}) [{alpha}{sub V{beta}3}-negative] and HEK293({beta}{sub 3}) [{alpha}{sub V{beta}3}-overexpressing] and human glioblastoma U87MG [naturally expressing {alpha}{sub V{beta}3}], together with their subcutaneous xenografts in athymic nude mice, were used for the present study. The expression levels of {alpha}{sub V{beta}3} on these cell lines and tumor xenografts were analyzed by flow cytometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography, respectively. Results: {sup 64}Cu-cyclam-RAFT-c(-RGDfK-){sub 4} demonstrated the in vitro and in vivo specificity for the {alpha}{sub V{beta}3} integrin and displayed rapid blood clearance, predominantly renal excretion and low uptake in nontumor tissues. Tumor uptake of {sup 64}Cu-cyclam-RAFT-c(-RGDfK-){sub 4} (3 h postinjection) in HEK293({beta}{sub 3}) (high levels of {alpha}{sub V{beta}3}), U87MG (moderate levels of {alpha}{sub V{beta}3}) and HEK293({beta}{sub 1}) (undetectable levels of {alpha}{sub V{beta}3}) tumors was 9.35%{+-}1.19%, 3.46%{+-}0.45% and 1.18%{+-}0.30% injected dose per gram, respectively, with a strong and positive correlation with the tumor {alpha}{sub V{beta}3} expression levels

Functions of Tenascin-C and Integrin alpha9beta1 in Mediating Prostate Cancer Bone Metastasis

Science.gov (United States)

2017-10-01

AWARD  NUMBER:          W81XWH-16-1-0523 TITLE:  Functions of Tenascin- C and Integrin alpha9beta1 in Mediating Prostate Cancer Bone Metastasis...Prostat Prostate Cancer  Bone Metastasis   5a.  CONTRACT  NUMBER   Functions of Tenascin- C and Integrin alpha9beta1 in Mediating Prostate Cancer...SUPPLEMENTARY  NOTES 14. ABSTRACT The purpose of this work is to dissect mechanisms responsible for interactions between integrin a9b1 and tenascin- C that are

hnRNP L regulates differences in expression of mouse integrin alpha2beta1.

Science.gov (United States)

Cheli, Yann; Kunicki, Thomas J

2006-06-01

There is a 2-fold variation in platelet integrin alpha2beta1 levels among inbred mouse strains. Decreased alpha2beta1 in 4 strains carrying Itga2 haplotype 2 results from decreased affinity of heterogeneous ribonucleoprotein L (hnRNP L) for a 6 CA repeat sequence (CA6) within intron 1. Seven strains bearing haplotype 1 and a 21 CA repeat sequence at this position (CA21) express twice the level of platelet alpha2beta1 and exhibit an equivalent gain of platelet function in vitro. By UV crosslinking and immunoprecipitation, hnRNP L binds more avidly to CA21, relative to CA6. By cell-free, in vitro mRNA splicing, decreased binding of hnRNP L results in decreased splicing efficiency and an increased proportion of alternatively spliced product. The splicing enhancer activity of CA21 in vivo is abolished by prior treatment with hnRNP L-specific siRNA. Thus, decreased surface alpha2beta1 results from decreased Itga2 pre-mRNA splicing regulated by hnRNP L and depends on CA repeat length at a specific site in intron 1.

Improved targeting of the alpha(v)beta (3) integrin by multimerisation of RGD peptides.

NARCIS (Netherlands)

Dijkgraaf, I.; Kruijtzer, J.A.; Liu, S.; Soede, A.C.; Oyen, W.J.G.; Corstens, F.H.M.; Liskamp, R.M.; Boerman, O.C.

2007-01-01

PURPOSE: The integrin alpha(v)beta(3) is expressed on sprouting endothelial cells and on various tumour cell types. Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3) binding

Evidence for a prolonged role of alpha 4 integrin throughout active experimental allergic encephalomyelitis.

Science.gov (United States)

Keszthelyi, E; Karlik, S; Hyduk, S; Rice, G P; Gordon, G; Yednock, T; Horner, H

1996-10-01

The leukocyte integrin receptor, alpha 4 beta 1, and its endothelial cell ligand, vascular cell adhesion molecule 1, appear to be of critical importance in the leukocyte trafficking that accompanies CNS damage in experimental allergic encephalomyelitis (EAE). In this study, the persistence of the role for alpha 4 beta 1/VCAM-1 in EAE was established by observing antibody-mediated disease reversal up to 1 month following disease onset. Limited treatment with a monoclonal antibody against alpha 4 integrin, GG5/3, resulted in a significant decrease in both clinical and histopathologic signs. This was not observed in isotype control experiments. In the latter phase of progressive disease, widespread demyelination occurred in the animals that did not respond to 6 days of anti-alpha 4 treatment. These results demonstrate an essential role for alpha 4 beta 1 interactions throughout active EAE and illustrate the difference between reversible clinical deficits caused by edema and irreversible deficits associated with demyelination.

Interactions of the integrin subunit beta1A with protein kinase B/Akt, p130Cas and paxillin contribute to regulation of radiation survival

DEFF Research Database (Denmark)

Seidler, Julia; Durzok, Rita; Brakebusch, Cord

2005-01-01

25beta1B cells, which express mutant beta1B-integrins, were compared in terms of radiation survival and beta1-integrin signaling. MATERIALS AND METHODS: Cells grown on fibronectin, collagen-III, laminin, vitronectin, anti-beta1-integrin-IgG (beta1-IgG) or poly-l-lysine were irradiated with 0-6Gy...... and phosphorylation were analyzed by Western blot technique. RESULTS: Adhesion of GD25beta1A cells to extracellular matrix proteins or beta1-IgG resulted in growth factor-independent radiation survival. In contrast, serum starved GD25beta1B cells showed a significant (Pradiation survival on all...... phosphorylation. Phosphorylated p130Cas and paxillin subsequently prevented activation of cell death-regulating JNK. CONCLUSIONS: The data show that beta1-integrin-mediated signaling through the cytoplasmic integrin domains is critical for efficient pro-survival regulation after irradiation. Profound knowledge...

Inhibiting Vimentin or beta 1-integrin Reverts Prostate Tumor Cells in IrECM and Reduces Tumor Growth

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xueping; Fournier, Marcia V.; Ware, Joy L.; Bissell, Mina J.; Zehner, Zendra E.

2009-07-27

Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphological changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional (3D) lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the parental prostate epithelial P69 cell line by selection in nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin or {alpha}6-, {beta}4- and {beta}1-integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via siRNA interference or {beta}1-integrin expression by the addition of the blocking antibody, AIIB2, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by subcutaneous injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in 3D lrECM gels. These studies suggest that the levels of vimentin and {beta}1-integrin play a key role in the homeostasis of the normal acini in prostate and that their dysregulation may lead to tumorigenesis.

Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

DEFF Research Database (Denmark)

Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

2003-01-01

decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

Targeting ILK and β4 integrin abrogates the invasive potential of ovarian cancer

International Nuclear Information System (INIS)

Choi, Yoon Pyo; Kim, Baek Gil; Gao, Ming-Qing; Kang, Suki; Cho, Nam Hoon

2012-01-01

Highlights: ► The potential of targeting ILK and integrins for highly aggressive ovarian cancer. ► Unanticipated synergistic effect for the combination of ILK/β4 integrin. ► Combination of ILK/β4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. ► Targeting of β4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of β1 and β4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of β1 and β4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of β4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of β4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting β4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

Immunohistochemical localization of integrin alpha V beta 3 and osteopontin suggests that they do not interact during embryo implantation in ruminants

Directory of Open Access Journals (Sweden)

MacLaren Leslie A

2004-04-01

Full Text Available Abstract Background It has been suggested that trophoblast attachment requires co-expression of integrin alpha V beta 3 and its ligand osteopontin at the fetal-maternal interface. Until now the expression patterns of integrin alpha V beta 3 and osteopontin in the pregnant bovine uterus were unknown. The objectives of this study were to localize integrin alpha V beta 3 and osteopontin in bovine and sheep endometrium during the periimplantation period and to compare the distribution patterns using antibodies that had not yet been tested in sheep. Methods Cell compartments within endometrial tissue sections were scored for immunohistochemical staining intensity and data were analyzed to determine the effects of day of pregnancy or cycle. Results In pregnant bovine endometrium, integrin alpha V beta 3 was detected in luminal epithelium, stroma, myometrium and smooth muscle. A strong band of immunoreactivity was observed in the subepithelial stroma of intercaruncular regions, but there was reduced reactivity in the caruncles and glands. Bovine trophoblast did not express integrin alpha V beta 3 at any stage of pregnancy. In ovine endometrium a different pattern of staining for integrin alpha V beta 3 was observed. Reactivity was not present in the luminal epithelium or trophoblast. There was strong staining of the deep glands and no reactivity in the superficial glands. Osteopontin distribution was similar for sheep and cattle. For both species, apical staining was present on the luminal epithelium and glands and on embryonic tissues. Conclusion In ruminants, integrin alpha V beta 3 and osteopontin do not co-localize at the fetal-maternal interface indicating that these proteins could not interact to facilitate embryo attachment as has been proposed in other species.

T-cell-mediated immunity to lymphocytic choriomeningitis virus in beta2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice

DEFF Research Database (Denmark)

Christensen, Jan Pravsgaard; Marker, O; Thomsen, Allan Randrup

1996-01-01

The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild-type...... mice. T-cell-mediated inflammation, assessed as primary footpad swelling and susceptibility to intracerebral infection, was slightly compromised only in beta2-integrin-deficient mice. However, adoptive immunization of mutant mice soon after local infection did reveal a reduced capacity to support...... the inflammatory reaction, indicating that under conditions of more limited immune activation both molecules do play a role in formation of the inflammatory exudate. Finally, virus control was found to be somewhat impaired in both mutant strains. In conclusion, our results indicate that although LFA-1-ICAM-1...

The loss of alpha2beta1 integrin suppresses joint inflammation and cartilage destruction in mouse models of rheumatoid arthritis.

NARCIS (Netherlands)

Peters, M.E.W.J.; Wendholt, D.; Strietholt, S.; Frank, S.; Pundt, N.; Korb-Pap, A.; Joosten, L.A.B.; Berg, W.B. van den; Kollias, G.; Eckes, B.; Pap, T.

2012-01-01

OBJECTIVE: Integrin alpha2beta1 functions as a major receptor for type I collagen on different cell types, including fibroblasts and inflammatory cells. Although in vitro data suggest a role for alpha2beta1 integrin in regulating both cell attachment and expression of matrix-degrading enzymes such

Integrin beta3 Leu33Pro polymorphism and risk of hip fracture: 25 years follow-up of 9233 adults from the general population

DEFF Research Database (Denmark)

Tofteng, Charlotte L; Bach-Mortensen, Pernille; Bojesen, Stig E

2007-01-01

for the integrin beta3 Leu33Pro polymorphism have a two-fold risk of hip fracture, mainly confined to postmenopausal women. Integrin beta3 Leu33Pro homozygosity could prove a useful marker for risk of future hip fracture and may contribute to pharmacogenetic variation in effects of integrin alphavbeta3 antagonists....

The Effect of dcEFs on migration behavior of A549 cells and Integrin beta1 expression

Directory of Open Access Journals (Sweden)

Yunjie WANG

2008-04-01

Full Text Available Background and objective The effect of direct-current electric fields (dcEFs on cells attracted extensive attention. Moreover the metastasis and its potential are considered to be related to dcEFs. The aim is to study the effect of dcEFs on migration behavior of A549 cells, Integrin ?1 and its signal pathways. Methods According to exposure to 5 V/cm dcEFs or not and the time of exposure, the A549 cells were divided into 4 groups. Images were taken per 5 min within 2 h to recode the migration of the cells. The data of results were analyzed statistically. Results Most of A549cells exposed to the dcEFs aligned and elongated perpendicularly to the electric field lines and migrated to the cathode continually during 2 h. On the contrary, cells unexposed to dcEFs showed slightly random movements. Immunofluorescence showed that Integrin ?1 on plasma membrane polarized to the cathode of the dcEFs. Western blot showed that Integrin beta1 downstream signal pathways p-FAK and p-ERK were overexpressed in the dcEFs. Conclusion A549 cells have a galvanotatic feature of cathodal directed migration while exposed to the dcEFs. The polarization of Integrin beta1 and the promotion of its downstream signal pathways may play an important roles in the galvanotaxis of A549 cells.

Diverse roles of integrin receptors in articular cartilage.

Science.gov (United States)

Shakibaei, M; Csaki, C; Mobasheri, A

2008-01-01

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

Science.gov (United States)

Djaffar, I; Chen, Y P; Creminon, C; Maclouf, J; Cieutat, A M; Gayet, O; Rosa, J P

1994-05-15

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.

ADAM disintegrin-like domain recognition by the lymphocyte integrins α4β1 and α4β7

Science.gov (United States)

Bridges, Lance C.; Sheppard, Dean; Bowditch, Ron D.

2004-01-01

The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin α4β1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins α4β1, α4β7 and α9β1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported α4β1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin α4β7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with α4β7. All three disintegrin domains supported α9β1-dependent cell adhesion. Recognition by both α4β1 and α4β7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins α4β1, α4β7 and α9β1. ADAM33 exclusively supported only α9β1-dependent adhesion. PMID:15504110

PKCbeta-dependent activation of RhoA by syndecan-4 during focal adhesion formation

DEFF Research Database (Denmark)

Dovas, Athanassios; Yoneda, Atsuko; Couchman, John R

2006-01-01

Syndecan-4 is a ubiquitously expressed transmembrane heparan sulphate proteoglycan acting in concert with integrins in the formation of focal adhesions and stress fibres. Signalling events studied thus far suggest the formation of a ternary complex between syndecan-4, phosphatidylinositol 4...... necessary for the formation and maintenance of stress fibres in primary rat embryo fibroblasts. Inhibition of PKCalpha activity through the use of specific pharmacological inhibitors, a dominant-negative construct, or siRNA downregulation of protein levels, attenuated focal adhesion formation...

Localization of integrin alpha(v)beta3 and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in cutaneous and oral melanomas of dog.

Science.gov (United States)

Rawlings, N G; Simko, E; Bebchuk, T; Caldwell, S J; Singh, B

2003-07-01

Melanomas are common neoplasms of dogs and arise from pigment-producing cells called melanocytes or melanoblasts. Melanomas of skin are often easily cured by surgical excision, but those of oral mucosa are aggressive, metastasize to the regional lymph nodes and lungs, and respond poorly to conventional therapy. Tumor growth is sustained by proliferation of microvessels via a process called angiogenesis. Integrin alpha(v)beta3 is expressed in proliferating but not in quiescent microvessels suggesting a role in angiogenesis. Vascular endothelial growth factor (VEGF) manifests its mitogenic and angiogenic effects mainly via VEGF receptor-2 (VEGFR-2/Flk-1). We conducted this immunocytochemical study to investigate the expression of integrin alpha(v)beta3 and VEGFR-2 in archival and fresh samples from cases of canine melanomas. Results show that integrin alpha(v)beta3 was expressed in 72% and 88% of cutaneous and oral melanomas, respectively, and the expression was restricted to and immediately around the melanocytes and endothelial cells. VEGFR-2 staining of selected cases of melanoma revealed that its expression overlapped with the alpha(v)beta3 integrin. Dual immuno-gold electron microscopy confirmed co-localization of integrin alpha(v)beta3 and VEGFR-2 in melanocytes and endothelial cells. These data demonstrate expression and co-localization of integrin alpha(v)beta3 and VEGFR-2 in cutaneous and oral melanomas of dogs.

The Interaction of Src Kinase with beta 3 Integrin Tails : A Potential Therapeutic Target in Thrombosis and Cancer

NARCIS (Netherlands)

Huveneers, Stephan; Danen, Erik H. J.

2010-01-01

Activation of Src family kinases is an important event downstream of integrin adhesion signaling in many cell types. A particularly intriguing connection between an integrin and a Src family kinase was first discovered in platelets, where the selective direct interaction of alpha IIb beta 3

In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

Science.gov (United States)

Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

2013-01-01

SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

ADAM12 and alpha9beta1 integrin are instrumental in human myogenic cell differentiation

DEFF Research Database (Denmark)

Lafuste, Peggy; Sonnet, Corinne; Chazaud, Bénédicte

2005-01-01

of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion...

A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

OpenAIRE

Djaffar, I; Chen, Y P; Creminon, C; Maclouf, J; Cieutat, A M; Gayet, O; Rosa, J P

1994-01-01

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residu...

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

Science.gov (United States)

deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

2003-02-01

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the

Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

Science.gov (United States)

Wanten, G J; Geijtenbeek, T B; Raymakers, R A; van Kooyk, Y; Roos, D; Jansen, J B; Naber, A H

2000-01-01

To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

Lack of beta1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype

DEFF Research Database (Denmark)

Breau, Marie A; Pietri, Thomas; Eder, Olivier

2006-01-01

The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed beta1 integrins in the neural crest cells when they emerge from the neural tube. beta1-null enteric neural...

Differential expression of integrins and laminin-5 in normal oral epithelia

DEFF Research Database (Denmark)

Thorup, A K; Dabelsteen, Erik; Schou, S

1997-01-01

beta 1 and beta 4 integrins are receptors on epithelial cells mediating cell-extracellular matrix adhesion. Furthermore, alpha 2 beta 1 and alpha 3 beta 1 contribute to cell-cell adhesion. Laminin-5 in epithelial basement membranes (BMs) is a ligand for alpha 6 beta 4 and alpha 3 beta 1. Expressi...

Molecular imaging of alpha v beta3 integrin expression in atherosclerotic plaques with a mimetic of RGD peptide grafted to Gd-DTPA.

Science.gov (United States)

Burtea, Carmen; Laurent, Sophie; Murariu, Oltea; Rattat, Dirk; Toubeau, Gérard; Verbruggen, Alfons; Vansthertem, David; Vander Elst, Luce; Muller, Robert N

2008-04-01

The integrin alpha v beta3 is highly expressed in atherosclerotic plaques by medial and intimal smooth muscle cells and by endothelial cells of angiogenic microvessels. In this study, we have assessed non-invasive molecular magnetic resonance imaging (MRI) of plaque-associated alpha v beta3 integrin expression on transgenic ApoE-/- mice with a low molecular weight peptidomimetic of Arg-Gly-Asp (mimRGD) grafted to gadolinium diethylenetriaminepentaacetate (Gd-DTPA-g-mimRGD). The analogous compound Eu-DTPA-g-mimRGD was employed for an in vivo competition experiment and to confirm the molecular targeting. The specific interaction of mimRGD conjugated to Gd-DTPA or to 99mTc-DTPA with alpha v beta3 integrin was furthermore confirmed on Jurkat T lymphocytes. The mimRGD was synthesized and conjugated to DTPA. DTPA-g-mimRGD was complexed with GdCl3.6H2O, EuCl3.6H2O, or with [99mTc(CO)3(H2O)3]+. MRI evaluation was performed on a 4.7 T Bruker imaging system. Blood pharmacokinetics of Gd-DTPA-g-mimRGD were assessed in Wistar rats and in c57bl/6j mice. The presence of angiogenic blood vessels and the expression of alpha v beta3 integrin were confirmed in aorta specimens by immunohistochemistry. Gd-DTPA-g-mimRGD produced a strong enhancement of the external structures of the aortic wall and of the more profound layers (possibly tunica media and intima). The aortic lumen seemed to be restrained and distorted. Pre-injection of Eu-DTPA-g-mimRGD diminished the Gd-DTPA-g-mimRGD binding to atherosclerotic plaque and confirmed the specific molecular targeting. A slower blood clearance was observed for Gd-DTPA-g-mimRGD, as indicated by a prolonged elimination half-life and a diminished total clearance. The new compound is potentially useful for the diagnosis of vulnerable atherosclerotic plaques and of other pathologies characterized by alpha v beta3 integrin expression, such as cancer and inflammation. The delayed blood clearance, the significant enhancement of the signal

Synthesis and biological evaluation of potent alphavbeta3-integrin receptor antagonists.

NARCIS (Netherlands)

Dijkgraaf, I.; Kruijtzer, J.A.; Frielink, C.; Soede, A.C.; Hilbers, H.W.; Oyen, W.J.G.; Corstens, F.H.M.; Liskamp, R.M.; Boerman, O.C.

2006-01-01

INTRODUCTION: alpha(v)beta(3) Integrin is expressed in sprouting endothelial cells in growing tumors, whereas it is absent in quiescent blood vessels. In addition, various tumor cell types express alpha(v)beta(3) integrin. alpha(v)beta(3) Integrin, a transmembrane heterodimeric protein, binds to the

Syndecan-4 and integrins: combinatorial signaling in cell adhesion

DEFF Research Database (Denmark)

Couchman, J R; Woods, A

1999-01-01

It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal...... during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding...... site for protein kinase C(&agr;) and phosphatidylinositol 4, 5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein...

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

LENUS (Irish Health Repository)

Krause-Gruszczynska, Malgorzata

2011-12-28

Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

Science.gov (United States)

Saher, G; Hildt, E

1999-09-24

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

Science.gov (United States)

Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik

2017-03-01

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.

Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo

DEFF Research Database (Denmark)

Grüner, Sabine; Prostredna, Miroslava; Schulte, Valerie

2003-01-01

by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in alpha2-null mice and even in mice with a Cre/loxP-mediated loss of all beta1 integrins on their platelets. In contrast, inhibition of alphaIIbbeta3...... integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, alphaIIbbeta3 inhibition had a comparable effect in alpha2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional alpha2beta1...... and alphaIIbbeta3. These were identified to be alpha5beta1 and/or alpha6beta1 as alphaIIbbeta3 inhibition abrogated platelet adhesion in beta1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin...

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

DEFF Research Database (Denmark)

Nieswandt, B; Brakebusch, C; Bergmeier, W

2001-01-01

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subs...

A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair

DEFF Research Database (Denmark)

Grose, Richard; Hutter, Caroline; Bloch, Wilhelm

2002-01-01

of beta 1 integrins in keratinocytes caused a severe defect in wound healing. beta 1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate was not reduced in early wounds and even increased in late wounds....... The failure in re-epithelialisation resulted in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, beta 1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate...

Effect of integrin α5β1 inhibition on SDF-l/CXCR4- mediated choroidal neovascularization

Directory of Open Access Journals (Sweden)

Yang Lyu

2018-05-01

Full Text Available AIM: To investigate the roles of integrins in choroidal neovascularization (CNV and their associations with the stromal cell-derived factor-1 (SDF-1/CXCR4 axis. METHODS: CNV lesions were induced in mice using laser photocoagulation. After CNV induction, all animals were randomly assigned to: control, SDF-1, SDF-1+age-related macular degeneration (AMD 3100 (CXCR4 inhibitor, and SDF-1+ATN161 (integrin α5β1 inhibitor groups; their effects on CNV progression were observed using hematoxylin eosin (HE staining, fundus fluorescein angiography (FFA grading and optical coherence tomography (OCT, and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining. Hypoxia-exposed endothelial cells (ECs were used to simulate CNV in vitro, they were treated with SDF-1, combined with CXCR4 siRNA/AMD3100 or ATN161, and expression of integrin α5, cell migration and tube formation were analyzed. RESULTS: Integrin subunit α5 increased at 3rd and 7th day and decreased at 14th day in CNV mice, with no significant change of β1-integrin. CXCR4 expression in CNV mice had persistent increase within 14d after induction. SDF-1 treatment significantly promoted the CNV progression during 3-14d. The mean CNV length in AMD3100 and ATN161 group at day 7 was 270.13 and 264.23 μm in HE images, significantly lower than the mean length in SDF-1 (345.70 μm group. AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1. Mean integrin α5 positive area in SDF-1 group reached 2.31×104 μm2, significantly higher than control (1.25×104 μm2, which decreased to 1.78×104 μm2 after AMD3100 treatment. About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group, which significantly decreased to 43.12% after AMD3100 treatment. In vitro, integrin α5 peaked by 6 folds after 6h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24h of hypoxia. Approximately 25

The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

Science.gov (United States)

Bolduc, Gilles R; Madoff, Lawrence C

2007-12-01

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

Directory of Open Access Journals (Sweden)

Krause-Gruszczynska Malgorzata

2011-12-01

Full Text Available Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

Directory of Open Access Journals (Sweden)

Stefanie Löffek

Full Text Available The hemidesmosomal transmembrane component collagen XVII (ColXVII plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

Small molecule antagonists of integrin receptors.

Science.gov (United States)

Perdih, A; Dolenc, M Sollner

2010-01-01

The complex and widespread family of integrin receptors is involved in numerous physiological processes, such as tissue remodeling, angiogenesis, development of the immune response and homeostasis. In addition, their key role has been elucidated in important pathological disorders such as cancer, cardiovascular diseases, osteoporosis, autoimmune and inflammatory diseases and in the pathogenesis of infectious diseases, making them highly important targets for modern drug design campaigns. In this review we seek to present a concise overview of the small molecule antagonists of this diverse and highly complex receptor family. Integrin antagonists are classified according to the targeted integrin receptor and are discussed in four sections. First we present the fibrinogen alpha(IIb)beta3 and the vitronectin alpha (V)beta(3) receptor antagonists. The remaining selective integrin antagonists are examined in the third section. The final section is dedicated to molecules with dual or multiple integrin activity. In addition, the use of antibodies and peptidomimetic approaches to modulate the integrin receptors are discussed, as well providing the reader with an overall appreciation of the field.

Integrin β4 Regulates Migratory Behavior of Keratinocytes by Determining Laminin-332 Organization*s

Science.gov (United States)

Sehgal, Bernd U.; DeBiase, Phillip J.; Matzno, Sumio; Chew, Teng-Leong; Claiborne, Jessica N.; Hopkinson, Susan B.; Russell, Alan; Marinkovich, M. Peter; Jones, Jonathan C. R.

2010-01-01

Whether α6β4 integrin regulates migration remains controversial. β4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express β4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type β4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with α6β4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the α6β4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells. PMID:16973601

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

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Tóth, Beáta; Garabuczi, Eva; Sarang, Zsolt; Vereb, György; Vámosi, György; Aeschlimann, Daniel; Blaskó, Bernadett; Bécsi, Bálint; Erdõdi, Ferenc; Lacy-Hulbert, Adam; Zhang, Ailiang; Falasca, Laura; Birge, Raymond B; Balajthy, Zoltán; Melino, Gerry; Fésüs, László; Szondy, Zsuzsa

2009-02-15

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

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Annika Sommerfeld

2015-05-01

Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

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Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

2009-08-15

To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

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Salter, D M; Godolphin, J L; Gourlay, M S

1995-04-01

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

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Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second

Functional inhibition of NF-kappa B signal transduction in alpha v alpha beta 3 integrin expressing endothelial cells by using RGD-PEG-modified adenovirus with a mutant I kappa B gene

NARCIS (Netherlands)

Ogawara, K; Kuldo, JM; Oosterhuis, K; Kroesen, BJ; Rots, MG; Trautwein, C; Kimura, T; Haisma, HJ; Molema, G

2006-01-01

In order to selectively block nuclear factor kappa B (NF-kappa B)-dependent signal transduction in angiogenic endothelial cells, we constructed an alpha v beta 3 integrin specific adenovirus encoding dominant negative I kappa B (dnI kappa B) as a therapeutic gene. By virtue of RGD modification of

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.

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Wooten, D K; Teague, T K; McIntyre, B W

1999-01-01

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

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Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Integrin Beta 3 Regulates Cellular Senescence by Activating the TGF-β Pathway

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Valentina Rapisarda

2017-03-01

Full Text Available Cellular senescence is an important in vivo mechanism that prevents the propagation of damaged cells. However, the precise mechanisms regulating senescence are not well characterized. Here, we find that ITGB3 (integrin beta 3 or β3 is regulated by the Polycomb protein CBX7. β3 expression accelerates the onset of senescence in human primary fibroblasts by activating the transforming growth factor β (TGF-β pathway in a cell-autonomous and non-cell-autonomous manner. β3 levels are dynamically increased during oncogene-induced senescence (OIS through CBX7 Polycomb regulation, and downregulation of β3 levels overrides OIS and therapy-induced senescence (TIS, independently of its ligand-binding activity. Moreover, cilengitide, an αvβ3 antagonist, has the ability to block the senescence-associated secretory phenotype (SASP without affecting proliferation. Finally, we show an increase in β3 levels in a subset of tissues during aging. Altogether, our data show that integrin β3 subunit is a marker and regulator of senescence.

In vivo formation of beta-oxidized metabolites of leukotriene E4 in the rat

International Nuclear Information System (INIS)

Perrin, P.; Zirrolli, J.; Stene, D.O.; Lellouche, J.P.; Beaucourt, J.P.; Murphy, R.C.

1989-01-01

Intraperitoneal administration of [ 3 H]-leukotriene E4 in the rat resulted in the appearance of radiolabel in urine and feces. Separation of polar urinary metabolites and chromatographic comparison of synthetic metabolites indicated the in vivo formation of omega-oxidized metabolites of LTE4 with sequential beta-oxidation. Furthermore, the metabolite identified as 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl-LTE4 substantiates the biochemical pathway of beta-oxidation in vivo involving the 2,4-dienoyl CoA reductase as an integral step. These results substantiate beta-oxidation of sulfidopeptide leukotrienes in vivo and these metabolites account for some of the major urinary metabolites of this class of lipid mediator

Ligand-Occupied Integrin Internalization Links Nutrient Signaling to Invasive Migration

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Elena Rainero

2015-01-01

Full Text Available Integrin trafficking is key to cell migration, but little is known about the spatiotemporal organization of integrin endocytosis. Here, we show that α5β1 integrin undergoes tensin-dependent centripetal movement from the cell periphery to populate adhesions located under the nucleus. From here, ligand-engaged α5β1 integrins are internalized under control of the Arf subfamily GTPase, Arf4, and are trafficked to nearby late endosomes/lysosomes. Suppression of centripetal movement or Arf4-dependent endocytosis disrupts flow of ligand-bound integrins to late endosomes/lysosomes and their degradation within this compartment. Arf4-dependent integrin internalization is required for proper lysosome positioning and for recruitment and activation of mTOR at this cellular subcompartment. Furthermore, nutrient depletion promotes subnuclear accumulation and endocytosis of ligand-engaged α5β1 integrins via inhibition of mTORC1. This two-way regulatory interaction between mTORC1 and integrin trafficking in combination with data describing a role for tensin in invasive cell migration indicate interesting links between nutrient signaling and metastasis.

Integrin beta3 Leu33Pro polymorphism and risk of hip fracture: 25 years follow-up of 9233 adults from the general population

DEFF Research Database (Denmark)

Tofteng, Charlotte L; Bach-Mortensen, Pernille; Bojesen, Stig E

2007-01-01

OBJECTIVE: Integrin alphavbeta3 is essential for mature osteoclast function and therefore important for the development of osteoporosis and osteoporotic fractures. Integrin alphavbeta3 antagonists have antiresorptive effects in bone. We tested the hypothesis that the Leu33Pro polymorphism...... in the integrin beta3-subunit associates with risk of hip fracture. METHODS: We included 9233 men and women selected at random to represent the Danish general population as participants in the Copenhagen City Heart Study. First-ever hip fractures (n=267) were registered during 25 years follow-up. Log...

ADAM12/syndecan-4 signaling promotes beta 1 integrin-dependent cell spreading through protein kinase Calpha and RhoA

DEFF Research Database (Denmark)

Thodeti, Charles Kumar; Albrechtsen, Reidar; Grauslund, Morten

2002-01-01

The ADAMs (a disintegrin and metalloprotease) comprise a large family of multidomain proteins with cell-binding and metalloprotease activities. The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell attachment using syndecan-4 as a primary cell surface receptor that subsequently triggers beta...

RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

Science.gov (United States)

De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini

2017-01-10

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

Science.gov (United States)

Maxson, Michelle E; Naj, Xenia; O'Meara, Teresa R; Plumb, Jonathan D; Cowen, Leah E

2018-01-01

Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae. PMID:29553370

ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function

DEFF Research Database (Denmark)

Kawaguchi, Nobuko; Sundberg, Christina; Kveiborg, Marie

2003-01-01

-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary...

Loss of ADAM9 expression impairs β1 integrin endocytosis, focal adhesion formation and cancer cell migration

DEFF Research Database (Denmark)

Mygind, Kasper J; Schwarz, Jeanette; Sahgal, Pranshu

2018-01-01

knockdown increases β1 integrin levels through mechanisms that are independent of its protease activity. In ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with β1 integrin......, and both internalization and subsequent degradation of β1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on β1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction...

Feasibility of [18F]-RGD for ex vivo imaging of atherosclerosis in detection of alpha v beta 3 integrin expression

NARCIS (Netherlands)

Golestani, Reza; Mirfeizi, Leila; Zeebregts, Clark J.; Westra, Johanna; de Haas, Hans J.; Glaudemans, Andor W. J. M.; Koole, Michel; Luurtsema, Gert; Tio, Rene A.; Dierckx, Rudi A. J. O.; Boersma, Hendrikus H.; Elsinga, Philip H.; Slart, Riemer H. J. A.

2015-01-01

Background. Inflammation and angiogenesis play an important role in atherosclerotic plaque rupture. Therefore, molecular imaging of these processes could be used for determination of rupture-prone atherosclerotic plaques. alpha v beta 3 integrin is involved in the process of angiogenesis. Targeted

The integrin α6β4 as a signaling membrane protein for a damage response to ionizing radiation in human prostate cancer cell lines

International Nuclear Information System (INIS)

Woo, Charles; Nagle, Ray B.; Stea, Baldassarre; Cress, Anne E.

1996-01-01

Purpose/Object: Integrins are cell surface receptors that exist as heterodimers. The integrin α6β4 is a receptor for laminin and is present in normal human prostate tissue. In prostate carcinoma however, there is loss of β4 expression. Prior studies demonstrated that when a low β4 expressing rectal carcinoma cell line was transfected with β4, the cells underwent apoptosis. We investigated the effects that the β4 integrin had on DNA damage responses in a human prostate carcinoma line. Materials and Methods: DU-145 human prostate carcinoma cells previously selected by us for α6β1 expression were transfected with either a full length β4 construct or vector only. Both cell lines were grown simultaneously and maintained in geneticin for selection purposes. Cells were grown on glass coverslips in 60mm tissue culture dishes under optimal growth conditions. Radiation was delivered using a Co-60 machine with a dose rate of 35 Gy/hr. The cells were given 0, 2, 5, and 10 Gy. Three different radiation damage responses were assayed and include micronuclei (MN) formation, cell cycle distribution, and cell survival. 24 hours after irradiation, the cells were fixed and stained with propidium iodide. Micronuclei formation was detected using a Zeiss LSM10 confocal microscope, and the resulting digital images were analyzed using the NIH Image program. The observed MN were detected without the use of cytochalasin B, but were noted to contain nuclear histone and DNA and were morphologically distinct from apoptotic or necrotic bodies. Results: The quantitative analysis of MN formation revealed a radiation dose dependence of MN formation in both the α6β4 and α6β1 expressing cell lines. The presence of MN 24 hours after irradiation was observed at clinically significant doses (2 Gy) with the largest effect occurring at 5 Gy. The α6β4 expressing cells consistently produced approximately two fold more MN as compared to the α6β1 expressing cells at all radiation doses. The

MFAP4 Promotes Vascular Smooth Muscle Migration, Proliferation and Accelerates Neointima Formation

DEFF Research Database (Denmark)

Schlosser, Anders; Pilecki, Bartosz; Hemstra, Line E.

2016-01-01

in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation. APPROACH AND RESULTS: We produced Mfap4-deficient (Mfap4(-/-)) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular...... kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin αVβ3-dependent manner. CONCLUSIONS: MFAP4 regulates integrin αVβ3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular...

A novel role for integrin-linked kinase in epithelial sheet morphogenesis.

Science.gov (United States)

Vespa, Alisa; D'Souza, Sudhir J A; Dagnino, Lina

2005-09-01

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.

Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

Science.gov (United States)

Mattheij, Nadine J A; Gilio, Karen; van Kruchten, Roger; Jobe, Shawn M; Wieschhaus, Adam J; Chishti, Athar H; Collins, Peter; Heemskerk, Johan W M; Cosemans, Judith M E M

2013-05-10

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

DEFF Research Database (Denmark)

Bergmeier, W; Bouvard, D; Eble, J A

2001-01-01

Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the v...

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

Science.gov (United States)

Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

2013-01-01

Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

Science.gov (United States)

Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

2009-09-01

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

Conservation of the human integrin-type beta-propeller domain in bacteria.

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Bhanupratap Chouhan

Full Text Available Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca(2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca(2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and

Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells.

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Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

2017-01-01

The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth

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Carlstrom Lucas P

2011-11-01

Full Text Available Abstract Background Chemotropic factors in the extracellular microenvironment guide nerve growth by acting on the growth cone located at the tip of extending axons. Growth cone extension requires the coordination of cytoskeleton-dependent membrane protrusion and dynamic adhesion to the extracellular matrix, yet how chemotropic factors regulate these events remains an outstanding question. We demonstrated previously that the inhibitory factor myelin-associated glycoprotein (MAG triggers endocytic removal of the adhesion receptor β1-integrin from the growth cone surface membrane to negatively remodel substrate adhesions during chemorepulsion. Here, we tested how a neurotrophin might affect integrin adhesions. Results We report that brain-derived neurotropic factor (BDNF positively regulates the formation of substrate adhesions in axonal growth cones during stimulated outgrowth and prevents removal of β1-integrin adhesions by MAG. Treatment of Xenopus spinal neurons with BDNF rapidly triggered β1-integrin clustering and induced the dynamic formation of nascent vinculin-containing adhesion complexes in the growth cone periphery. Both the formation of nascent β1-integrin adhesions and the stimulation of axon extension by BDNF required cytoplasmic calcium ion signaling and integrin activation at the cell surface. Exposure to MAG decreased the number of β1-integrin adhesions in the growth cone during inhibition of axon extension. In contrast, the BDNF-induced adhesions were resistant to negative remodeling by MAG, correlating with the ability of BDNF pretreatment to counteract MAG-inhibition of axon extension. Pre-exposure to MAG prevented the BDNF-induced formation of β1-integrin adhesions and blocked the stimulation of axon extension by BDNF. Conclusions Altogether, these findings demonstrate the neurotrophin-dependent formation of integrin-based adhesions in the growth cone and reveal how a positive regulator of substrate adhesions can block

PI3-kinase γ promotes Rap1a-mediated activation of myeloid cell integrin α4β1, leading to tumor inflammation and growth.

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Michael C Schmid

Full Text Available Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4β1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX, while activated Rap (RapV12 promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4β1. Similar to blockade of PI3Kγ or integrin α4β1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.

Cross-talk between integrins α1β1 and α2β1 in renal epithelial cells

International Nuclear Information System (INIS)

Abair, Tristin D.; Sundaramoorthy, Munirathinam; Chen, Dong; Heino, Jyrki; Ivaska, Johanna; Hudson, Billy G.; Sanders, Charles R.; Pozzi, Ambra; Zent, Roy

2008-01-01

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function

Integrin Beta 1 Suppresses Multilayering of a Simple Epithelium

Science.gov (United States)

Chen, Jichao; Krasnow, Mark A.

2012-01-01

Epithelia are classified as either simple, a single cell layer thick, or stratified (multilayered). Stratified epithelia arise from simple epithelia during development, and transcription factor p63 functions as a key positive regulator of epidermal stratification. Here we show that deletion of integrin beta 1 (Itgb1) in the developing mouse airway epithelium abrogates airway branching and converts this monolayer epithelium into a multilayer epithelium with more than 10 extra layers. Mutant lung epithelial cells change mitotic spindle orientation to seed outer layers, and cells in different layers become molecularly and functionally distinct, hallmarks of normal stratification. However, mutant lung epithelial cells do not activate p63 and do not switch to the stratified keratin profile of epidermal cells. These data, together with previous data implicating Itgb1 in regulation of epidermal stratification, suggest that the simple-versus-stratified developmental decision may involve not only stratification inducers like p63 but suppressors like Itgb1 that prevent simple epithelia from inappropriately activating key steps in the stratification program. PMID:23285215

Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin

DEFF Research Database (Denmark)

Hui, Tommy; Sørensen, Esben Skipper; Rittling, Susan R.

2015-01-01

Osteopontin (OPN) is a ligand for the α4 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of posttranslational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines, and compared OPN interaction...

ADAM2 interactions with mouse eggs and cell lines expressing α4/α9 (ITGA4/ITGA9 integrins: implications for integrin-based adhesion and fertilization.

Directory of Open Access Journals (Sweden)

Ulyana V Desiderio

2010-10-01

Full Text Available Integrins are heterodimeric cell adhesion molecules, with 18 α (ITGA and eight β (ITGB subunits forming 24 heterodimers classified into five families. Certain integrins, especially the α(4/α(9 (ITGA4/ITGA9 family, interact with members of the ADAM (a disintegrin and metalloprotease family. ADAM2 is among the better characterized and also of interest because of its role in sperm function. Having shown that ITGA9 on mouse eggs participates in mouse sperm-egg interactions, we sought to characterize ITGA4/ITGA9-ADAM2 interactions.An anti-β(1/ITGB1 function-blocking antibody that reduces sperm-egg binding significantly inhibited ADAM2 binding to mouse eggs. Analysis of integrin subunit expression indicates that mouse eggs could express at least ten different integrins, five in the RGD-binding family, two in the laminin-binding family, two in the collagen-binding family, and ITGA9-ITGB1. Adhesion assays to characterize ADAM2 interactions with ITGA4/ITGA9 family members produced the surprising result that RPMI 8866 cell adhesion to ADAM2 was inhibited by an anti-ITGA9 antibody, noteworthy because ITGA9 has only been reported to dimerize with ITGB1, and RPMI 8866 cells lack detectable ITGB1. Antibody and siRNA studies demonstrate that ITGB7 is the β subunit contributing to RPMI 8866 adhesion to ADAM2.These data indicate that a novel integrin α-β combination, ITGA9-ITGB7 (α(9β(7, in RPMI 8866 cells functions as a binding partner for ADAM2. ITGA9 had previously only been reported to dimerize with ITGB1. Although ITGA9-ITGB7 is unlikely to be a widely expressed integrin and appears to be the result of "compensatory dimerization" occurring in the context of little/no ITGB1 expression, the data indicate that ITGA9-ITGB7 functions as an ADAM binding partner in certain cellular contexts, with implications for mammalian fertilization and integrin function.

Natalizumab plus interferon beta-1a for relapsing multiple sclerosis.

NARCIS (Netherlands)

Rudick, R.A.; Stuart, W.H.; Calabresi, P.A.; Confavreux, C.; Galetta, S.L.; Radue, E.W.; Lublin, F.D.; Weinstock-Guttman, B.; Wynn, D.R.; Lynn, F.; Panzara, M.A.; Sandrock, A.W.

2006-01-01

BACKGROUND: Interferon beta is used to modify the course of relapsing multiple sclerosis. Despite interferon beta therapy, many patients have relapses. Natalizumab, an alpha4 integrin antagonist, appeared to be safe and effective alone and when added to interferon beta-1a in preliminary studies.

Cellular and substrate adhesion molecules (integrins) and their ligands in cerebral amyloid plaques in Alzheimer's disease

NARCIS (Netherlands)

Eikelenboom, P.; Zhan, S. S.; Kamphorst, W.; van der Valk, P.; Rozemuller, J. M.

1994-01-01

Integrins belonging to different subfamilies can be identified immunohistochemically in cerebral amyloid plaques. Monoclonal antibodies against the VLA family beta 1-integrins show staining of the corona of classical amyloid plaques for beta 1, alpha 3 and alpha 6. Immunostaining reveal also the

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

Science.gov (United States)

Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

1999-03-01

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even

Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

Science.gov (United States)

El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

2002-01-01

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key

Integrin α4 Enhances Metastasis and May Be Associated with Poor Prognosis in MYCN-low Neuroblastoma.

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Shanique A Young

Full Text Available High-risk neuroblastoma is associated with an overall survival rate of 30-50%. Neuroblastoma-expressed cell adhesion receptors of the integrin family impact cell adhesion, migration, proliferation and survival. Integrin α4 is essential for neural crest cell motility during development, is highly expressed on leukocytes, and is critical for transendothelial migration. Thus, cancer cells that express this receptor may exhibit increased metastatic potential. We show that α4 expression in human and murine neuroblastoma cell lines selectively enhances in vitro interaction with the alternatively spliced connecting segment 1 of fibronectin, as well as vascular cell adhesion molecule-1 and increases migration. Integrin α4 expression enhanced experimental metastasis in a syngeneic tumor model, reconstituting a pattern of organ involvement similar to that seen in patients. Accordingly, antagonism of integrin α4 blocked metastasis, suggesting adhesive function of the integrin is required. However, adhesive function was not sufficient, as mutants of integrin α4 that conserved the matrix-adhesive and promigratory function in vitro were compromised in their metastatic capacity in vivo. Clinically, integrin α4 is more frequently expressed in non-MYNC amplified tumors, and is selectively associated with poor prognosis in this subset of disease. These results reveal an unexpected role for integrin α4 in neuroblastoma dissemination and identify α4 as a potential prognostic indicator and therapeutic target.

Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Caneva Soumetz, Federico [Department of Communication, Computer and System Sciences, University of Genova, Via Opera Pia, 13-16145 Genova (Italy); Saenz, Jose F. [Biophysical and Electronic Engineering Department, University of Genova, Via All' Opera Pia 11a, 16145 Genova (Italy); Pastorino, Laura; Ruggiero, Carmelina [Department of Communication, Computer and System Sciences, University of Genova, Via Opera Pia, 13-16145 Genova (Italy); Nosi, Daniele [Department of Anatomy, Histology and Forensic Medicine, Bio-photonic Laboratory, University of Florence, viale Morgagni, 85 Firenze, CAP 50134 Florence (Italy); Raiteri, Roberto, E-mail: rr@unige.it [Biophysical and Electronic Engineering Department, University of Genova, Via All' Opera Pia 11a, 16145 Genova (Italy)

2010-03-15

The transforming growth factor {beta}1 (TGF-{beta}1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-{beta}1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalised with monoclonal antibodies specific to the {beta}1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-{beta}1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the {beta}1 integrin subunit was enhanced by TGF-{beta}1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-{beta}1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.

Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

Science.gov (United States)

Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K

1997-11-01

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

ADAM disintegrin-like domain recognition by the lymphocyte integrins α4β1 and α4β7

OpenAIRE

Bridges, Lance C.; Sheppard, Dean; Bowditch, Ron D.

2005-01-01

The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin α4β1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins α4β1, α4β7 and α9β1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 d...

Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin ?5?1 and Fibronectin

Energy Technology Data Exchange (ETDEWEB)

Nam, Jin-Min; Onodera, Yasuhito; Bissell, Mina J; Park, Catherine C

2010-04-07

Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that {beta}1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific {alpha} heterodimer of {beta}1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare {alpha}-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of {alpha}5{beta}1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of {alpha}5{beta}1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of {alpha}5{beta}1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of {alpha}5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and {alpha}5{beta}1-integrin as targets for breast cancer therapy.

Cross-talk between Integrin α6β4 and Insulin-like Growth Factor-1 Receptor (IGF1R) through Direct α6β4 Binding to IGF1 and Subsequent α6β4-IGF1-IGF1R Ternary Complex Formation in Anchorage-independent Conditions*

OpenAIRE

Fujita, Masaaki; Ieguchi, Katsuaki; Davari, Parastoo; Yamaji, Satoshi; Taniguchi, Yukimasa; Sekiguchi, Kiyotoshi; Takada, Yoko K.; Takada, Yoshikazu

2012-01-01

Background: Integrin αvβ3-extracellular matrix interaction and/or αvβ3 binding to insulin-like growth factor-1 (IGF1; and integrin-IGF1-IGF1 receptor ternary complex formation) is critical for IGF signaling.

Entamoeba histolytica: a beta 1 integrin-like fibronectin receptor assembles a signaling complex similar to those of mammalian cells.

Science.gov (United States)

Flores-Robles, Donaciano; Rosales, Carlos; Rosales-Encina, José Luis; Talamás-Rohana, Patricia

2003-01-01

During tissue invasion, Entamoeba histolytica trophozoites interact with endothelial cells and extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, and laminin. It has been demonstrated that trophozoites interact with FN through a beta1 integrin-like FN receptor (beta 1EhFNR), activating tyrosine kinases. In order to characterize the signaling process triggered by the amoebic receptor, activation, and association of tyrosine kinases and structural proteins were determined. As a result of FN binding by the beta 1EhFNR, the receptor itself, FAK, and paxillin were phosphorylated in tyrosine. Co-immunoprecipitation experiments showed that a multimolecular signaling complex was formed by the amoebic FN receptor, FAK, paxillin, and vinculin. These results strongly suggest that a signaling pathway, similar to the one used in mammalian cells, is activated when E. histolytica trophozoites adhere to FN.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

Science.gov (United States)

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Indium-labelled human gut-derived T cells from healthy subjects with strong in vitro adhesion to MAdCAM-1 show no detectable homing to the gut in vivo

DEFF Research Database (Denmark)

Rømer, Johanne Lade

2004-01-01

Integrin alpha4beta 7 is the principal gut-homing receptor, and it is assumed that expression of this specific integrin directs lymphocytes to the gut in vivo. Adoptive cellular immunotherapy against inflammatory bowel disease (IBD) may depend on the expression of integrin alpha4beta 7...

The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.

OpenAIRE

Briesewitz, R; Kern, A; Smilenov, L B; David, F S; Marcantonio, E E

1996-01-01

Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor l...

Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

OpenAIRE

Case, Lindsay B.; Waterman, Clare M.

2011-01-01

At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the ex...

Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: evidence of a role for alpha 5 beta 1 integrin as a chondrocyte mechanoreceptor.

Science.gov (United States)

Wright, M O; Nishida, K; Bavington, C; Godolphin, J L; Dunne, E; Walmsley, S; Jobanputra, P; Nuki, G; Salter, D M

1997-09-01

Mechanical stimuli influence chondrocyte metabolism, inducing changes in intracellular cyclic adenosine monophosphate and proteoglycan production. We have previously demonstrated that primary monolayer cultures of human chondrocytes have an electrophysiological response after intermittent pressure-induced strain characterised by a membrane hyperpolarisation of approximately 40%. The mechanisms responsible for these changes are not fully understood but potentially involve signalling molecules such as integrins that link extracellular matrix with cytoplasmic components. The results reported in this paper demonstrate that the transduction pathways involved in the hyperpolarisation response of human articular chondrocytes in vitro after cyclical pressure-induced strain involve alpha 5 beta 1 integrin. We have demonstrated, using pharmacological inhibitors of a variety of intracellular signalling pathways, that the actin cytoskeleton, the phospholipase C calmodulin pathway, and both tyrosine protein kinase and protein kinase C activities are important in the transduction of the electrophysiological response. These results suggest that alpha 5 beta 1 is an important chondrocyte mechanoreceptor and a potential regulator of chondrocyte function.

Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

Energy Technology Data Exchange (ETDEWEB)

Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

1998-06-29

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9, CD81, and CD98.

Science.gov (United States)

Takahashi, Y; Bigler, D; Ito, Y; White, J M

2001-04-01

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-alpha6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-alpha6 mAb, or by mAbs against either the alphav or beta3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other beta1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg beta1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface "tetraspan web" facilitates fertilization and that it may do so by fostering ADAM-integrin interactions.

The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype.

Science.gov (United States)

Shekaran, Asha; Shoemaker, James T; Kavanaugh, Taylor E; Lin, Angela S; LaPlaca, Michelle C; Fan, Yuhong; Guldberg, Robert E; García, Andrés J

2014-11-01

-lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the osteocalcin-specific β1 integrin deletion had only minor effects on skeletal phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

Alterations in integrin expression modulates invasion of pancreatic cancer cells.

LENUS (Irish Health Repository)

Walsh, Naomi

2009-01-01

BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

BMP-7 enhances cell migration and αvβ3 integrin expression via a c-Src-dependent pathway in human chondrosarcoma cells.

Directory of Open Access Journals (Sweden)

Jui-Chieh Chen

Full Text Available Bone morphogenic protein (BMP-7 is a member of the transforming growth factor (TGF-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

DEFF Research Database (Denmark)

Whiteford, James; Behrends, Volker; Kirby, Hishani

2007-01-01

and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any...... epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable...

Gd-EDDA/HYNIC-RGD as an MR molecular probe imaging integrin {alpha}{nu}{beta}3 receptor-expressed tumor-MR molecular imaging of angiogenesis

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Huo Tianlong [Peking University People' s Hospital, Radiology Department, 11 Xizhimen South Street, Xicheng District, Beijing 100044 (China)], E-mail: huotianlong@bjmu.edu.cn; Du Xiangke [Peking University People' s Hospital, Radiology Department, 11 Xizhimen South Street, Xicheng District, Beijing 100044 (China)], E-mail: duxk@263.net; Zhang Sen [Peking University People' s Hospital, Radiology Department, 11 Xizhimen South Street, Xicheng District, Beijing 100044 (China)], E-mail: skagerrak_s@yahoo.com.cn; Liu Xia [Peking University People' s Hospital, Radiology Department, 11 Xizhimen South Street, Xicheng District, Beijing 100044 (China)], E-mail: iamliuxia@126.com; Li Xubing [Peking University People' s Hospital, Radiology Department, 11 Xizhimen South Street, Xicheng District, Beijing 100044 (China)], E-mail: lixb@bjmu.edu.cn

2010-02-15

Rationale and objective: The aim of this study is to develop a novel MR probe containing arginine-glycine-aspartic acid (RGD) motif for imaging integrin {alpha}{nu}{beta}3 receptor-expressed tumor. Materials and methods: Commercially available HYNIC-RGD conjugated with co-ligand EDDA was labeled with Gd{sup 3+}, and the mixture was isolated and purified by solid phase extract (SPE) to get the entire probe Gd-EDDA/HYNIC-RGD. Human hepatocellular carcinoma (HHCC) cell line BEL-7402 was cultured and the cells harvested and suspended in serum-free Dulbecco's modified Eagle medium (DMEM) were subcutaneously inoculated into athymic nude mice for tumor growth. In vitro cell binding assay to integrin {alpha}{nu}{beta}3 receptor and cell viability experiments were conducted. The in vivo imaging of the three arms of xenografts were performed by MR scan with a dedicated animal coil at time points of 0, 30, 60, 90 min and 24-h post-intravenous injection (p.i.). Three arms of nude mice then were sacrificed for histological examination to confirm the imaging results. Results: Gd-EDDA/HYNIC-RGD was successfully isolated by SPE and validity was verified on signal enhancement through in vitro and in vivo experiments. The nude mice model bearing HHCC was well established. There was approx. 30% signal enhancement on T1WI FSE images at 90 min post-intravenous injection of the Gd-EDDA/HYNIC-RGD compared with baseline, and the signal to time curve is straightforward over time in the span of 0-90 min p.i., while the control arms do not show this tendency. Conclusion: Gd-EDDA/HYNIC-RGD has the potential to serve as an MR probe detecting integrin {alpha}{nu}{beta}3 receptor-expressed tumor.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms.

Science.gov (United States)

Lee, JeHoon; Banu, Sakhila K; Burghardt, Robert C; Starzinski-Powitz, Anna; Arosh, Joe A

2013-03-01

Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.

beta 1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts

International Nuclear Information System (INIS)

Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.

2008-01-01

β1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of β1 integrin signaling. We showed previously that β1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and β1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo

Integrin β1 regulates leiomyoma cytoskeletal integrity and growth

Science.gov (United States)

Malik, Minnie; Segars, James; Catherino, William H.

2014-01-01

Uterine leiomyomas are characterized by an excessive extracellular matrix, increased mechanical stress, and increased active RhoA. Previously, we observed that mechanical signaling was attenuated in leiomyoma, but the mechanisms responsible remain unclear. Integrins, especially integrin β1, are transmembrane adhesion receptors that couple extracellular matrix stresses to the intracellular cytoskeleton to influence cell proliferation and differentiation. Here we characterized integrin and laminin to signaling in leiomyoma cells. We observed a 2.25 ± 0.32 fold increased expression of integrin β1 in leiomyoma cells, compared to myometrial cells. Antibody-mediated inhibition of integrin β1 led to significant growth inhibition in leiomyoma cells and a loss of cytoskeletal integrity. Specifically, polymerization of actin filaments and formation of focal adhesions were reduced by inhibition of integrin p1. Inhibition of integrin β1 in leiomyoma cells led to 0.81 ± 0.02 fold decrease in active RhoA, and resembled levels found in serum-starved cells. Likewise, inhibition of integrin β1 was accompanied by a decrease in phospho-ERK. Compared to myometrial cells, leiomyoma cells demonstrated increased expression of integrin α6 subunit to laminin receptor (1.91 ± 0.11 fold), and increased expression of laminin 5α (1.52±0.02), laminin 5β (3.06±0.92), and laminin 5γ (1.66 ± 0.06). Of note, leiomyoma cells grown on laminin matrix appear to realign themselves. Taken together, the findings reveal that the attenuated mechanical signaling in leiomyoma cells is accompanied by an increased expression and a dependence on integrin β1 signaling in leiomyoma cells, compared to myometrial cells. PMID:23023061

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state.

Science.gov (United States)

Zhang, Y; Chen, M; Venugopal, S; Zhou, Y; Xiang, W; Li, Y-H; Lin, Q; Kini, R M; Chong, Y-S; Ge, R

2011-05-05

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel 'RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

Endothelial-Rac1 is not required for tumor angiogenesis unless alphavbeta3-integrin is absent.

Directory of Open Access Journals (Sweden)

Gabriela D'Amico

2010-03-01

Full Text Available Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial beta3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in beta3-null mice are all Rac1-dependent. These data indicate that in the presence of alphavbeta3-integrin Rac1 is not required for tumor angiogenesis.

Function of Etk in Growth Factor Receptor Signaling to Integrins in Breast Cancer

National Research Council Canada - National Science Library

Shimizu, Yoji

2001-01-01

The central hypothesis in this IDEA Award is that increased integrin-mediated adhesiveness and migration of breast cancer cells in response to stimulation by the growth factors heregulin beta (HRG beta...

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

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Barja-Fidalgo C.

2005-01-01

Full Text Available Extracellular matrix proteins and cell adhesion receptors (integrins play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

Science.gov (United States)

Mierke, Claudia Tanja

2013-01-01

The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock

The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

International Nuclear Information System (INIS)

Mierke, Claudia Tanja

2013-01-01

The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3 high or αvβ3 low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3 high cells showed a threefold increased cell invasion compared to αvβ3 low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3 high cells but not in αvβ3 low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3 low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3 high cells, whereas the invasiveness of β3 specific knock

Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

Science.gov (United States)

Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

1999-01-01

Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

Effect of adduct formation on valent state of cerium in its. beta. -diketonates

Energy Technology Data Exchange (ETDEWEB)

Spitsyn, V.I.; Martynenko, L.I.; Pechurova, N.I.; Snezhko, N.I.; Murav' eva, I.A.; Anufrieva, S.I. (Moskovskij Gosudarstvennyj Univ. (USSR))

1982-04-01

Physicochemical investigation of the system cerium (III, IV)-..beta..-diketone-additional ligand shows that ..beta..-diketonate ability to adduct formation decreases in the series tenoyltrifluoro-acetonate > acetylacetonate > dibenzoylmethanate > benzoylmethanate. Adduct formation of the cerium (III, IV) ..beta..-diketonates stabilizes cerium in trivalent condition, while oxidation degree 4+ is stable in tetrakis-..beta..-diketonates. The additional ligands are arranged in the series: tributhylphosphate < trioctyl-phosphineoxide < triphenylphosphineoxide < ..cap alpha.., ..cap alpha..'-dipyridyl < o-phenanthroline by the effect on cerium (III) stabilization in its ..beta..-diketonates.

Alpha-enolase (ENO1 controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis

Directory of Open Access Journals (Sweden)

Moitza Principe

2017-01-01

Full Text Available Abstract Background We have previously shown that in pancreatic ductal adenocarcinoma (PDA cells, the glycolytic enzyme alpha-enolase (ENO1 also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1 PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM. The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN via urokinase plasminogen activator receptor (uPAR. Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell

Inhibition of cell migration by focal adhesion kinase: Time-dependent difference in integrin-induced signaling between endothelial and hepatoblastoma cells.

Science.gov (United States)

Yu, Hongchi; Gao, Min; Ma, Yunlong; Wang, Lijuan; Shen, Yang; Liu, Xiaoheng

2018-05-01

angiogenesis plays an important role in the development and progression of tumors, and it involves a series of signaling pathways contributing to the migration of endothelial cells for vascularization and to the invasion of cancer cells for secondary tumor formation. Among these pathways, the focal adhesion kinase (FAK) signaling cascade has been implicated in a variety of human cancers in connection with cell adhesion and migration events leading to tumor angiogenesis, metastasis and invasion. Therefore, the inhibition of FAK in endothelial and/or cancer cells is a potential target for anti‑angiogenic therapy. In the present study, a small‑molecule FAK inhibitor, 1,2,4,5-benzenetetramine tetrahydrochloride (Y15), was used to study the effects of FAK inhibition on the adhesion and migration behaviors of vascular endothelial cells (VECs) and human hepatoblastoma cells. Furthermore, the time-dependent differences in proteins associated with the integrin-mediated FAK/Rho GTPases signaling pathway within 2 h were examined. The results indicated that the inhibition of FAK significantly decreased the migration ability of VECs and human hepatoblastoma cells in a dose-dependent manner. Inhibition of FAK promoted cell detachment by decreasing the expression of focal adhesion components, and blocked cell motility by reducing the level of Rho GTPases. However, the expression of crucial proteins involved in integrin-induced signaling in two cell lines exhibited a time-dependent difference with increased duration of FAK inhibitor treatment, suggesting different mechanisms of FAK-mediated cell migration behavior. These results suggest that the mechanism underlying FAK-mediated adhesion and migration behavior differs among various cells, which is expected to provide evidence for future FAK therapy targeted against tumor angiogenesis.

Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbβ3 integrin ligand binding affinity.

Directory of Open Access Journals (Sweden)

Joel Raborn

Full Text Available The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala(252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala(252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

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Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina

2012-10-01

Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

The recognition of adsorbed and denatured proteins of different topographies by β2 integrins and effects on leukocyte adhesion and activation

DEFF Research Database (Denmark)

Brevig, T.; Holst, B.; Ademovic, Z.

2005-01-01

Leukocyte beta(2) integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography...

The role of mechanical force and ROS in integrin-dependent signals.

Directory of Open Access Journals (Sweden)

Kathrin S Zeller

Full Text Available Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5β1 or αvβ3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and

Catfish thrombocytes express an integrin-like CD41/CD61 complex.

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Passer, B J; Chen, C H; Miller, N W; Cooper, M D

1997-08-01

A thrombocyte-specific antigen was identified in two closely related catfish, Ictalurus punctatus and Ictalurus furcatus, by monoclonal antibodies 4-20 and 7-2. The antibodies immunoprecipitate two noncovalently associated glycoprotein chains of Mr 180,000 and Mr 95,000. Under reducing conditions the Mr 180,000 chain is resolved into Mr 150,000 and 32,000 subcomponents. Analysis of N-terminal amino acid sequences indicates homology of the Mr 95,000 chain with the beta3 integrin subunit and homology of the Mr 150,000 chain with the alphaIIb integrin subunit. These antibodies induce catfish thrombocyte aggregation and alteration of cell shape. The data indicate conservation of the megakaryocyte/platelet-restricted CD41/CD61 complex in bony fish.

Upregulated expression of integrin α1 in mesangial cells and integrin α3 and vimentin in podocytes of Col4a3-null (Alport mice.

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Brooke M Steenhard

Full Text Available Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV networks found in mature kidney glomerular basement membrane (GBM. The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV. This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type, and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

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David Garciadiego-Cázares

Full Text Available The Integrin β1 family is the major receptors of the Extracellular matrix (ECM, and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA. In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β Superfamily, such as Growth differentiation factor 5 (Gdf-5 and Bone morphogenetic protein 7 (Bmp-7, play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh, Gdf-5 and α5 integrin to maintain articular cartilage and prevent

Noninvasive imaging of tumor integrin expression using 18F-labeled RGD dimer peptide with PEG4 linkers

International Nuclear Information System (INIS)

Liu, Zhaofei; Liu, Shuanglong; Wang, Fan; Liu, Shuang; Chen, Xiaoyuan

2009-01-01

Various radiolabeled Arg-Gly-Asp (RGD) peptides have been previously investigated for tumor integrin α v β 3 imaging. To further develop RGD radiotracers with enhanced tumor-targeting efficacy and improved in vivo pharmacokinetics, we designed a new RGD homodimeric peptide with two PEG 4 spacers (PEG 4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) between the two monomeric RGD motifs and one PEG 4 linker on the glutamate α-amino group ( 18 F-labeled PEG 4 -E[PEG 4 -c(RGDfK)] 2 , P-PRGD2), as a promising agent for noninvasive imaging of integrin expression in mouse models. P-PRGD2 was labeled with 18 F via 4-nitrophenyl 2- 18 F-fluoropropionate ( 18 F-FP) prosthetic group. In vitro and in vivo characteristics of the new dimeric RGD peptide tracer 18 F-FP-P-PRGD2 were investigated and compared with those of 18 F-FP-P-RGD2 ( 18 F-labeled RGD dimer without two PEG 4 spacers between the two RGD motifs). The ability of 18 F-FP-P-PRGD2 to image tumor vascular integrin expression was evaluated in a 4T1 murine breast tumor model. With the insertion of two PEG 4 spacers between the two RGD motifs, 18 F-FP-P-PRGD2 showed enhanced integrin α v β 3 -binding affinity, increased tumor uptake and tumor-to-nontumor background ratios compared with 18 F-FP-P-RGD2 in U87MG tumors. MicroPET imaging with 18 F-FP-P-PRGD2 revealed high tumor contrast and low background in tumor-bearing nude mice. Biodistribution studies confirmed the in vivo integrin α v β 3 -binding specificity of 18 F-FP-P-RGD2. 18 F-FP-P-PRGD2 can specifically image integrin α v β 3 on the activated endothelial cells of tumor neovasculature. 18 F-FP-P-PRGD2 can provide important information on integrin expression on the tumor vasculature. The high integrin binding affinity and specificity, excellent pharmacokinetic properties and metabolic stability make the new RGD dimeric tracer 18 F-FP-P-PRGD2 a promising agent for PET imaging of tumor angiogenesis and for monitoring the efficacy of antiangiogenic

Integrin α6Bβ4 inhibits colon cancer cell proliferation and c-Myc activity

International Nuclear Information System (INIS)

Dydensborg, Anders Bondo; Teller, Inga C; Groulx, Jean-François; Basora, Nuria; Paré, Fréderic; Herring, Elizabeth; Gauthier, Rémy; Jean, Dominique; Beaulieu, Jean-François

2009-01-01

Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin β4 subunit is up-regulated in primary colon cancer. Its partner, the integrin α6 subunit, exists as two different mRNA splice variants, α6A and α6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these α6 splice variants is still lacking. In this work, we first analyzed the expression of integrin α6A and α6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of α6A and α6B on the regulation of cell proliferation in a colon cancer cell line. Using variant-specific antibodies, we observed that α6A and α6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express α6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed α6B. A relative decrease of α6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the α6A/α6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the α6A/α6B balance in favor of α6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc. The findings that the α6Bβ4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its α6Aβ4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this α6Bβ4 integrin. Taken together, these findings point out the importance of integrin

Study of formation of deep trapping mechanism by UV, beta and gamma irradiated Eu(3+) activated SrY2O4 and Y4Al2O9 phosphors.

Science.gov (United States)

Dubey, Vikas; Kaur, Jagjeet; Parganiha, Yogita; Suryanarayana, N S; Murthy, K V R

2016-04-01

This paper reports the thermoluminescence properties of Eu(3+) doped different host matrix phosphors (SrY2O4 and Y4Al2O9). The phosphor is prepared by high temperature solid state reaction method. The method is suitable for large scale production and fixed concentration of boric acid using as a flux. The prepared samples were characterized by X-ray diffraction technique and the crystallite size calculated by Scherer's formula. The prepared phosphor characterized by Scanning Electron Microscopic (SEM), Fourier Transform Infrared (FTIR), Energy Dispersive X-ray analysis (EDX), thermoluminescence (TL) and Transmission Electron Microscopic (TEM) techniques. The prepared phosphors for different concentration of Eu(3+) ions were examined by TL glow curve for UV, beta and gamma irradiation. The UV 254nm source used for UV irradiation, Sr(90) source was used for beta irradiation and Co(60) source used for gamma irradiation. SrY2O4:Eu(3+)and Y4Al2O9:Eu(3+) phosphors which shows both higher temperature peaks and lower temperature peaks for UV, beta and gamma irradiation. Here UV irradiated sample shows the formation of shallow trap (surface trapping) and the gamma irradiated sample shows the formation of deep trapping. The estimation of trap formation was evaluated by knowledge of trapping parameters. The trapping parameters such as activation energy, order of kinetics and frequency factor were calculated by peak shape method. Here most of the peak shows second order of kinetics. The effect of gamma, beta and UV exposure on TL studies was also examined and it shows linear response with dose which indicate that the samples may be useful for TL dosimetry. Formation of deep trapping mechanism by UV, beta and gamma irradiated Eu(3+) activated SrY2O4 and Y4Al2O9 phosphors is discussed in this paper. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enthalpies of combustion and formation of {alpha}-D-glucoheptono-1,4-lactone and {alpha},{beta}-glucooctanoic-1,4-lactone

Energy Technology Data Exchange (ETDEWEB)

Amador, Patricia [Facultad de Ciencias Qui' micas, Benemerita Universidad Autonoma de Puebla, 14 Sur y Av. San Claudio, Col. Manuel, C.P. 72570 Puebla Pue (Mexico)], E-mail: cs000721@siu.buap.mx; Mata, Marian Y.; Flores, Henoc [Facultad de Ciencias Qui' micas, Benemerita Universidad Autonoma de Puebla, 14 Sur y Av. San Claudio, Col. Manuel, C.P. 72570 Puebla Pue (Mexico)

2008-05-15

The standard molar energies of combustion, {delta}{sub c}U{sub m}{sup 0}(cr,298.15K), of {alpha}-D-glucoheptono-1,4-lactone (GH) and {alpha},{beta}-glucooctanoic-1,4-lactone (GO) were obtained by micro-combustion calorimetry. The obtained values are -(2924.6 {+-} 2.3) kJ . mol{sup -1} and -(3459.5 {+-} 2.5) kJ . mol{sup -1}, respectively. From combustion energies, the standard molar enthalpies of formation in crystalline phase, {delta}{sub f}H{sub m}{sup 0}(cr,298.15K), for GH and GO were determined as -(1546.2 {+-} 2.5) kJ . mol{sup -1} and -(1690.6 {+-} 2.7) kJ . mol{sup -1}, respectively. Also it was found that when the hydroxyl group number increases in the aldonolactones their standard molar enthalpies of formation increase too.

The newcomer in the integrin family: Integrin α9 in biology and cancer

DEFF Research Database (Denmark)

Høye, Anette Melissa; Couchman, John Robert; Wewer, Ulla M.

2012-01-01

Integrins are heterodimeric transmembrane receptors regulating cell-cell and cell-extracellular matrix interactions. Of the 24 integrin heterodimers identified in humans, a9ß1 integrin is one of the least studied. a9, together with a4, comprise a more recent evolutionary sub-family of integrins...... of cell types, interacts with many ligands for example fibronectin, tenascin-C and ADAM12, and has been shown to have important functions in processes such as cell adhesion and migration, lung development, lymphatic and venous valve development, and in wound healing. This has sparked an interest...

Optimized formation of detergent micelles of beta-carotene and retinal production using recombinant human beta,beta-carotene 15,15'-monooxygenase.

Science.gov (United States)

Kim, Nam-Hee; Kim, Yeong-Su; Kim, Hye-Jung; Oh, Deok-Kun

2008-01-01

The formation of beta-carotene detergent micelles and their conversion into retinal by recombinant human beta,beta-carotene 15,15'-monooxygenase was optimized under aqueous conditions. Toluene was the most hydrophobic among the organic solvents tested; thus, it was used to dissolve beta-carotene, which is a hydrophobic compound. Tween 80 was selected as the detergent because it supported the highest level of retinal production among all of the detergents tested. The maximum production of retinal was achieved in detergent micelles containing 200 mg/L of beta-carotene and 2.4% (w/v) Tween 80. Under these conditions, the recombinant enzyme produced 97 mg/L of retinal after 16 h with a conversion yield of 48.5% (w/w). The amount of retinal produced, which is the highest ever reported, is a result of the ability of our system to dissolve large amounts of beta-carotene.

Design and synthesis of novel platelet fibrinogen receptor antagonists with 2H-1,4-benzoxazine-3(4H)-one scaffold. A systematic study.

Science.gov (United States)

Anderluh, Marko; Cesar, Jozko; Stefanic, Petra; Kikelj, Danijel; Janes, Damjan; Murn, Jernej; Nadrah, Kristina; Tominc, Mojca; Addicks, Elisabeth; Giannis, Athanassios; Stegnar, Mojca; Dolenc, Marija Sollner

2005-01-01

New platelet glycoprotein IIb/IIIa (GP IIb/IIIa, integrin alpha(IIb)beta3) antagonists were prepared on a 2H-1,4-benzoxazine-3(4H)-one scaffold. Their anti-aggregatory activities in human platelet rich plasma and their affinity towards alpha(IIb)beta3 and alpha(V)beta3 integrins were assessed. Various substitution positions and side chain variations were studied. In contrast to the generally accepted model, compounds containing ethyl esters as aspartate mimetics were in general more active than the corresponding free acids. We suggest an explanation for the observed behaviour of these new compounds.

Muscle-specific integrins in masseter muscle fibers of chimpanzees: an immunohistochemical study.

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Gianluigi Vaccarino

2010-05-01

Full Text Available Most notably, recent comparative genomic analyses strongly indicate that the marked differences between modern human and chimpanzees are likely due more to changes in gene regulation than to modifications of the genes. The most peculiar aspect of hominoid karyotypes is that human have 46 chromosomes whereas gorillas and chimpanzees have 48. Interestingly, human and chimpanzees do share identical inversions on chromosome 7 and 9 that are not evident in the gorilla karyotype. Thus, the general phylogeny suggests that humans and chimpanzees are sister taxa; based on this, it seems that human-chimpanzee sequence similarity is an astonishing 99%. At this purpose, of particular interest is the inactivation of the myosin heavy chain 16 (MYH16 gene, most prominently expressed in the masticatory muscle of mammals. It has been showed that the loss of this gene in humans may have resulted in smaller masticatory muscle and consequential changes to cranio-facial morphology and expansion of the human brain case. Powerful masticatory muscles are found in most primates; contrarily, in both modern and fossil member Homo, these muscles are considerably smaller. The evolving hominid masticatory apparatus shifted towards a pattern of gracilization nearly simultaneously with accelerated encephalization in early Homo. To better comprehend the real role of the MYH16 gene, we studied the primary proteins present in the muscle fibers of humans and non-humans, in order to understand if they really can be influenced by MYH16 gene. At this aim we examined the muscle-specific integrins, alpha 7B and beta 1D-integrins, and their relative fetal isoforms, alpha 7A and beta 1A-integrins, analyzing, by immunohistochemistry, muscle biopsies of two components of a chimpanzee's group in captivity, an alpha male and a non-alpha male subjects; all these integrins participate in vital biological processes such as maintenance of tissue integrity, embryonic development, cell

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†

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Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.

2017-01-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031

Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

Science.gov (United States)

Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

2017-05-01

Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25 min -1 , threefold faster than α3 integrin (1.0 min -1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min -1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min -1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge*

Science.gov (United States)

Puklin-Faucher, Eileen; Vogel, Viola

2009-01-01

Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII10-bound αVβ3 integrin headpiece how the binding pocket and interdomain βA/hybrid domain hinge on the distal end of the βA domain are allosterically linked via a hydrophobic T-junction between the middle of the α1 helix and top of the α7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca2+ in place of Mg2+ at the site adjacent to the metal ion-dependent adhesion site (“ADMIDAS”). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca2+ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated. PMID:19762919

Beta response of CaSO4:Dy based TLD badge and its angular dependence studies for personnel monitoring applications

International Nuclear Information System (INIS)

Kumar, Munish; Rakesh, R.B.; Sneha, C.; Ratna, P.; Bakshi, A.K.; Datta, D.

2016-01-01

In India, shallow/skin doses received by radiation workers from beta particles are measured using CaSO 4 :Dy based Teflon embedded TLD badge. The beta particles having maximum energy E max > 0.6 MeV - ≥ 3.54 MeV are monitored. The ratio of the response of discs under open and plastic regions (D Open/ D Perspex ) is used to estimate the energy of the beta source and to apply response correction factor. This is required as the disc dosimeters are thick and exhibit energy dependent response. Due to lack of comprehensive information regarding disc ratios and associated beta multiplication/response correction factors, present study was performed

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture.

Science.gov (United States)

Wallace, Charles S; Strike, Sophie A; Truskey, George A

2007-09-01

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

beta1 integrin maintains integrity of the embryonic neocortical stem cell niche.

Directory of Open Access Journals (Sweden)

Karine Loulier

2009-08-01

Full Text Available During embryogenesis, the neural stem cells (NSC of the developing cerebral cortex are located in the ventricular zone (VZ lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM, which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin alpha2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs

Nano-beta-tricalcium phosphates synthesis and biodegradation: 2. Biodegradation and apatite layer formation on nano-{beta}-TCP synthesized via microwave treatment

Energy Technology Data Exchange (ETDEWEB)

Abdel-Fattah, Wafa I; Elkhooly, Tarek A, E-mail: nrcfifi@yahoo.co [Department of Biomaterials, National Research Center, Cairo (Egypt)

2010-06-01

The degradation and/or apatite layer precipitation ability of porous {beta}-tricalcium phosphate ({beta}-TCP) samples treated and untreated with microwave radiation during synthesis is investigated. Microwave heating was used to accelerate the formation of CDHA with the Ca/P ratio 1.5 in a shorter processing time which later forms {beta}-TCP at around 650 {sup 0}C. Soaking in simulated body fluid (SBF) for several periods (4, 8, 12, 24, 36, 48, 60 and 72 h) is performed in a cumulative manner. The deposition of an apatite layer is followed through diffuse reflected FT-IR, SEM and EDS. A microwave-treated sample having a smaller particle size than its parent induces the formation of a homogeneous carbonated apatite layer on its surface. On the other hand, the parent {beta}-TCP sample exhibited less ability to induce Ca-P formation after being soaked in SBF. The formation of an apatite layer is attributed to the increase in surface area consequent to reduced particle and grain sizes besides the presence of a minor amount of hydroxyapatite phase in the microwave-treated {beta}-TCP sample. The results prove that it is possible to control the biodegradation and apatite layer formation on sintered {beta}-TCP porous disks through controlling the particle size.

Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and beta1 integrin receptors.

Science.gov (United States)

Smith, Julie A; Samayawardhena, Lionel A; Craig, Andrew W B

2010-03-01

Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.

Protein kinase C involvement in focal adhesion formation

DEFF Research Database (Denmark)

Woods, A; Couchman, J R

1992-01-01

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

Directory of Open Access Journals (Sweden)

Takahashi Takashi

2007-04-01

Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

Formation and release of. beta. -glucosidase by Aspergillus niger ZIMET 43 746 in correlation to process operations

Energy Technology Data Exchange (ETDEWEB)

Kerns, G; Dalchow, E; Klappach, G; Meyer, D

1986-01-01

The total formation of ..beta..-glucosidase by the wild strain of Aspergillus niger ZIMET 43 746 is non-growth-associated. In discontinuous culture the total ..beta..-glucosidase activity related to the mycelium is increasing with the age of the mycelium. The complete release of the remaining mycelial-associated ..beta..-glucosidase is dependent on the structure of the mycelium. In the cases of the mycelium forms pellets throughout the growth phase than the release of ..beta..-glucosidase is accelerated compared to the release from loosly branched mycelium. Increasing shear stress caused by increasing of the impeller speed promotes the formation of pellets.

Interactions of foot-and-mouth disease virus with soluble bovine alphaVbeta3 and alphaVbeta6 integrins.

Science.gov (United States)

Duque, Hernando; LaRocco, Michael; Golde, William T; Baxt, Barry

2004-09-01

At least four members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, alphaVbeta6, and alphaVbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Our investigators have recently shown that the efficiency of receptor usage appears to be related to the viral serotype and may be influenced by structural differences on the viral surface (H. Duque and B. Baxt, J. Virol. 77:2500-2511, 2003). To further examine these differences, we generated soluble alphaVbeta3 and alphaVbeta6 integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences encoding the subunits' signal peptide and ectodomain, resulting in subunits lacking transmembrane and cytoplasmic domains. COS-1 cells were transfected with plasmids encoding the soluble alphaV subunit and either the soluble beta3 or beta6 subunit and labeled with [35S]methionine-cysteine. Complete subunit heterodimeric integrins were secreted into the medium, as determined by radioimmunoprecipitation with specific monoclonal and polyclonal antibodies. For the examination of the integrins' biological activities, stable cell lines producing the soluble integrins were generated in HEK 293A cells. In the presence of divalent cations, soluble alphaVbeta6 bound to representatives of type A or O viruses, immobilized on plastic dishes, and significantly inhibited viral replication, as determined by plaque reduction assays. In contrast, soluble alphaVbeta3 was unable to bind to immobilized virus of either serotype; however, virus bound to the immobilized integrin, suggesting that FMDV binding to alphaVbeta3 is a low-affinity interaction. In addition, soluble alphaVbeta3 did not neutralize virus infectivity. Incubation of soluble alphaVbeta6 with labeled type A12 or O1 resulted in a significant inhibition of virus adsorption to BHK cells, while soluble alphaVbeta3 caused a low (20 to 30%), but consistent, inhibition of virus

Extracellular Membrane-proximal Domain of HAb18G/CD147 Binds to Metal Ion-dependent Adhesion Site (MIDAS) Motif of Integrin β1 to Modulate Malignant Properties of Hepatoma Cells*

Science.gov (United States)

Li, Yong; Wu, Jiao; Song, Fei; Tang, Juan; Wang, Shi-Jie; Yu, Xiao-Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2012-01-01

Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp179 in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells. PMID:22130661

Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

International Nuclear Information System (INIS)

Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya; Hirose, Takahisa; Kawamori, Ryuzo; Fujitani, Yoshio; Watada, Hirotaka

2009-01-01

Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.

Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

Energy Technology Data Exchange (ETDEWEB)

Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Hirose, Takahisa [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Center for Therapeutic Innovations in Diabetes, Juntendo University School of Medicine, Tokyo (Japan); Kawamori, Ryuzo [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Center for Therapeutic Innovations in Diabetes, Juntendo University School of Medicine, Tokyo (Japan); Center for Beta Cell Biology and Regeneration, Juntendo University School of Medicine, Tokyo (Japan); Sportology Center, Juntendo University School of Medicine, Tokyo (Japan); Fujitani, Yoshio, E-mail: fujitani@juntendo.ac.jp [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Center for Therapeutic Innovations in Diabetes, Juntendo University School of Medicine, Tokyo (Japan); Watada, Hirotaka, E-mail: hwatada@juntendo.ac.jp [Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, Tokyo (Japan); Sportology Center, Juntendo University School of Medicine, Tokyo (Japan)

2009-12-18

Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4 daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.

Non-redundant roles of phosphoinositide 3-kinase isoforms alpha and beta in glycoprotein VI-induced platelet signaling and thrombus formation.

Science.gov (United States)

Gilio, Karen; Munnix, Imke C A; Mangin, Pierre; Cosemans, Judith M E M; Feijge, Marion A H; van der Meijden, Paola E J; Olieslagers, Servé; Chrzanowska-Wodnicka, Magdalena B; Lillian, Rivka; Schoenwaelder, Simone; Koyasu, Shigeo; Sage, Stewart O; Jackson, Shaun P; Heemskerk, Johan W M

2009-12-04

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cgamma2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kalpha or -beta strongly and selectively suppressed GPVI-induced Ca(2+) mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cgamma2 by PI3Kalpha/beta. That PI3Kalpha and -beta have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca(2+) increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kalpha/beta was blocked or p85alpha was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kdelta or -gamma. Furthermore, PI3Kalpha/beta, but not PI3Kgamma, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kalpha and -beta isoforms are required for full GPVI-dependent platelet Ca(2+) signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kalpha an interesting new target for anti-platelet therapy.

Molecular nuclear imaging of tumoral angio genesis using a rgd-containing tracer, Raft-RGD, targeted at the neo vessel-specific integrin {alpha}{sub v}{beta}{sub 3}; Evaluation d'un radioligand de l'integrine {alpha}{sub v}{beta}{sub 3} (RAFT-RGD) pour l'imagerie moleculaire de l'angiogenese tumorale

Energy Technology Data Exchange (ETDEWEB)

Sancey, L

2006-06-15

Tumoral neo-angio genesis targeting is currently a major field of research for the diagnostic and treatment of solid tumors. Endothelial cells from neo vessels over express several specific markers such as the {alpha}{sub v}{beta}{sub 3} integrin, which binds RGD (-Arg-Gly-Asp-)- containing peptides. We evaluated the potential of a novel radiotracer - RAFT-RGD - for the molecular nuclear imaging of neo vessels. In vitro, the coupling of 4 c(RGDfK) to the RAFT platform resulted in an increased cellular uptake of the tracer by {alpha}{sub v}{beta}{sub 3} positive cells when compared to c(RGDfK). Furthermore, RAFTRGD has a higher affinity than c(RGDfK) and similar properties for angio genesis inhibition. In vivo, both {alpha}{sub v}{beta}{sub 3} positive and negative tumors were visible by non invasive whole body planar and tomographic imaging from 30 min to 24 h post-injection, using a gamma camera dedicated to small animal imaging. Despite a lack of significant contrast improvement compare with c(RGDfK), RAFT-RGD could represent a promising tracer for tumoral angio genesis since it could provide invaluable information about tumor development and treatment efficacy in Nuclear Medicine departments. Furthermore, thanks to its chemical structure, RAFT-RGD can be labelled with a variety of radioisotopes including {gamma} and {beta}{sup -} emitters, allowing interesting therapeutical applications such as internal targeted radiotherapy. (author)

Force via integrins but not E-cadherin decreases Oct3/4 expression in embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Uda, Yuhei [Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aramaki-aoba, Aoba-ward, Sendai City (Japan); Poh, Yeh-Chuin; Chowdhury, Farhan; Wu, Douglas C. [Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Tanaka, Tetsuya S. [Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Sato, Masaaki [Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aramaki-aoba, Aoba-ward, Sendai City (Japan); Wang, Ning, E-mail: nwangrw@illinois.edu [Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

2011-11-18

Highlights: Black-Right-Pointing-Pointer Force via integrins or cadherins induces similar cell stiffening responses. Black-Right-Pointing-Pointer Force via integrins but not cadherins induces cell spreading. Black-Right-Pointing-Pointer Force via integrins but not cadherins induces differentiation of embryonic stem cells. -- Abstract: Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.

Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability

Science.gov (United States)

Li, Shuoran; Nih, Lina R.; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S. Thomas; Barker, Thomas H.; Segura, Tatiana

2017-09-01

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer

International Nuclear Information System (INIS)

Samardzija, Chantel; Luwor, Rodney B.; Quinn, Michael A.; Kannourakis, George; Findlay, Jock K.; Ahmed, Nuzhat

2016-01-01

Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis. The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice. We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34

SPARC regulates extracellular matrix organization through its modulation of integrin-linked kinase activity.

Science.gov (United States)

Barker, Thomas H; Baneyx, Gretchen; Cardó-Vila, Marina; Workman, Gail A; Weaver, Matt; Menon, Priya M; Dedhar, Shoukat; Rempel, Sandra A; Arap, Wadih; Pasqualini, Renata; Vogel, Viola; Sage, E Helene

2005-10-28

SPARC, a 32-kDa matricellular glycoprotein, mediates interactions between cells and their extracellular matrix, and targeted deletion of Sparc results in compromised extracellular matrix in mice. Fibronectin matrix provides provisional tissue scaffolding during development and wound healing and is essential for the stabilization of mature extracellular matrix. Herein, we report that SPARC expression does not significantly affect fibronectin-induced cell spreading but enhances fibronectin-induced stress fiber formation and cell-mediated partial unfolding of fibronectin molecules, an essential process in fibronectin matrix assembly. By phage display, we identify integrin-linked kinase as a potential binding partner of SPARC and verify the interaction by co-immunoprecipitation and colocalization in vitro. Cells lacking SPARC exhibit diminished fibronectin-induced integrin-linked kinase activation and integrin-linked kinase-dependent cell-contractile signaling. Furthermore, induced expression of SPARC in SPARC-null fibroblasts restores fibronectin-induced integrin-linked kinase activation, downstream signaling, and fibronectin unfolding. These data further confirm the function of SPARC in extracellular matrix organization and identify a novel mechanism by which SPARC regulates extracellular matrix assembly.

Tumour exosome integrins determine organotropic metastasis.

Science.gov (United States)

Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Tesic Mark, Milica; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M; Dumont-Cole, Vanessa D; Kramer, Kimberly; Wexler, Leonard H; Narendran, Aru; Schwartz, Gary K; Healey, John H; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H; Grandgenett, Paul M; Hollingsworth, Michael A; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K; Jarnagin, William R; Brady, Mary S; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J; Bissell, Mina J; Garcia, Benjamin A; Kang, Yibin; Rajasekhar, Vinagolu K; Ghajar, Cyrus M; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

2015-11-19

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

CCM proteins control endothelial β1 integrin dependent response to shear stress

Directory of Open Access Journals (Sweden)

Zuzana Macek Jilkova

2014-11-01

Full Text Available Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that β1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated “shear-stress-like” phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

International Nuclear Information System (INIS)

Rossier, Olivier; Giannone, Grégory

2016-01-01

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

Science.gov (United States)

Rossier, Olivier; Giannone, Grégory

2016-04-10

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

Energy Technology Data Exchange (ETDEWEB)

Rossier, Olivier; Giannone, Grégory [Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France); CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France)

2016-04-10

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

Cell adhesion to fibrillin-1: identification of an Arg-Gly-Asp-dependent synergy region and a heparin-binding site that regulates focal adhesion formation

DEFF Research Database (Denmark)

Bax, Daniel V; Mahalingam, Yashithra; Cain, Stuart

2007-01-01

We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated...... a requirement for upstream domains for integrin-alpha(5)beta(1)-mediated cell adhesion and migration. An adjacent heparin-binding site, which supports focal adhesion formation, was mapped to the fibrillin-1 TB5 motif. Site-directed mutagenesis revealed two arginine residues that are crucial for heparin binding...

Oscillatory fluid flow elicits changes in morphology, cytoskeleton and integrin-associated molecules in MLO-Y4 cells, but not in MC3T3-E1 cells

Directory of Open Access Journals (Sweden)

Huiyun Xu

2012-01-01

Full Text Available Interstitial fluid flow stress is one of the most important mechanical stimulations of bone cells under physiological conditions. Osteocytes and osteoblasts act as primary mechanosensors within bones, and in vitro are able to respond to fluid shear stress, both morphologically and functionally. However, there is little information about the response of integrin-associated molecules using both osteoblasts and osteocytes. In this study, we investigated the changes in response to 2 hours of oscillatory fluid flow stress in the MLO-Y4 osteocyte-like cell line and the MC3T3-E1 osteoblast-like cell line. MLO-Y4 cells exhibited a significant increase in the expression of integrin-associated molecules, including OPN, CD44, vinculin and integrin avp3. However, there was no or limited increase observed in MC3T3-E1 osteoblast-like cells. Cell area and fiber stress formation were also markedly promoted by fluid flow only in MLO-Y4 cells. But the numbers of processes per cell remain unaffected in both cell lines.

Role of Integrin-Beta1 in Polycystic Kidney Disease

Science.gov (United States)

2012-04-01

interactions affect proliferation and cell differentiation2. We previously showed an increase of integrin-!2ŕ expression in polycystin-1 (PC1) deficient...that since the Cre activity in TgAqp2-Cre transgenic mouse was observed also in testes, our breeding scheme was devised to ensure maternal ...inheritance of the transgene, as paternal transmission would lead to gene recombination not only in the kidneys, but also in sperm and the resulting

Dual role of beta-carotene in combination with cigarette smoke aqueous extract on the formation of mutagenic lipid peroxidation products in lung membranes: dependence on pO2.

Science.gov (United States)

Palozza, P; Serini, S; Trombino, S; Lauriola, L; Ranelletti, F O; Calviello, G

2006-12-01

Results from some intervention trials indicated that supplemental beta-carotene enhanced lung cancer incidence and mortality in chronic smokers. The aim of this study was to verify the hypothesis that high concentrations of the carotenoid, under the pO2 present in lung (100-150 mmHg), may exert deleterious effects through a prooxidant mechanism. To test this hypothesis, we examined the interactions of beta-carotene and cigarette smoke condensate (tar) on the formation of lipid peroxidation products in rat lung microsomal membranes enriched in vitro with varying beta-carotene concentrations (from 1 to 10 nmol/mg prot) and then incubated with tar (6-25 microg/ml) under different pO2. As markers of lipid peroxidation, we evaluated the levels of conjugated dienes and malondialdehyde, possessing mutagenic and pro-carcinogenic activity. The exposure of microsomal membranes to tar induced a dose-dependent enhancement of lipid peroxidation, which progressively increased as a function of pO2. Under a low pO2 (15 mmHg), beta-carotene acted clearly as an antioxidant, inhibiting tar-induced lipid peroxidation. However, the carotenoid progressively lost its antioxidant efficiency by increasing pO2 (50-100 mmHg) and acted as a prooxidant at pO2 ranging from 100 to 760 mmHg in a dose-dependent manner. Consistent with this finding, the addition of alpha-tocopherol (25 microM) prevented the prooxidant effects of the carotenoid. beta-Carotene auto-oxidation, measured as formation of 5,6-epoxy-beta,beta-carotene, was faster at high than at low pO2 and the carotenoid was more rapidly consumed in the presence of tar. These data point out that the carotenoid may enhance cigarette smoke-induced oxidative stress and exert potential deleterious effects at the pO2 normally present in lung tissue.

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

NARCIS (Netherlands)

Stroeken, Peter J. M.; Alvarez, Belén; van Rheenen, Jacco; Wijnands, Yvonne M.; Geerts, Dirk; Jalink, Kees; Roos, Ed

2006-01-01

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By

Hierarchy of ADAM12 binding to integrins in tumor cells

DEFF Research Database (Denmark)

Thodeti, Charles Kumar; Fröhlich, Camilla; Nielsen, Christian Kamp

2005-01-01

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADA...

Modulation of type II TGF-β receptor degradation by integrin-linked kinase.

Science.gov (United States)

Vi, Linda; Boo, Stellar; Sayedyahossein, Samar; Singh, Randeep K; McLean, Sarah; Di Guglielmo, Gianni M; Dagnino, Lina

2015-03-01

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Kindlin-3 Is Essential for the Resting α4β1 Integrin-mediated Firm Cell Adhesion under Shear Flow Conditions.

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Lu, Ling; Lin, ChangDong; Yan, ZhanJun; Wang, Shu; Zhang, YouHua; Wang, ShiHui; Wang, JunLei; Liu, Cui; Chen, JianFeng

2016-05-06

Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin α4β1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin β tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting α4β1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting α4β1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to β1 and down-regulated the binding affinity of the resting α4β1 to soluble VCAM-1. Notably, it converted the resting α4β1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn(2+)-activated α4β1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting α4β1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting α4β1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting α4β1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Kindlin-3 Is Essential for the Resting α4β1 Integrin-mediated Firm Cell Adhesion under Shear Flow Conditions*

Science.gov (United States)

Lu, Ling; Lin, ChangDong; Yan, ZhanJun; Wang, Shu; Zhang, YouHua; Wang, ShiHui; Wang, JunLei; Liu, Cui; Chen, JianFeng

2016-01-01

Integrin-mediated rolling and firm cell adhesion are two critical steps in leukocyte trafficking. Integrin α4β1 mediates a mixture of rolling and firm cell adhesion on vascular cell adhesion molecule-1 (VCAM-1) when in its resting state but only supports firm cell adhesion upon activation. The transition from rolling to firm cell adhesion is controlled by integrin activation. Kindlin-3 has been shown to bind to integrin β tails and trigger integrin activation via inside-out signaling. However, the role of kindlin-3 in regulating resting α4β1-mediated cell adhesion is not well characterized. Herein we demonstrate that kindlin-3 was required for the resting α4β1-mediated firm cell adhesion but not rolling adhesion. Knockdown of kindlin-3 significantly decreased the binding of kindlin-3 to β1 and down-regulated the binding affinity of the resting α4β1 to soluble VCAM-1. Notably, it converted the resting α4β1-mediated firm cell adhesion to rolling adhesion on VCAM-1 substrates, increased cell rolling velocity, and impaired the stability of cell adhesion. By contrast, firm cell adhesion mediated by Mn2+-activated α4β1 was barely affected by knockdown of kindlin-3. Structurally, lack of kindlin-3 led to a more bent conformation of the resting α4β1. Thus, kindlin-3 plays an important role in maintaining a proper conformation of the resting α4β1 to mediate both rolling and firm cell adhesion. Defective kindlin-3 binding to the resting α4β1 leads to a transition from firm to rolling cell adhesion on VCAM-1, implying its potential role in regulating the transition between integrin-mediated rolling and firm cell adhesion. PMID:26994136

Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-11

Science.gov (United States)

Hyun, Young-Min; Chung, Hung-Li; McGrath, James L.; Waugh, Richard E.; Kim, Minsoo

2009-01-01

Lymphocyte migration from blood into lymphoid tissues or to sites of inflammation occurs through interactions between cell surface integrins and their ligands expressed on the vascular endothelium and the extracellular matrix. Very Late Antigen-4 (VLA-4, α4β1) is a key integrin in the effective trafficking of lymphocytes. Although it has been well established that integrins undergo functionally significant conformational changes to mediate cell adhesion, there is no mechanistic information that explains how these are dynamically and spatially regulated during lymphocyte polarization and migration. Using dynamic fluorescence resonance energy transfer (FRET) analysis of a novel VLA-4 FRET sensor under total internal reflection fluorescence (TIRF) microscopy, we show that VLA-4 activation localizes to the lamellipodium in living cells. During T cell migration on VCAM-1, VLA-4 activation concurs with spatial redistribution of chemokine receptor and active Rap1 at the leading edge. Selective inhibition of the activated VLA-4 at leading edge with a small molecule inhibitor is sufficient to block T cell migration. These data suggest that a subpopulation of activated VLA-4 is mainly localized to the leading edge of polarized human T cells, and is critical for T cell migration on VCAM-1. PMID:19542447

Integrin-directed modulation of macrophage responses to biomaterials.

Science.gov (United States)

Zaveri, Toral D; Lewis, Jamal S; Dolgova, Natalia V; Clare-Salzler, Michael J; Keselowsky, Benjamin G

2014-04-01

Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials. Copyright © 2014 Elsevier Ltd. All rights reserved.

Stabilization of beta-catenin induces pancreas tumor formation.

Science.gov (United States)

Heiser, Patrick W; Cano, David A; Landsman, Limor; Kim, Grace E; Kench, James G; Klimstra, David S; Taketo, Maketo M; Biankin, Andrew V; Hebrok, Matthias

2008-10-01

beta-Catenin signaling within the canonical Wnt pathway is essential for pancreas development. However, the pathway is normally down-regulated in the adult organ. Increased cytoplasmic and nuclear localization of beta-catenin can be detected in nearly all human solid pseudopapillary neoplasms (SPN), a rare tumor with low malignant potential. Conversely, pancreatic ductal adenocarcinoma (PDA) accounts for the majority of pancreatic tumors and is among the leading causes of cancer death. Whereas activating mutations within beta-catenin and other members of the canonical Wnt pathway are rare, recent reports have implicated Wnt signaling in the development and progression of human PDA. Here, we sought to address the role of beta-catenin signaling in pancreas tumorigenesis. Using Cre/lox technology, we conditionally activated beta-catenin in a subset of murine pancreatic cells in vivo. Activation of beta-catenin results in the formation of large pancreatic tumors at a high frequency in adult mice. These tumors resemble human SPN based on morphologic and immunohistochemical comparisons. Interestingly, stabilization of beta-catenin blocks the formation of pancreatic intraepithelial neoplasia (PanIN) in the presence of an activating mutation in Kras that is known to predispose individuals to PDA. Instead, mice in which beta-catenin and Kras are concurrently activated develop distinct ductal neoplasms that do not resemble PanIN lesions. These results demonstrate that activation of beta-catenin is sufficient to induce pancreas tumorigenesis. Moreover, they indicate that the sequence in which oncogenic mutations are acquired has profound consequences on the phenotype of the resulting tumor.

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

Science.gov (United States)

Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

2018-05-10

Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms.

NARCIS (Netherlands)

Schmidt, S.; Friedl, P.H.A.

2010-01-01

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In

Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

Science.gov (United States)

Maza, Paloma K; Suzuki, Erika

2016-01-01

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

Cardiac integrins the ties that bind.

Science.gov (United States)

Simpson, D G; Reaves, T A; Shih, D T; Burgess, W; Borg, T K; Terracio, L

1998-01-01

An elaborate series of morphogenetic events must be precisely coordinated during development to promote the formation of the elaborate three-dimensional structure of the normal heart. In this study we focus on discussing how interconnections between the cardiac myocyte and its surrounding environment regulate cardiac form and function. In vitro experiments from our laboratories provide direct evidence that cardiac cell shape is regulated by a dynamic interaction between constituents of the extracellular matrix (ECM) and by specific members of the integrin family of matrix receptors. Our data indicates that phenotypic information is stored in the tertiary structure and chemical identity of the ECM. This information appears to be actively communicated and transduced by the α1β1 integrin molecule into an intracellular signal that regulates cardiac cell shape and myofibrillar organization. In this study we have assessed the phenotypic consequences of suppressing the expression and accumulation of the α1 integrin molecule in aligned cultures of cardiac myocytes. In related experiments we have examined how the overexpression of α2 and α5 integrin, integrins normally not present or present at very low copy number on the cell surface of neonatal cardiac myocytes, affect cardiac protein metabolism. We also consider how biochemical signals and the mechanical signals mediated by the integrins may converge on common intracellular signaling pathways in the heart. Experiments with the whole embryo culture system indicate that angiotensin II, a peptide that carries information concerning cardiac load, plays a role in controling cardiac looping and the proliferation of myofibrils during development.

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

DEFF Research Database (Denmark)

Sasaki, T; Brakebusch, C; Engel, J

1998-01-01

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

Oncofetal Chondroitin Sulfate Glycosaminoglycans are Key Players in Integrin Signaling and Tumor Cell Motility

Science.gov (United States)

Clausen, Thomas Mandel; Pereira, Marina Ayres; Al Nakouzi, Nader; Oo, Htoo Zarni; Agerbæk, Mette Ø; Lee, Sherry; Ørum-Madsen, Maj Sofie; Christensen, Anders Riis; El-Naggar, Amal; Grandgenett, Paul M.; Grem, Jean L.; Hollingsworth, Michael A.; Holst, Peter J.; Theander, Thor; Sorensen, Poul H.; Daugaard, Mads; Salanti, Ali

2016-01-01

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum. We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion and anchorage-independent growth of tumor cells in vitro. Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns, revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin β1 (ITGB1) and integrin α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core CS synthesis enzymes Beta-1,3-Glucuronyltransferase 1 (B3GAT1) and Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and pre-incubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. Implications The cancer specific expression of oncofetal chondroitin sulfate aids in metastatic phenotypes and is a candidate target for therapy. PMID:27655130

Targeted Gene Deletion Demonstrates that Cell Adhesion MoleculeICAM-4 is Critical for Erythroblastic Island Formation

Energy Technology Data Exchange (ETDEWEB)

Lee, Gloria; Lo, Annie; Short, Sarah A.; Mankelow, Tosti J.; Spring, Frances; Parsons, Stephen F.; Mohandas, Narla; Anstee, David J.; Chasis, Joel Anne

2006-02-15

Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule-4 (ICAM-4), animmunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha V integrins as ICAM-4 counter receptors. Since macrophages express alpha V, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knockout mice and developed quantitative, live cell techniques for harvesting intact islands and for reforming islands in vitro. We observed a 47 percent decrease in islands reconstituted from ICAM-4 null marrow compared to wild type. We also found a striking decrease in islands formed in vivo in knockout mice. Further, peptides that block ICAM-4 alpha V adhesion produced a 53-57 percent decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha V functions in island integrity. Importantly, we documented that alpha V integrin is expressed in macrophages isolated from erythro blastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.

Dependence of the DIII-D beta limit on the current profile

Energy Technology Data Exchange (ETDEWEB)

Strait, E.J.; Chu, M.S.; Ferron, J.R.; Lao, L.L.; Osborne, T.H.; Taylor, T.S.; Turnbull, A.D. (General Atomics, San Diego, CA (United States)); Lazarus, E.A. (Oak Ridge National Lab., TN (United States))

1991-01-01

The maximum beta values achieved in DIII-D are not fully described by the simple scaling law [beta][sub max][proportional to]I/aB. There is, in addition, a dependence on the form of the current profile as parameterized by the safety factor q and internal inductance l[sub i]. The maximum experimentally achieved value of normalized beta [beta][sub N] = [beta]/(I/aB) varies from 3.5 at low safety factor q (q[sub 95]<3) to 5 at higher values of q. At low q, discharges are terminated by disruptions at high [beta][sub N] and at both the low and high l[sub i] boundaries of the stable range. These disruptions are attributed to external and global kink modes. At higher q, such disruptions are much less frequent, and beta is limited by slowly growing resistive modes, fishbones, and possibly by ballooning modes. At each value of q, the maximum beta tends to increase with internal inductance l[sub i]. A numerical study of kink mode stability has shown a similar trend for optimized pressure profiles. These observations have suggested a new scaling law for the operational beta limit: [beta][sub max]=4l[sub i](I/aB), which fits the DIII-D data well. (author) 13 refs., 4 figs.

Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

Science.gov (United States)

Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

2017-06-01

The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

Copper(II) 12-metallacrown-4 complexes of alpha-, beta- and gamma-aminohydroxamic acids: a comparative thermodynamic study in aqueous solution.

Science.gov (United States)

Tegoni, Matteo; Remelli, Maurizio; Bacco, Dimitri; Marchiò, Luciano; Dallavalle, Francesco

2008-05-28

A complete thermodynamic study of the protonation and Cu(II) complex formation equilibria of a series of alpha- and beta-aminohydroxamic acids in aqueous solution was performed. The thermodynamic parameters obtained for the protonation of glycine-, (S)-alpha-alanine-, (R,S)-valine-, (S)-leucine-, beta-alanine- and (R)-aspartic-beta-hydroxamic acids were compared with those previously reported for gamma-amino- and (S)-glutamic-gamma-hydroxamic acids. The enthalpy/entropy parameters calculated for the protonation microequilibria of these three types of ligands are in very good agreement with the literature values for simple amines and hydroxamic acids. The pentanuclear complexes [Cu5L4H(-4)]2+ contain the ligands acting as (NH2,N-)-(O,O-) bridging bis-chelating and correspond to 12-metallacrown-4 (12-MC-4) which are formed by self-assembly between pH 4 and 6 with alpha-aminohydroxamates (HL), while those with beta- and gamma-derivatives exist in a wider pH range (4-11). The stability order of these metallomacrocycles is beta- > alpha- > gamma-aminohydroxamates. The formation of 12-MC-4 with alpha-aminohydroxamates is entropy-driven, and that with beta-derivatives is enthalpy-driven, while with gamma-GABAhydroxamate both effects occur. These results are interpreted on the basis of specific enthalpies or entropy contributions related to chelate ring dimensions, charge neutralization and solvation-desolvation effects. The enthalpy/entropy parameters of 12-MC-4 with alpha-aminohydroxamic acids considered are also dependent on the optical purity of the ligands. Actually, that with (R,S)-valinehydroxamic acid presents an higher entropy and a lower enthalpy value than those of enantiopure ligands, although the corresponding stabilities are almost equivalent. Moreover, DFT calculations are in agreement with a more exothermic enthalpy found for metallacrowns with enantiomerically pure ligands.

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

Science.gov (United States)

Fong, J. H.; Ingber, D. E.

1996-01-01

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

Endocytosis of Integrin-Binding Human Picornaviruses

Directory of Open Access Journals (Sweden)

Pirjo Merilahti

2012-01-01

Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

β1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain

Directory of Open Access Journals (Sweden)

Teppei Fujioka

2017-02-01

Full Text Available Cerebral ischemic stroke is a main cause of chronic disability. However, there is currently no effective treatment to promote recovery from stroke-induced neurological symptoms. Recent studies suggest that after stroke, immature neurons, referred to as neuroblasts, generated in a neurogenic niche, the ventricular-subventricular zone, migrate toward the injured area, where they differentiate into mature neurons. Interventions that increase the number of neuroblasts distributed at and around the lesion facilitate neuronal repair in rodent models for ischemic stroke, suggesting that promoting neuroblast migration in the post-stroke brain could improve efficient neuronal regeneration. To move toward the lesion, neuroblasts form chain-like aggregates and migrate along blood vessels, which are thought to increase their migration efficiency. However, the molecular mechanisms regulating these migration processes are largely unknown. Here we studied the role of β1-class integrins, transmembrane receptors for extracellular matrix proteins, in these migrating neuroblasts. We found that the neuroblast chain formation and blood vessel-guided migration critically depend on β1 integrin signaling. β1 integrin facilitated the adhesion of neuroblasts to laminin and the efficient translocation of their soma during migration. Moreover, artificial laminin-containing scaffolds promoted neuroblast chain formation and migration toward the injured area. These data suggest that laminin signaling via β1 integrin supports vasculature-guided neuronal migration to efficiently supply neuroblasts to injured areas. This study also highlights the importance of vascular scaffolds for cell migration in development and regeneration.

Dual antagonists of integrins.

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Nadrah, K; Dolenc, M Sollner

2005-01-01

The roles of integrins in pathologies have been studied intensively and only partially explained. This has resulted in the development of several nanomolar antagonists to certain integrins. In most cases, the aim was to produce compounds which are highly selective towards specific integrins. This paradigm has recently shifted a little. Targeting two or more integrins with one compound has become a very attractive concept, especially since it has become clear that several severe disorders, such as pathological angiogenesis, cannot be treated just with highly specific integrin antagonists. This review is aimed to elucidate some aspects regarding the design of drugs with dual activity towards integrins. Integrin structure and tissue distribution will first be described, in order to provide the basis for their functions in various pathologies which will follow. Inhibitors of several pairs of integrins will be described.

Formation of {beta}-FeSi{sub 2} thin films by partially ionized vapor deposition

Energy Technology Data Exchange (ETDEWEB)

Harada, Noriyuki; Takai, Hiroshi

2003-05-01

The partially ionized vapor deposition (PIVD) is proposed as a new method to realize low temperature formation of {beta}-FeSi{sub 2} thin films. In this method, Fe is evaporated by E-gun and a few percents of Fe atoms are ionized. We have investigated influences of the ion content and the accelerating voltage of Fe ions on the structural properties of {beta}-FeSi{sub 2} films deposited on Si substrates. It was confirmed that {beta}-FeSi{sub 2} can be formed on Si(1 0 0) substrate by PIVD even at substrate temperature as low as 350, while FeSi by the conventional vacuum deposition. It was concluded that the influence of Fe ions on preferential orientation of {beta}-FeSi{sub 2} depends strongly on the content and the acceleration energy of ions.

Suppression of β3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis

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Tim S. Ellison

2015-09-01

Full Text Available Anti-angiogenic treatments against αvβ3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvβ3-integrin-independent mechanisms. Here, we show that suppression of β3-integrin in mice leads to the activation of a neuropilin-1 (NRP1-dependent cell migration pathway in endothelial cells via a mechanism that depends on NRP1's mobilisation away from mature focal adhesions following VEGF-stimulation. The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours. These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

Formation of a stable oligomer of beta-2 microglobulin requires only transient encounter with Cu(II).

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Calabrese, Matthew F; Miranker, Andrew D

2007-03-16

Beta-2 Microglobulin (beta2m) is a small, globular protein, with high solubility under conditions comparable to human serum. A complication of hemodialysis in renal failure patients is the deposition of unmodified beta2m as amyloid fibers. In vitro, exposure of beta2m to equimolar Cu(2+) under near-physiological conditions can result in self-association leading to amyloid fiber formation. Previously, we have shown that the early steps in this process involve a catalyzed structural rearrangement followed by formation of discrete oligomers. These oligomers, however, have a continued requirement for Cu(2+) while mature fibers are resistant to addition of metal chelate. Here, we report that the transition from Cu(2+) dependent to chelate resistant states occurs in the context of small oligomers, dimeric to hexameric in size. These species require Cu(2+) to form, but once generated, do not need metal cation for stability. Importantly, this transition occurs gradually over several days and the resulting oligomers are isolatable and kinetically stable on timescales exceeding weeks. In addition, formation is enhanced by levels of urea similar to those found in hemodialysis patients. Our results are consistent with our hypothesis that transient encounter of full-length wild-type beta2m with transition metal cation at the dialysis membrane interface is causal to dialysis related amyloidosis.

Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

Energy Technology Data Exchange (ETDEWEB)

Selmi, Anna [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Malinowski, Mariusz [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Brutkowski, Wojciech [Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw (Poland); Bednarek, Radoslaw [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland)

2012-08-15

Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

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Chen, Shulian; Peng, Chuandu; Wei, Xin; Luo, Deyi; Lin, Yifei; Yang, Tongxin; Jin, Xi; Gong, Lina; Li, Hong; Wang, Kunjie

2017-08-01

To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

Role of focal adhesion tyrosine kinases in GPVI-dependent platelet activation and reactive oxygen species formation.

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Naadiya Carrim

Full Text Available We have previously shown the presence of a TRAF4/p47phox/Hic5/Pyk2 complex associated with the platelet collagen receptor, GPVI, consistent with a potential role of this complex in GPVI-dependent ROS formation. In other cell systems, NOX-dependent ROS formation is facilitated by Pyk2, which along with its closely related homologue FAK are known to be activated and phosphorylated downstream of ligand binding to GPVI.To evaluate the relative roles of Pyk2 and FAK in GPVI-dependent ROS formation and to determine their location within the GPVI signaling pathway.Human and mouse washed platelets (from WT or Pyk2 KO mice were pre-treated with pharmacological inhibitors targeting FAK or Pyk2 (PF-228 and Tyrphostin A9, respectively and stimulated with the GPVI-specific agonist, CRP. FAK, but not Pyk2, was found to be essential for GPVI-dependent ROS production and aggregation. Subsequent human platelet studies with PF-228 confirmed FAK is essential for GPVI-mediated phosphatidylserine exposure, α-granule secretion (P-selectin (CD62P surface expression and integrin αIIbβ3 activation. To determine the precise location of FAK within the GPVI pathway, we analyzed the effect of PF-228 inhibition in CRP-stimulated platelets in conjunction with immunoprecipitation and pulldown analysis to show that FAK is downstream of Lyn, Spleen tyrosine kinase (Syk, PI3-K and Bruton's tyrosine kinase (Btk and upstream of Rac1, PLCγ2, Ca2+ release, PKC, Hic-5, NOX1 and αIIbβ3 activation.Overall, these data suggest a novel role for FAK in GPVI-dependent ROS formation and platelet activation and elucidate a proximal signaling role for FAK within the GPVI pathway.

Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and β1-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in vitro

International Nuclear Information System (INIS)

Cordes, N.; Beinke, C.; Beuningen, D. van; Plasswilm, L.

2004-01-01

Background and purpose: interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance-mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. Material and methods: the human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 μM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 μM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-β 1 -integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC 50 for irradiation (2 Gy; IC 50 = 2.2 Gy), cisplatin (2 μM), paclitaxel (5 nM), or mitomycin (7 μM) were performed. Results: attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of β 1 -integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following β 1 -integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC 50 of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. Conclusion: for the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of β 1 -integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin

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Figueira, Cláudio Pereira; Carvalhal, Djalma Gomes Ferrão; Almeida, Rafaela Andrade; Hermida, Micely d’ El-Rei; Touchard, Dominique; Robert, Phillipe; Pierres, Anne; Bongrand, Pierre; dos-Santos, Washington LC

2015-01-01

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis. PMID:26249106

Selective Modulation of Integrin-mediated Cell Migration by Distinct ADAM Family MembersV⃞

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Huang, Jing; Bridges, Lance C.; White, Judith M.

2005-01-01

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin or on its α4β1 or α5β1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the α4β1 but not the α5β1 integrin. ADAM17 had the reciprocal effect; it inhibited α5β1- but not α4β1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both α4β1 and α5β1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the α4β1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains. PMID:16079176

Dwarfism in mice lacking collagen-binding integrins alpha 2 beta 1 and alpha 11 beta 1 is caused by severely diminished IGF-1 levels

OpenAIRE

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F.; Ehlen, Harald W.A.; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C.; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-01-01

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggest...

Elucidating the role of select cytoplasmic proteins in altering diffusion of integrin receptors.

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Sander, Suzanne; Arora, Neha; Smith, Emily A

2012-06-01

Cytoplasmic proteins that affect integrin diffusion in the cell membrane are identified using a combination of fluorescence recovery after photobleaching (FRAP) and RNA interference. Integrin receptors are essential for many cellular events, and alterations in lateral diffusion are one mechanism for modulating their function. In cells expressing native cytoplasmic protein concentrations and spread on a slide containing integrin extracellular ligand, 45 ± 2% of the integrin is mobile with a time-dependent 5.2 ± 0.9 × 10(-9) cm(2)/s diffusion coefficient at 1 s. The time exponent is 0.90 ± 0.07, indicating integrin diffusion moderately slows at longer times. The role of a specific cytoplasmic protein in altering integrin diffusion is revealed through changes in the FRAP curve after reducing the cytoplasmic protein's expression. Decreased expression of cytoplasmic proteins rhea, focal adhesion kinase (FAK), or steamer duck decreases the integrin mobile fraction. For rhea and FAK, there is a concomitant shift to Brownian (i.e., time-independent) diffusion at reduced concentrations of these proteins. In contrast, when the expression of actin 42A, dreadlocks, paxillin, integrin-linked kinase (ILK), or vinculin is reduced, integrin diffusion generally becomes more constrained with an increase in the integrin mobile fraction. This same change in integrin diffusion is measured in the absence of integrin extracellular ligand. The results indicate breaking the extracellular ligand-integrin-cytoskeletal linkage alters integrin diffusion properties, and, in most cases, there is no correlation between integrin and lipid diffusion properties.

Pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3.

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Chen, Wei-Sheng; Cao, Zhiyi; Sugaya, Satoshi; Lopez, Maria J; Sendra, Victor G; Laver, Nora; Leffler, Hakon; Nilsson, Ulf J; Fu, Jianxin; Song, Jianhua; Xia, Lijun; Hamrah, Pedram; Panjwani, Noorjahan

2016-04-12

Lymphangiogenesis plays a pivotal role in diverse pathological conditions. Here, we demonstrate that a carbohydrate-binding protein, galectin-8, promotes pathological lymphangiogenesis. Galectin-8 is markedly upregulated in inflamed human and mouse corneas, and galectin-8 inhibitors reduce inflammatory lymphangiogenesis. In the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection. Further, in the murine model of herpes simplex virus keratitis, corneal pathology and lymphangiogenesis are ameliorated in Lgals8(-/-) mice. Mechanistically, VEGF-C-induced lymphangiogenesis is significantly reduced in the Lgals8(-/-) and Pdpn(-/-) mice; likewise, galectin-8-induced lymphangiogenesis is reduced in Pdpn(-/-) mice. Interestingly, knockdown of VEGFR-3 does not affect galectin-8-mediated lymphatic endothelial cell (LEC) sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtails both galectin-8- and VEGF-C-mediated LEC sprouting. Together, this study uncovers a unique molecular mechanism of lymphangiogenesis in which galectin-8-dependent crosstalk among VEGF-C, podoplanin and integrin pathways plays a key role.

Integrins as Modulators of Transforming Growth Factor Beta Signaling in Dermal Fibroblasts During Skin Regeneration After Injury.

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Boo, Stellar; Dagnino, Lina

2013-06-01

Abnormal wound repair results from disorders in granulation tissue remodeling, and can lead to hypertrophic scarring and fibrosis. Excessive scarring can compromise tissue function and decrease tissue resistance to additional injuries. The development of potential therapies to minimize scarring is, thus, necessary to address an important clinical problem. It has been clearly established that multiple cytokines and growth factors participate in the regulation of cutaneous wound healing. More recently, it has become apparent that these factors do not necessarily activate isolated signaling pathways. Rather, in some cases, there is cross-modulation of several cellular pathways involved in this process. Two of the key pathways that modulate each other during wound healing are activated by transforming growth factor-β and by extracellular matrix proteins acting through integrins. The pathogenesis of excessive scarring upon wound healing is not fully understood, as a result of the complexity of this process. However, the fact that many pathways combine to produce fibrosis provides multiple potential therapeutic targets. Some of them have been identified, such as focal adhesion kinase and integrin-linked kinase. Currently, a major challenge is to develop pharmacological inhibitors of these proteins with therapeutic value to promote efficient wound repair. The ability to better understand how different pathways crosstalk during wound repair and to identify and pharmacologically modulate key factors that contribute to the regulation of multiple wound-healing pathways could potentially provide effective therapeutic targets to decrease or prevent excessive scar formation and/or development of fibrosis.

The role of integrin αv in proliferation and differentiation of human dental pulp cell response to calcium silicate cement.

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Hung, Chi-Jr; Hsu, Hsin-I; Lin, Chi-Chang; Huang, Tsui-Hsien; Wu, Buor-Chang; Kao, Chia-Tze; Shie, Ming-You

2014-11-01

It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and β-tricalcium phosphate (TCP) cement. In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. The results indicate that CS releases Si ion-increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Origin of the various beta dependences of ELMy H-mode confinement properties

International Nuclear Information System (INIS)

Takizuka, T; Urano, H; Takenaga, H; Oyama, N

2006-01-01

Dependence of the energy confinement in ELMy H-mode tokamak on the beta has been investigated for a long time, but a common conclusion has not been obtained so far. Recent non-dimensional transport experiments in JT-60U demonstrated clearly the beta degradation. A database for JT-60U ELMy H-mode confinement was assembled. Analysis of this database is carried out, and the strong beta degradation consistent with the above experiments is confirmed. Two subsets of ASDEX Upgrade and JET data in the ITPA H-mode confinement database are analysed to find the origin of the various beta dependences. The shaping of the plasma cross section, as well as the fuelling condition, affects the confinement performance. The beta dependence is not identical for different devices and conditions. The shaping effect, as well as the fuelling effect, is a possible candidate for causing the variation of beta dependence

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

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Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

2014-04-01

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alterated integrin expression in lichen planopilaris

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Erriquez Roberta

2007-02-01

Full Text Available Abstract Background Lichen planopilaris (LPP is an inflammatory disease characterized by a lymphomononuclear infiltrate surrounding the isthmus and infundibulum of the hair follicle of the scalp, that evolves into atrophic/scarring alopecia. In the active phase of the disease hairs are easily plucked with anagen-like hair-roots. In this study we focused on the expression of integrins and basement membrane components of the hair follicle in active LPP lesions. Methods Scalp biopsies were taken in 10 patients with LPP and in 5 normal controls. Using monoclonal antibodies against α3β1 and α6β4 integrins we showed the expression of these integrins and of the basement membrane components of the hair follicle in active LPP lesions and in healthy scalp skin. Results In the LPP involved areas, α3β1 was distributed in a pericellular pattern, the α6 subunit was present with a basolateral distribution while the β4 subunit showed discontinuous expression at the basal pole and occasionally, basolateral staining of the hair follicle. Conclusion: An altered distribution of the integrins in active LPP lesions can explain the phenomenon of easy pulling-out of the hair with a "gelatinous" root-sheath.

Integrins and extracellular matrix in mechanotransduction

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Ramage L

2011-12-01

Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

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Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

2013-08-01

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.

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Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

2015-01-01

Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging. Copyright © 2014 Elsevier Ltd. All rights reserved.

Localization of thymosin beta-4 in tumors

DEFF Research Database (Denmark)

Larsson, L. -I.; Holck, Susanne

2007-01-01

in colorectal carcinomas. The degree of staining of breast cancer cells for thymosin beta-4 correlated neither to histological grade nor to endothelial cell staining. However, there was a tendency toward correlation (P = 0.07) between staining of endothelial cells and histological grade. Treatment of cultured......Overexpression of thymosin beta-4 has been linked to malignant progression but the localization of this polypeptide within tumors is incompletely known. We therefore examined breast cancers for thymosin beta-4 using immunofluorescence. Reactive cells were identified with monoclonal cell marker...... breast cancer cells (SK-BR-3) with 1-4 microg thymosin beta-4/mL significantly increased cell numbers, as determined by MTT-assays. These data reveal an unexpected cellular heterogeneity of thymosin beta-4 expression in breast and colonic carcinomas and suggest that local release of this polypeptide...

Apoptosis following interleukin-2 withdrawal from T cells: evidence for a regulatory role of CD18 (beta 2-integrin) molecules

DEFF Research Database (Denmark)

Röpke, C; Gladstone, P; Nielsen, M

1996-01-01

, these findings suggest that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following cytokine withdrawal, and that the regulation is mediated, at least partly, through T-T cell interactions. Thus, apoptotic death following IL-2 deprivation appears to be under "social" control...... activated T cells. Thus, removal of IL-2 from proliferating T cells not only induces growth arrest, but triggers a massive cell death due to apoptosis. While the apoptotic response involves a series of well-described events, it remains less clear how apoptosis is regulated following IL-2 withdrawal. Here...... molecules (CD28, CD29, CD49d, CD80, CD86) did not. Secondly, IL-2 withdrawal resulted in a retarded apoptotic response in LFA-1 (CD11a/CD18) negative T cells obtained from a leukocyte adhesion deficiency (LAD) patient, as compared to LFA-1 positive T cell lines. Thirdly, co-culture of LFA-1 positive...

Mechanical control of cyclic AMP signalling and gene transcription through integrins

Science.gov (United States)

Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

2000-01-01

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

Outside-In Signal Transmission by Conformational Changes in Integrin Mac-11

Science.gov (United States)

Lefort, Craig T.; Hyun, Young-Min; Schultz, Joanne B.; Law, Foon-Yee; Waugh, Richard E.; Knauf, Philip A.; Kim, Minsoo

2010-01-01

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the αM and β2 subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals. PMID:19864611

Glycation induces formation of amyloid cross-beta structure in albumin.

Science.gov (United States)

Bouma, Barend; Kroon-Batenburg, Loes M J; Wu, Ya-Ping; Brünjes, Bettina; Posthuma, George; Kranenburg, Onno; de Groot, Philip G; Voest, Emile E; Gebbink, Martijn F B G

2003-10-24

Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.

Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.

Science.gov (United States)

El Ghalbzouri, Abdoelwaheb; Jonkman, Marcel F; Dijkman, Remco; Ponec, Maria

2005-01-01

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

Genetic analysis of beta1 integrin function: confirmed, new and revised roles for a crucial family of cell adhesion molecules

DEFF Research Database (Denmark)

Brakebusch, C; Hirsch, E; Potocnik, A

1997-01-01

Integrins are heterodimeric cell adhesion proteins connecting the extracellular matrix to the cytoskeleton and transmitting signals in both directions. These integrins are suggested to be involved in many different biological processes such as growth, differentiation, migration, and cell death. O...

Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

Science.gov (United States)

Zhang, Kai; Ding, Wei; Sun, Wei; Sun, Xiao-jiang; Xie, You-zhuan; Zhao, Chang-qing; Zhao, Jie

2016-01-01

Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

Entrainment of prefrontal beta oscillations induces an endogenous echo and impairs memory formation.

Science.gov (United States)

Hanslmayr, Simon; Matuschek, Jonas; Fellner, Marie-Christin

2014-04-14

Brain oscillations across all frequency bands play a key role for memory formation. Specifically, desynchronization of local neuronal assemblies in the left inferior prefrontal cortex (PFC) in the beta frequency (∼18 Hz) has been shown to be central for encoding of verbal memories. However, it remains elusive whether prefrontal beta desynchronization is causally relevant for memory formation and whether these endogenous beta oscillations can be entrained by external stimulation. By using combined EEG-TMS (transcranial magnetic stimulation), we here address these fundamental questions in human participants performing a word-list learning task. Confirming our predictions, memory encoding was selectively impaired when the left inferior frontal gyrus (IFG) was driven at beta (18.7 Hz) compared to stimulation at other frequencies (6.8 Hz and 10.7 Hz) and to ineffective sham stimulation (18.7 Hz). Furthermore, a sustained oscillatory "echo" in the left IFG, which outlasted the stimulation period by approximately 1.5 s, was observed solely after beta stimulation. The strength of this beta echo was related to memory impairment on a between-subjects level. These results show endogenous oscillatory entrainment effects and behavioral impairment selectively in beta frequency for stimulation of the left IFG, demonstrating an intimate causal relationship between prefrontal beta desynchronization and memory formation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

Science.gov (United States)

Sun, Hao; Lagarrigue, Frederic; Gingras, Alexandre R; Fan, Zhichao; Ley, Klaus; Ginsberg, Mark H

2018-04-02

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development. © 2018 Sun et al.

Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

1995-12-01

Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma

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W. Theunissen

2014-03-01

Full Text Available Thymosin beta 4 (Tβ4 and thymosin beta 10 (Tβ10 are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tβ4 and Tβ10 in hepatocellular carcinoma (HCC. To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tβ4 and Tβ10. Immunoreactivity for Tβ4 and Tβ10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tβ4 reactivity was detected in 7/23 cases (30% and Tβ10 reactivity in 22/23 (97% cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tβ10 showed a strong homogeneous expression, was Tβ4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tβ10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tβ10 in HCC progression.

Integrins as Therapeutic Targets: Successes and Cancers

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Sabine Raab-Westphal

2017-08-01

Full Text Available Integrins are transmembrane receptors that are central to the biology of many human pathologies. Classically mediating cell-extracellular matrix and cell-cell interaction, and with an emerging role as local activators of TGFβ, they influence cancer, fibrosis, thrombosis and inflammation. Their ligand binding and some regulatory sites are extracellular and sensitive to pharmacological intervention, as proven by the clinical success of seven drugs targeting them. The six drugs on the market in 2016 generated revenues of some US$3.5 billion, mainly from inhibitors of α4-series integrins. In this review we examine the current developments in integrin therapeutics, especially in cancer, and comment on the health economic implications of these developments.

Laminin-binding integrins and their tetraspanin partners as potential antimetastatic targets

Science.gov (United States)

Stipp, Christopher S.

2010-01-01

Within the integrin family of cell adhesion receptors, integrins α3β1, α6β1, α6β4 and α7β1 make up a laminin-binding subfamily. The literature is divided on the role of these laminin-binding integrins in metastasis, with different studies indicating either pro- or antimetastatic functions. The opposing roles of the laminin-binding integrins in different settings might derive in part from their unusually robust associations with tetraspanin proteins. Tetraspanins organise integrins into multiprotein complexes within discrete plasma membrane domains termed tetraspanin-enriched microdomains (TEMs). TEM association is crucial to the strikingly rapid cell migration mediated by some of the laminin-binding integrins. However, emerging data suggest that laminin-binding integrins also promote the stability of E-cadherin-based cell–cell junctions, and that tetraspanins are essential for this function as well. Thus, TEM association endows the laminin-binding integrins with both pro-invasive functions (rapid migration) and anti-invasive functions (stable cell junctions), and the composition of TEMs in different cell types might help determine the balance between these opposing activities. Unravelling the tetraspanin control mechanisms that regulate laminin-binding integrins will help to define the settings where inhibiting the function of these integrins would be helpful rather than harmful, and may create opportunities to modulate integrin activity in more sophisticated ways than simple functional blockade. PMID:20078909

Fibronectin regulates the activation of THP-1 cells by TGF-beta1.

Science.gov (United States)

Wang, A C; Fu, L

2001-03-01

To determine how fibronectin regulates the immunomodulatory effects of transforming growth factor (TGF)-beta on THP-1 cells. THP-1 monocytic cell line. THP-1 cells were primed for 48 h in the presence or absence of 250 pM TGF-beta1. Assays or assessments carried out, together with statistical test applied. We found that adherence to fibronectin dramatically modulates the effects of TGF-beta1 on the human monocytic cell line THP-1. TGF-beta did not significantly affect constitutive interleukin (IL)-8 secretion or IL-1beta-induced IL-8 secretion from suspended cells. In contrast, TGF-beta stimulated IL-8 secretion as well as augmented IL-1beta-induced IL-8 secretion from adherent cells. The differential effects of TGF-beta1 on IL-8 secretion from suspended and adherent cells could not be explained by differences in IL-1 receptor antagonist production. The effects of fibronectin on TGF-beta1 induced IL-8 secretion from THP-1 cells were mimicked by adhesion to immobilized anti-a4beta1 integrin antibody and to a fibronectin fragment containing the CS-1 domain. These results indicate that alpha4beta1-mediated adhesion to fibronectin may play a key role during inflammation by profoundly influencing the effects of TGF-beta1 on monocytes.

PET imaging of angiogenesis after myocardial infarction/reperfusion using a one-step labeled integrin-targeted tracer {sup 18}F-AlF-NOTA-PRGD2

Energy Technology Data Exchange (ETDEWEB)

Gao, Haokao [The Fourth Military Medical University, Department of Cardiology, Xijing Hospital, Xi' an (China); National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), Bethesda, MD (United States); Lang, Lixin; Guo, Ning; Quan, Qimeng; Hu, Shuo; Kiesewetter, Dale O.; Niu, Gang; Chen, Xiaoyuan [National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), Bethesda, MD (United States); Cao, Feng [The Fourth Military Medical University, Department of Cardiology, Xijing Hospital, Xi' an (China)

2012-04-15

The {alpha}{sub v}{beta}{sub 3} integrin represents a potential target for noninvasive imaging of angiogenesis. The purpose of this study was to evaluate a novel one-step labeled integrin {alpha}{sub v}{beta}{sub 3}-targeting positron emission tomography (PET) probe, {sup 18}F-AlF-NOTA-PRGD2, for angiogenesis imaging in a myocardial infarction/reperfusion (MI/R) animal model. Male Sprague-Dawley rats underwent 45-min transient left coronary artery occlusion followed by reperfusion. The myocardial infarction was confirmed by ECG, {sup 18}F-fluorodeoxyglucose (FDG) imaging, and cardiac ultrasound. In vivo PET imaging was used to determine myocardial uptake of {sup 18}F-AlF-NOTA-PRGD2 at different time points following reperfusion. The control peptide RAD was labeled with a similar procedure and used to confirm the specificity. Ex vivo autoradiographic analysis and CD31/CD61 double immunofluorescence staining were performed to validate the PET results. Myocardial origin of the {sup 18}F-AlF-NOTA-PRGD2 accumulation was confirmed by {sup 18}F-FDG and autoradiography. PET imaging demonstrated increased focal accumulation of {sup 18}F-AlF-NOTA-PRGD2 in the infarcted area which started at day 3 (0.28 {+-} 0.03%ID/g, p < 0.05) and peaked between 1 and 3 weeks (0.59 {+-} 0.16 and 0.55 {+-} 0.13%ID/g, respectively). The focal accumulation decreased but still kept at a higher level than the sham group after 4 months of reperfusion (0.31 {+-} 0.01%ID/g, p < 0.05). Pretreatment with unlabeled arginine-glycine-aspartic acid (RGD) peptide significantly decreased tracer uptake, indicating integrin specificity of this tracer. At 1 week after MI/R, uptake of the control tracer {sup 18}F-AlF-NOTA-RAD that does not bind to integrin, in the infarcted area, was only 0.21 {+-} 0.01%ID/g. Autoradiographic imaging showed the same trend of uptake in the myocardial infarction area. The time course of focal tracer uptake was consistent with the pattern of vascular density and integrin {beta

Integrins Regulate Apical Constriction via Microtubule Stabilization in the Drosophila Eye Disc Epithelium

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Vilaiwan M. Fernandes

2014-12-01

Full Text Available During morphogenesis, extracellular signals trigger actomyosin contractility in subpopulations of cells to coordinate changes in cell shape. To illuminate the link between signaling-mediated tissue patterning and cytoskeletal remodeling, we study the progression of the morphogenetic furrow (MF, the wave of apical constriction that traverses the Drosophila eye imaginal disc preceding photoreceptor neurogenesis. Apical constriction depends on actomyosin contractility downstream of the Hedgehog (Hh and bone morphogenetic protein (BMP pathways. We identify a role for integrin adhesion receptors in MF progression. We show that Hh and BMP regulate integrin expression, the loss of which disrupts apical constriction and slows furrow progression; conversely, elevated integrins accelerate furrow progression. We present evidence that integrins regulate MF progression by promoting microtubule stabilization, since reducing microtubule stability rescues integrin-mediated furrow acceleration. Thus, integrins act as a genetic link between tissue-level signaling events and morphological change at the cellular level, leading to morphogenesis and neurogenesis in the eye.

Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

Science.gov (United States)

del Pozo, Miguel A.; Alderson, Nazilla B.; Grande-García, Araceli; Balasubramanian, Nagaraj; Schwartz, Martin A.; Kiosses, William B.; Anderson, Richard G.W.

2005-01-01

Growth of normal cells is anchorage-dependent because signalling through multiple pathways including Erk, PI 3-kinase and Rac requires integrin-mediated cell adhesion 1. Components of these pathways localize to low density, cholesterol-rich domains in the plasma membrane named “lipid rafts” 2,3 or “cholesterol enriched membrane microdomains” (CEMM) 4. We previously reported that integrin-mediated adhesion regulates CEMM trafficking such that cell detachment from the extracellular matrix (ECM) triggers CEMM internalisation and clearance from the plasma membrane 5. We now report that this internalisation is mediated by dynamin-2 and caveolin-1. Internalisation requires phosphorylation of caveolin-1 on tyrosine 14. A shift in localisation of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalisation upon cell detachment, which mediates inhibition of Erk, PI 3-kinase and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1. PMID:16113676

Dual integrin and gastrin-releasing peptide receptor targeted tumor imaging using 18F-labeled PEGylated RGD-bombesin heterodimer 18F-FB-PEG3-Glu-RGD-BBN.

Science.gov (United States)

Liu, Zhaofei; Yan, Yongjun; Chin, Frederic T; Wang, Fan; Chen, Xiaoyuan

2009-01-22

Radiolabeled RGD and bombesin peptides have been extensively investigated for tumor integrin alpha(v)beta(3) and GRPR imaging, respectively. Due to the fact that many tumors are both integrin and GRPR positive, we designed and synthesized a heterodimeric peptide Glu-RGD-BBN, which is expected to be advantageous over the monomeric peptides for dual-receptor targeting. A PEG(3) spacer was attached to the glutamate alpha-amino group of Glu-RGD-BBN to enhance the (18)F labeling yield and to improve the in vivo kinetics. PEG(3)-Glu-RGD-BBN possesses the comparable GRPR and integrin alpha(v)beta(3) receptor-binding affinities as the corresponding monomers, respectively. The dual-receptor targeting properties of (18)F-FB-PEG(3)-Glu-RGD-BBN were observed in PC-3 tumor model. (18)F-FB-PEG(3)-Glu-RGD-BBN with high tumor contrast and favorable pharmacokinetics is a promising PET tracer for dual integrin and GRPR positive tumor imaging. This heterodimer strategy may also be an applicable method to develop other molecules with improved in vitro and in vivo characterizations for tumor diagnosis and therapy.

Alteration of Pituitary Tumor Transforming Gene-1 Regulates Trophoblast Invasion via the Integrin/Rho-Family Signaling Pathway.

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Seung Mook Lim

Full Text Available Trophoblast invasion ability is an important factor in early implantation and placental development. Recently, pituitary tumor transforming gene 1 (PTTG1 was shown to be involved in invasion and proliferation of cancer. However, the role of PTTG1 in trophoblast invasion remains unknown. Thus, in this study we analyzed PTTG1 expression in trophoblasts and its effect on trophoblast invasion activity and determined the mechanism through which PTTG1 regulates trophoblast invasion. Trophoblast proliferation and invasion abilities, regardless of PTTG1 expression, were analyzed by quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting analysis, invasion assay, western blot, and zymography after treatment with small interfering RNA against PTTG1 (siPTTG1. Additionally, integrin/Rho-family signaling in trophoblasts by PTTG1 alteration was analyzed. Furthermore, the effect of PTTG1 on trophoblast invasion was evaluated by microRNA (miRNA mimic and inhibitor treatment. Trophoblast invasion was significantly reduced through decreased matrix metalloproteinase (MMP-2 and MMP-9 expression when PTTG1 expression was inhibited by siPTTG1 (p < 0.05. Furthermore, knockdown of PTTG1 increased expression of integrin alpha 4 (ITGA4, ITGA5, and integrin beta 1 (ITGB1; otherwise, RhoA expression was significantly decreased (p < 0.05. Treatment of miRNA-186-5p mimic and inhibitor controlled trophoblast invasion ability by altering PTTG1 and MMP expression. PTTG1 can control trophoblast invasion ability via regulation of MMP expression through integrin/Rho-family signaling. In addition, PTTG1 expression and its function were regulated by miRNA-186-5p. These results help in understanding the mechanism through which PTTG1 regulates trophoblast invasion and thereby implantation and placental development.

Integrins and small GTPases as modulators of phagocytosis.

Science.gov (United States)

Sayedyahossein, Samar; Dagnino, Lina

2013-01-01

Phagocytosis is the mechanism whereby cells engulf large particles. This process has long been recognized as a critical component of the innate immune response, which constitutes the organism's defense against microorganisms. In addition, phagocytic internalization of apoptotic cells or cell fragments plays important roles in tissue homeostasis and remodeling. Phagocytosis requires target interactions with receptors on the plasma membrane of the phagocytic cell. Integrins have been identified as important mediators of particle clearance, in addition to their well-established roles in cell adhesion, migration and mechanotransduction. Indeed, these ubiquitously expressed proteins impart phagocytic capacity to epithelial, endothelial and mesenchymal cell types. The importance of integrins in particle internalization is emphasized by the ability of microbial and viral pathogens to exploit their signaling pathways to invade host cells, and by the wide variety of disorders that arise from abnormalities in integrin-dependent phagocytic uptake. Copyright © 2013 Elsevier Inc. All rights reserved.

Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of β1 integrin-null cells

International Nuclear Information System (INIS)

Kikkawa, Yamato; Yu, Hao; Genersch, Elke; Sanzen, Noriko; Sekiguchi, Kiyotoshi; Faessler, Reinhard; Campbell, Kevin P.; Talts, Jan F.; Ekblom, Peter

2004-01-01

The presence of many laminin receptors of the β1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin α6β4 and dystroglycan. We therefore tested the binding of a β1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin α6Aβ4A variant. GD25 β1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin α6 antibody, but not by a dystroglycan antibody. Hence, integrin α6Aβ4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin α6Aβ4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin α6Aβ4A

Construction of hydroxypropyl-{beta}-cyclodextrin copolymer nanoparticles and targeting delivery of paclitaxel

Energy Technology Data Exchange (ETDEWEB)

Miao Qinghua; Li Suping; Han Siyuan [National Center for Nanoscience and Technology of China, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety (China); Wang Zhi, E-mail: wangzhi@jlu.edu.cn [Jilin University, Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education (China); Wu Yan, E-mail: wuy@nanoctr.cn; Nie Guangjun, E-mail: niegj@nanoctr.cn [National Center for Nanoscience and Technology of China, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety (China)

2012-08-15

A novel amphiphilic copolymer with p-maleimidophenyl isocyanate-hydroxypropyl-{beta}-cyclodextrin-polylactide-1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine to generate copolymer nanoparticles (NPs) has been designed. In order to develop an active targeting system, integrin {alpha}{sub v}{beta}{sub 3}-specific targeting peptide cyclo(Arg-Gly-Asp-D-Phe-Cys), cRGD, was conjugated to the surface of NPs (NPs-RGD). These NPs were used to encapsulate anti-tumor drug, paclitaxel. The resulting NPs exhibited high drug-loading capacity and controlled drug release in vitro at acidic pH. In vitro cytotoxicity assay demonstrates that paclitaxel-loaded NPs-RGD significantly inhibited B16 tumor cell (high {alpha}{sub v}{beta}{sub 3}) proliferation relative to free paclitaxel and paclitaxel-loaded NPs at high concentrations. Paclitaxel-loaded NPs-RGD localized mainly in lysosomes in B16 cells as revealed by confocal microscopy. These results suggest a novel strategy for fabrication-functionalizing hydroxypropyl-{beta}-cyclodextrin copolymer nanoparticles for targeting delivery of paclitaxel to integrin {alpha}{sub v}{beta}{sub 3}-rich tumor cells. These nanocarriers can be readily extended to couple other bioactive molecules for active targeting and delivery of various chemotherapeutic drugs.

Ouabain interactions with the α4 isoform of the sodium pump trigger non-classical steroid hormone signaling and integrin expression in spermatogenic cells.

Science.gov (United States)

Upmanyu, Neha; Dietze, Raimund; Kirch, Ulrike; Scheiner-Bobis, Georgios

2016-11-01

In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones. Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, β3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and β3 is also seen in cultures of seminiferous tubules exposed to 10nM ouabain. At the protein level we observed a significant stimulation of β3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of β3 integrin. The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology. Copyright © 2016 Elsevier B.V. All rights reserved.

Leukocyte integrins and their ligand interactions

Science.gov (United States)

Hyun, Young-Min; Lefort, Craig T.; Kim, Minsoo

2010-01-01

Although critical for cell adhesion and migration during normal immune-mediated reactions, leukocyte integrins are also involved in the pathogenesis of diverse clinical conditions including autoimmune diseases and chronic inflammation. Leukocyte integrins therefore have been targets for anti-adhesive therapies to treat the inflammatory disorders. Recently, the therapeutic potential of integrin antagonists has been demonstrated in psoriasis and multiple sclerosis. However, current therapeutics broadly affect integrin functions and, thus, yield unfavorable side effects. This review discusses the major leukocyte integrins and the anti-adhesion strategies for treating immune diseases. PMID:19184539

Dopamine receptor D4 internalization requires a beta-arrestin and a visual arrestin.

Science.gov (United States)

Deming, Janise D; Shin, Jung-A; Lim, Kayleen; Lee, Eun-Jin; Van Craenenbroeck, Kathleen; Craft, Cheryl Mae

2015-10-01

The G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors. To test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4. Immunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation. The results have functional

Analysis of interleukin (IL)-1 beta and transforming growth factor (TGF)-beta-induced signal transduction pathways in IL-2 and TGF-beta secretion and proliferation in the thymoma cell line EL4.NOB-1.

Science.gov (United States)

Siese, A; Jaros, P P; Willig, A

1999-02-01

In the present study we investigated the interleukin (IL)-1beta and transforming growth factor-beta1 (TGF-beta1)-mediated proliferation, and production of IL-2 and TGF-beta, in the murine T-cell line, EL4.NOB-1. This cell line is resistant to TGF-beta concerning growth arrest but not autoinduction or suppression of IL-1-induced IL-2 production. When cocultured with IL-1beta, TGF-beta showed growth-promoting activity that could be antagonized by adding the phosphatidyl choline-dependent phospholipase C (PC-PLC) inhibitor, D609. Using specific enzyme inhibitors of protein kinases (PK) C and A, mitogen-activated protein kinase (MAPK), phospholipase A2 (PLA2), phosphatidylinositol-dependent (PI)-PLC and PC-PLC, we showed that IL-1beta-induced IL-2 synthesis was dependent on all investigated kinases and phospholipases, except PC-PLC. TGF-beta1 was able to inhibit IL-2 synthesis by the activation of PKA and MAPK. The same kinases are involved in TGF-beta autoinduction that is accompanied by a secretion of the active but not the latent growth factor and is antagonized by IL-1beta. Addition of the PI-PLC inhibitor, ET 18OCH3, or the PLA2 inhibitor (quinacrine) alone, resulted in secretion of latent TGF-beta and, in the case of ET 18OCH3, active TGF-beta. These data implicate a role for PI-PLC and PLA2 in the control of latency and secretion. Analysis of specific tyrosine activity and c-Fos expression showed synergistic but no antagonistic effects. These events are therefore not involved in IL- and TGF-beta-regulated IL-2 and TGF-beta production, but might participate in IL-1/TGF-beta-induced growth promotion.

Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

Science.gov (United States)

Mills, E N; Huang, L; Noel, T R; Gunning, A P; Morris, V J

2001-06-11

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

Inhibition of vimentin or B1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xueping; Fournier, Marcia V; Ware, Joy L; Bissell, Mina J; Yacoub, Adly; Zehner, Zendra E

2008-06-12

Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphologic changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the immortalized, prostate epithelial P69 cell line by selection in athymic, nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the parental, nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin, or {alpha}6 and {beta}1 integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via small interfering RNA interference or the expression of {alpha}6 and {beta}1 integrins by the addition of blocking antibodies, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by s.c. injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in three-dimensional lrECM gels. These studies suggest that the levels of vimentin and {beta}1 integrin play a key role in the homeostasis of the normal acinus in prostate and that their dysregulation may lead to tumorigenesis. [Mol Cancer Ther 2009;8(3):499-508].

Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and β4 integrin function in MDA-MB-231 breast cancer cells

International Nuclear Information System (INIS)

Lee, W.-J.; Chen, W.-K.; Wang, C.-J.; Lin, W.-L.; Tseng, T.-H.

2008-01-01

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration and anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of β4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin β4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By

Tissue-engineered bone formation using human bone marrow stromal cells and novel {beta}-tricalcium phosphate

Energy Technology Data Exchange (ETDEWEB)

Liu Guangpeng [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Zhao Li [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cui Lei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Liu Wei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cao Yilin [National Tissue Engineering Research and Development Center, Shanghai 200235 (China)

2007-06-01

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel {beta}-tricalcium phosphate ({beta}-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous {beta}-TCP scaffolds with pores of 300-500 {mu}m in size were prepared by the polymeric sponge method. {beta}-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous {beta}-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In {beta}-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in {beta}-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous {beta}-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

Lamellipodia nucleation by filopodia depends on integrin occupancy and downstream Rac1 signaling

International Nuclear Information System (INIS)

Guillou, Herve; Depraz-Depland, Adeline; Planus, Emmanuelle; Vianay, Benoit; Chaussy, Jacques; Grichine, Alexei; Albiges-Rizo, Corinne; Block, Marc R.

2008-01-01

Time-lapse video-microscopy unambiguously shows that fibroblast filopodia are the scaffold of lamellipodia nucleation that allows anisotropic cell spreading. This process was dissected into elementary stages by monitoring cell adhesion on micropatterned extracellular matrix arrays of various pitches. Adhesion structures are stabilized by contact with the adhesive plots and subsequently converted into lamellipodia-like extensions starting at the filopodia tips. This mechanism progressively leads to full cell spreading. Stable expression of the dominant-negative Rac1 N17 impairs this change in membrane extension mode and stops cell spreading on matrix arrays. Similar expression of the dominant-negative Cdc42 N17 impairs cell spreading on homogenous and structured substrate, suggesting that filopodia extension is a prerequisite for cell spreading in this model. The differential polarity of the nucleation of lamellipodial structures by filopodia on homogenous and structured surfaces starting from the cell body and of filopodia tip, respectively, suggested that this process is triggered by areas that are in contact with extracellular matrix proteins for longer times. Consistent with this view, wild-type cells cannot spread on microarrays made of function blocking or neutral anti-β 1 integrin antibodies. However, stable expression of a constitutively active Rac1 mutant rescues the cell ability to spread on these integrin microarrays. Thereby, lamellipodia nucleation by filopodia requires integrin occupancy by matrix substrate and downstream Rac1 signaling

Wnt/beta-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation.

Science.gov (United States)

Mak, Kingston Kinglun; Chen, Miao-Hsueh; Day, Timothy F; Chuang, Pao-Tien; Yang, Yingzi

2006-09-01

Both the Wnt/beta-catenin and Ihh signaling pathways play essential roles in crucial aspects of endochondral ossification: osteoblast differentiation, chondrocyte proliferation and hypertrophy. To understand the genetic interaction between these two signaling pathways, we have inactivated the beta-catenin gene and upregulated Ihh signaling simultaneously in the same cells during endochondral skeletal development using beta-catenin and patched 1 floxed alleles. We uncovered previously unexpected roles of Ihh signaling in synovial joint formation and the essential function of Wnt/beta-catenin signaling in regulating chondrocyte survival. More importantly, we found that Wnt and Ihh signaling interact with each other in distinct ways to control osteoblast differentiation, chondrocyte proliferation, hypertrophy, survival and synovial joint formation in the developing endochondral bone. Beta-catenin is required downstream of Ihh signaling and osterix expression for osteoblast differentiation. But in chondrocyte survival, beta-catenin is required upstream of Ihh signaling to inhibit chondrocyte apoptosis. In addition, Ihh signaling can inhibit chondrocyte hypertrophy and synovial joint formation independently of beta-catenin. However, there is a strong synergistic interaction between Wnt/beta-catenin and Ihh signaling in regulating synovial joint formation.

VCAM-1 on peritoneum and α4β1 integrin in endometrium and their implications in endometriosis.

Science.gov (United States)

Schutt, Amy K; Atkins, Kristen A; Slack-Davis, Jill K; Stovall, Dale W

2015-01-01

The objective of the study is to investigate vascular cellular adhesion molecule-1 (VCAM-1) expression on peritoneal mesothelial cells and α4β1 integrin on eutopic endometrium as possible mechanisms in the pathogenesis of endometriosis. It is a case-control study carried out at an academic medical center. Participants are patients with (n=9) and without (n=15) endometriosis. The main outcome measures included VCAM-1 expression on peritoneal mesothelial cells and α4β1 expression on eutopic endometrium using immunohistochemistry and flow cytometry, respectively. Patients with endometriosis were more likely to express VCAM-1 on peritoneal mesothelial cells, both in areas with and without macroscopic disease, compared with patients without endometriosis (9/9 vs. 3/15, Pperitoneal mesothelial cells is associated with endometriosis. This field effect, in addition to the similarity found with regards to the expression of α4β1 integrin in eutopic endometrium between cases and controls, may implicate the expression of VCAM-1 in the peritoneum, and not changes in the eutopic endometrium, as a contributor to the pathogenesis of endometriosis.

Integrin Signalling

OpenAIRE

Schelfaut, Roselien

2005-01-01

Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins b...

Standard molar enthalpies of formation of copper(II) {beta}-diketonates and monothio-{beta}-diketonates

Energy Technology Data Exchange (ETDEWEB)

Ribeiro da Silva, Manuel A.V. [Centro de Investigacao em Quimica, Departamento de Quimica, Faculdade de Ciencias, Universidade do Porto, Rua do Campo Alegre, 687, P-4169-007 Porto (Portugal)]. E-mail: risilva@fc.up.pt; Santos, Luis M.N.B.F. [Centro de Investigacao em Quimica, Departamento de Quimica, Faculdade de Ciencias, Universidade do Porto, Rua do Campo Alegre, 687, P-4169-007 Porto (Portugal)

2006-07-15

The standard (p{sup o}=0.1MPa) molar enthalpies of formation of the crystalline complexes of dibenzoylmethane (Hdbm), thenoyltrifluoroacetone (Httfa), monothiodibenzoylmethane (HdbmS), and monothiothenoyltrifluoroacetone (HttfaS) of copper(II) were determined, at T=298.15K, by high precision solution-reaction calorimetry. The standard molar enthalpies of sublimation of the copper(II) {beta}-diketonate complexes were measured by high-temperature Calvet microcalorimetry. From the standard molar enthalpies of formation of the complexes in the gaseous state, the mean molar dissociation enthalpies copper(II)-ligand, (Cu-L), were derived. {delta}{sub f}H{sub m}{sup o} (cr){delta}{sub cr}{sup g}H{sub m}{sup o} kJ.mol{sup -1}kJ.mol{sup -1}Bis(dibenzoylmethanate)copper(II), Cu(dbm){sub 2}-364.0+/-3.9230.7+/-8.2Bis(thenoyltrifluoroacetonate)copper(II), Cu(ttfa){sub 2}-1824.3+/-8.3167.9+/-7.4Bis(monothiodibenzoylmethanate)copper(II), Cu(dbmS){sub 2}35.6+/-7.7[241+/-15]Bis(monothiothenoyltrifluoroacetonate) copper(II), Cu(ttfaS){sub 2}-1405.7+/-8.3[177+/-15].

MHC class II ligation induces CD58 (LFA-3)-mediated adhesion in human T cells

DEFF Research Database (Denmark)

Nielsen, M; Gerwien, J; Geisler, C

1998-01-01

ligation induces homotypic adhesion in both beta2-integrin-positive and negative, CD4-positive T cell lines. Anti-CD18 monoclonal antibody (mAb) weakly inhibited the adhesion response in beta2-integrin-positive T cells and had no effect on beta2-integrin-negative T cells. In contrast, an anti-CD58 (LFA-3...

Apolipoprotein M predicts pre-beta-HDL formation: studies in type 2 diabetic and nondiabetic subjects

DEFF Research Database (Denmark)

Plomgaard, P; Dullaart, R P F; de Vries, R

2009-01-01

protein (PLTP) activity and the ability of plasma to promote cholesterol efflux from cultured fibroblasts. RESULTS: ApoM was approximately 9% lower in patients with type 2 diabetes compared to controls (0.025 +/- 0.006 vs. 0.027 +/- 0.007 g L(-1), P = 0.01). The difference in apoM was largely attributable...... diabetes. Pre-beta-HDL and pre-beta-HDL formation are positively associated with apoM, supporting the hypothesis that apoM plays a role in HDL remodelling in humans. Lower apoM may provide a mechanism to explain why pre-beta-HDL formation is not increased in type 2 diabetes despite elevated PLTP activity.......OBJECTIVE: Studies in mice suggest that plasma apoM is lowered in hyperinsulinaemic diabetes and that apoM stimulates formation of pre-beta-HDL. Pre-beta-HDL is an acceptor of cellular cholesterol and may be critical for reverse cholesterol transport. Herein, we examined whether patients with type...

The impact of quercetin on wound healing relates to changes in αV and β1 integrin expression.

Science.gov (United States)

Doersch, Karen M; Newell-Rogers, M Karen

2017-08-01

Overly fibrotic wound healing can lead to excess scar formation, causing functional impairment and undesirable cosmetic results. However, there are few successful treatments available to prevent or remediate scars. This study sought to explore the molecular mechanisms by which quercetin, a naturally-occurring antifibrotic agent, diminishes scar formation. Using both mice and fibroblast cells, we examined quercetin's impact on fibrosis and the wound healing rate, and potential molecular mechanisms underlying the quercetin-mediated reduction of fibrosis. While cultured fibroblasts demonstrated normal growth in response to quercetin, quercetin increased surface αV integrin and decreased β1 integrin. These changes in surface integrin expression may impact factors that contribute to fibrosis including cell migration, proliferation, and extracellular matrix production. In both quercetin-treated and control mice, wounds healed in about 14 days. Masson's trichrome stain revealed diminished fibrosis at the wound site in quercetin-treated animals despite the normal healing rate, indicating the potential for better cosmetic results without delaying healing. An in vitro scratch wound model using cells plated on an artificial extracellular matrix demonstrated delayed closure following quercetin treatment. The extracellular matrix also ameliorated quercetin's effect on αV integrin. Thus, αV integrin recruitment in response to quercetin treatment may promote the quercetin-mediated decrease extracellular matrix because cells require less extracellular matrix to migrate into a wound. With added extracellular matrix, β1 integrin remained diminished in response to quercetin, indicating that quercetin's effect on β1 integrin expression is independent of extracellular matrix -mediated signaling and is likely driven by inhibition of the intracellular mechanisms driving β1 expression. These findings suggest that quercetin could alter the cells' interactions with the extracellular

Comparison of iodine-123 labelled 2{beta}-carbomethoxy-3{beta}-(4-iodophenyl)tropane and 2{beta}-carbomethoxy-3{beta}-(4-iodophenyl)-N-(3-fluoropropyl)nortropane for imaging of the dopamine transporter in the living human brain

Energy Technology Data Exchange (ETDEWEB)

Kuikka, J.T. [Dept. of Clinical Physiology, Kuopio Univ. Hospital (Finland); Bergstroem, K.A. [Dept. of Clinical Physiology, Kuopio Univ. Hospital (Finland); Ahonen, A. [Dept. of Clinical Chemistry, Oulu Univ. Central Hospital (Finland); Hiltunen, J. [MAP Medical Technologies Oy, Tikkakoski (Finland); Haukka, J. [MAP Medical Technologies Oy, Tikkakoski (Finland); Laensimies, E. [Dept. of Clinical Physiology, Kuopio Univ. Hospital (Finland); Wang Shaoyin [Research Biochemicals International (RBI), Natick, MA (United States); Neumeyer, J.L. [Research Biochemicals International (RBI), Natick, MA (United States)

1995-04-01

Several cocaine congeners are of potential for imaging the dopamine transporter (DAT). Previous studies have shown that iodine-123 labelled 2{beta}-carbomethoxy-3{beta}-(4-iodophenyl)tropane ([{sup 123}I]{beta}-CIT) is a promising radiotracer for imaging the serotonin (5-HT) and dopamine (DA) transporters in the living human brain with single-photon emission tomography (SPET). [{sup 123}I]{beta}-CIT was found to be not very practical for 1-day DAT imaging protocols since peak DAT uptake occurs later than 8 h. Here we report a pilot comparison of [{sup 123}I]{beta}-CIT and 2{beta}-carbomethoxy-3{beta}-(4-iodophenyl)-N-(3-fluoropropyl)nortropane ([{sup 123}I]{beta}-CIT-FP), using SPET imaging in four healthy male subjects. Peak uptake of [{sup 123}I]{beta}-CIT-FP into the basal ganglia occurred earlier (3-4 h after injection of tracer) than that of [{sup 123}I]{beta}-CIT (>8 h). However, the specific DAT binding of [{sup 123}I]{beta}-CIT-FP in the basal ganglia was somewhat less (0.813{+-}0.047) than that of [{sup 123}I]{beta}-CIT (0.922{+-}0.004). Imaging quality is excellent with both tracers and they are potentially of value for brain imaging in various neuropsychiatric disorders. (orig.)

α4-integrin receptor desaturation and disease activity return after natalizumab cessation.

Science.gov (United States)

Derfuss, Tobias; Kovarik, John M; Kappos, Ludwig; Savelieva, Marina; Chhabra, Richa; Thakur, Avinash; Zhang, Ying; Wiendl, Heinz; Tomic, Davorka

2017-09-01

To describe the time course of α4-integrin receptor desaturation and disease activity return in patients with relapsing-remitting MS who discontinued natalizumab and to investigate baseline and on-study predictors for the recurrence of disease activity. In the course of TOFINGO, a 32-week, patient- and rater-blinded multicenter, parallel-group study, we performed MRI, counted relapses, and measured α4-integrin receptor occupancy (RO) at baseline and 8, 12, 16, 20, and 24 weeks. The relationship between RO and total number of new T1 gadolinium-enhancing (Gd+) lesions was modeled using Poisson linear regression. Patients (N = 142) were randomized (1:1:1) to 8-, 12-, or 16-week washout (WO) groups. At randomization, the median RO in the 8-, 12-, and 16-week WO groups was 94.5%, 92.4%, and 90.9%, which declined to 79.8%, 30.7%, and 8.7% after 8, 12, and 16 weeks of WO, respectively. The percentage of patients with new T1 Gd+ lesions increased with longer WO period before commencing fingolimod: 2.1% (8 weeks), 9.1% (12 weeks), and 50.0% (16 weeks). Overall, 71% of patients with first relapse between weeks 6 and 18 had RO values below the time-matched population median. Higher T2 lesion volume (LV) at baseline predicted a higher number of new T1 Gd+ lesions. A faster decline in natalizumab RO, longer WO period, and higher T2 LV at baseline were associated with an increased risk for return of inflammatory disease activity. These results provide a mechanistic rationale and, together with the main outcomes of the TOFINGO study, support initiation of fingolimod within 8 weeks of natalizumab discontinuation. NCT01499667.

The Integrin Receptor in Biologically Relevant Bilayers

DEFF Research Database (Denmark)

Kalli, Antreas C.; Róg, Tomasz; Vattulainen, Ilpo

2017-01-01

/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study...... demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin....../talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction...

Tensin stabilizes integrin adhesive contacts in Drosophila.

Science.gov (United States)

Torgler, Catherine N; Narasimha, Maithreyi; Knox, Andrea L; Zervas, Christos G; Vernon, Matthew C; Brown, Nicholas H

2004-03-01

We report the functional characterization of the Drosophila ortholog of tensin, a protein implicated in linking integrins to the cytoskeleton and signaling pathways. A tensin null was generated and is viable with wing blisters, a phenotype characteristic of loss of integrin adhesion. In tensin mutants, mechanical abrasion is required during wing expansion to cause wing blisters, suggesting that tensin strengthens integrin adhesion. The localization of tensin requires integrins, talin, and integrin-linked kinase. The N-terminal domain and C-terminal PTB domain of tensin provide essential recruitment signals. The intervening SH2 domain is not localized on its own. We suggest a model where tensin is recruited to sites of integrin adhesion via its PTB and N-terminal domains, localizing the SH2 domain so that it can interact with phosphotyrosine-containing proteins, which stabilize the integrin link to the cytoskeleton.

Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice.

Science.gov (United States)

Han, H S; Jun, H S; Utsugi, T; Yoon, J W

1997-06-01

A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta

LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

Energy Technology Data Exchange (ETDEWEB)

Kawamoto, Eiji [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Takagi, Yoshimi [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Honda, Goichi [Medical Affairs Department, Asahi Kasei Pharma Corporation, 1-105 Kanda Jinbo-cho, Chiyoda-ku, Tokyo 101-8101 (Japan); Suzuki, Koji [Faculty of Pharmaceutical Science, Suzuka University of Medical Science, 3500-3, Minamitamagaki-cho, Suzuka, Mie 513-8679 (Japan); Imai, Hiroshi [Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Shimaoka, Motomu, E-mail: shimaoka@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan)

2016-05-13

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

Modeling the mechanism of CLN025 beta-hairpin formation

Science.gov (United States)

McKiernan, Keri A.; Husic, Brooke E.; Pande, Vijay S.

2017-09-01

Beta-hairpins are substructures found in proteins that can lend insight into more complex systems. Furthermore, the folding of beta-hairpins is a valuable test case for benchmarking experimental and theoretical methods. Here, we simulate the folding of CLN025, a miniprotein with a beta-hairpin structure, at its experimental melting temperature using a range of state-of-the-art protein force fields. We construct Markov state models in order to examine the thermodynamics, kinetics, mechanism, and rate-determining step of folding. Mechanistically, we find the folding process is rate-limited by the formation of the turn region hydrogen bonds, which occurs following the downhill hydrophobic collapse of the extended denatured protein. These results are presented in the context of established and contradictory theories of the beta-hairpin folding process. Furthermore, our analysis suggests that the AMBER-FB15 force field, at this temperature, best describes the characteristics of the full experimental CLN025 conformational ensemble, while the AMBER ff99SB-ILDN and CHARMM22* force fields display a tendency to overstabilize the native state.

The Relative Influence of Metal Ion Binding Sites in the I-like Domain and the Interface with the Hybrid Domain on Rolling and Firm Adhesion by Integrin α4β7*

Science.gov (United States)

Chen, JianFeng; Takagi, Junichi; Xie, Can; Xiao, Tsan; Luo, Bing-Hao; Springer, Timothy A.

2015-01-01

We examined the effect of conformational change at the β7 I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin α4β7. An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the α4β7 headpiece. Wild-type α4β7 mediates rolling adhesion in Ca2+ and Ca2+/Mg2+ but firm adhesion in Mg2+ and Mn2+. Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn2+, confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn2+. Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion. PMID:15448154

alpha(4)beta(7) independent pathway for CD8(+) T cell-mediated intestinal immunity to rotavirus.

Science.gov (United States)

Kuklin, N A; Rott, L; Darling, J; Campbell, J J; Franco, M; Feng, N; Müller, W; Wagner, N; Altman, J; Butcher, E C; Greenberg, H B

2000-12-01

Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

PET imaging of {alpha}{sub v}{beta}{sub 3} integrin expression in tumours with {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides

Energy Technology Data Exchange (ETDEWEB)

Dijkgraaf, Ingrid; Franssen, Gerben M.; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Yim, Cheng-Bin [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Utrecht University, Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht (Netherlands); Schuit, Robert C. [VU University Medical Centre, Department of Nuclear Medicine and PET Research, P.O. Box 7057, Amsterdam (Netherlands); Luurtsema, Gert [University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Hanzeplein 1, P.O. Box 30.001, Groningen (Netherlands); Liu, Shuang [Purdue University, School of Health Sciences, West Lafayette, IN (United States)

2011-01-15

Due to the restricted expression of {alpha}{sub v}{beta}{sub 3} in tumours, {alpha}{sub v}{beta}{sub 3} is considered a suitable receptor for tumour targeting. In this study the {alpha}{sub v}{beta}{sub 3}-binding characteristics of {sup 68}Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their {sup 111}In-labelled counterparts. A monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]{sub 2}) and a tetrameric (E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2}) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with {sup 68}Ga. In vitro {alpha}{sub v}{beta}{sub 3}-binding characteristics were determined in a competitive binding assay. In vivo {alpha}{sub v}{beta}{sub 3}-targeting characteristics of the compounds were assessed in mice with subcutaneously growing SK-RC-52 xenografts. In addition, microPET images were acquired using a microPET/CT scanner. The IC{sub 50} values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]{sub 2} and DOTA-E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2} were 23.9 {+-} 1.22, 8.99 {+-} 1.20 and 1.74 {+-} 1.18 nM, respectively, and were similar to those of the In(III)-labelled mono-, di- and tetrameric RGD peptides (26.6 {+-} 1.15, 3.34 {+-} 1.16 and 1.80 {+-} 1.37 nM, respectively). At 2 h post-injection, tumour uptake of the {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides (3.30 {+-} 0.30, 5.24 {+-} 0.27 and 7.11 {+-} 0.67%ID/g, respectively) was comparable to that of their {sup 111}In-labelled counterparts (2.70 {+-} 0.29, 5.61 {+-} 0.85 and 7.32 {+-} 2.45%ID/g, respectively). PET scans were in line with the biodistribution data. On all PET scans, the tumour could be clearly visualised. The integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The {sup 68}Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of {alpha}{sub v} {beta}{sub 3} expression with PET. (orig.)

Lumican alleviates hypertrophic scarring by suppressing integrin-FAK signaling

International Nuclear Information System (INIS)

Zhao, Yuqian; Li, Xueyong; Xu, Xiaoli; He, Zhi; Cui, Lei; Lv, Xiaoxing

2016-01-01

Hypertrophic scarring (HS) is an overcompensation of wound healing that increases the risk of cosmetic disfigurement and functional impairment. No gold standard has been established for the treatment or prevention of HS. Our study aims to elucidate the expression and function of lumican in the pathogenesis of HS as well as the underlying mechanism involved in this procedure. An animal model of HS (rabbit ear) was established, and the Ad-lumican vectors were locally injected. Primary fibroblasts isolated from patients with hypertrophic burn scars were used in vitro. Histological and molecular changes in HS pathogenesis were evaluated. The results showed that lumican is significantly reduced in HS tissues and fibroblasts from HS patients as compared to normal skin or cells. Lumican levels were further suppressed in response to TGF-β stimulation. However, lumican upregulation effectively thinned the scar area and inhibited fibroblast proliferation and the cell cycle. Meanwhile, Ad-lumican administration suppressed the deposition of extracellular matrix, such as collagen and CTGF. Ad-lumican injected animals or fibroblasts presented comparable integrin α 2 β 1 expression while greatly reduced phosphorylation of FAK compared to the negative control. Moreover, Ad-lumican administration largely enhanced the binding of lumican to integrin α 2 β 1 and may thus inhibit the signaling propagation of collagen-integrin α 2 β 1 . Overall, the restoration of lumican levels contributed to suppressing the HS progression by inhibiting collagen-integrin α 2 β 1 -FAK signaling. - Highlights: • Lumican is downregulated during hypertrophic scar formation. • Lumican inhibits fibroblast proliferation. • Lumican inhibits extracellular matrix deposition. • Lumican suppresses collagen-integrin-FAK signaling.

Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes.

Directory of Open Access Journals (Sweden)

Marius R Robciuc

Full Text Available Peroxisome proliferator-activated receptor (PPAR delta is an important regulator of fatty acid (FA metabolism. Angiopoietin-like 4 (Angptl4, a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR, PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

Science.gov (United States)

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

Science.gov (United States)

Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

2016-01-01

Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

International Nuclear Information System (INIS)

Crowe, David L; Ohannessian, Arthur

2004-01-01

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

Directory of Open Access Journals (Sweden)

Ohannessian Arthur

2004-05-01

Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

Comparative analyses of two thermophilic enzymes exhibiting both beta-1,4 mannosidic and beta-1,4 glucosidic cleavage activities from Caldanaerobius polysaccharolyticus.

Science.gov (United States)

Han, Yejun; Dodd, Dylan; Hespen, Charles W; Ohene-Adjei, Samuel; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

2010-08-01

The hydrolysis of polysaccharides containing mannan requires endo-1,4-beta-mannanase and 1,4-beta-mannosidase activities. In the current report, the biochemical properties of two endo-beta-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TM1 [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-1,4-beta-mannanase and endo-1,4-beta-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-1,4-beta-mannanase activity and little endo-1,4-beta-glucanase activity; however, this enzyme also exhibited 1,4-beta-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of beta-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed.

Inhibition of phosphatidylcholine-specific phospholipase C prevents bone marrow stromal cell senescence in vitro.

Science.gov (United States)

Sun, Chunhui; Wang, Nan; Huang, Jie; Xin, Jie; Peng, Fen; Ren, Yinshi; Zhang, Shangli; Miao, Junying

2009-10-01

Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS. (c) 2009 Wiley-Liss, Inc.

Phoyunnanin E inhibits migration of non-small cell lung cancer cells via suppression of epithelial-to-mesenchymal transition and integrin αv and integrin β3.

Science.gov (United States)

Petpiroon, Nareerat; Sritularak, Boonchoo; Chanvorachote, Pithi

2017-12-29

The conversion of the epithelial phenotype of cancer cells into cells with a mesenchymal phenotype-so-called epithelial-mesenchymal transition (EMT)-has been shown to enhance the capacity of the cells to disseminate throughout the body. EMT is therefore becoming a potential target for anti-cancer drug discovery. Here, we showed that phoyunnanin E, a compound isolated from Dendrobium venustum, possesses anti-migration activity and addressed its mechanism of action. The cytotoxic and proliferative effects of phoyunnanin E on human non-small cell lung cancer-derived H460, H292, and A549 cells and human keratinocyte HaCaT cells were investigated by MTT assay. The effect of phoyunnanin E on EMT was evaluated by determining the colony formation and EMT markers. The migration and invasion of H460, H292, A549 and HaCaT cells was evaluated by wound healing assay and transwell invasion assay, respectively. EMT markers, integrins and migration-associated proteins were examined by western blot analysis. Phoyunnanin E at the concentrations of 5 and 10 μM, which are non-toxic to H460, H292, A549 and HaCaT cells showed good potential to inhibit the migratory activity of three types of human lung cancer cells. The anti-migration effect of phoyunnanin E was shown to relate to the suppressed EMT phenotypes, including growth in anchorage-independent condition, cell motility, and EMT-specific protein markers (N-cadherin, vimentin, slug, and snail). In addition to EMT suppression, we found that phoyunnanin E treatment with 5 and 10 μM could decrease the cellular level of integrin αv and integrin β3, these integrins are frequently up-regulated in highly metastatic tumor cells. We further characterized the regulatory proteins in cell migration and found that the cells treated with phoyunnanin E exhibited a significantly lower level of phosphorylated focal adhesion kinase (p-FAK) and phosphorylated ATP-dependent tyrosine kinase (p-AKT), and their downstream effectors (including

Therapeutic Ultrasound Bypasses Canonical Syndecan-4 Signaling to Activate Rac1*S⃞

Science.gov (United States)

Mahoney, Claire M.; Morgan, Mark R.; Harrison, Andrew; Humphries, Martin J.; Bass, Mark D.

2009-01-01

The application of pulsed, low intensity ultrasound is emerging as a potent therapy for the treatment of complex bone fractures and tissue damage. Ultrasonic stimuli accelerate fracture healing by up to 40% and enhance tendon and ligament healing by promoting cell proliferation, migration, and matrix synthesis through an unresolved mechanism. Ultrasound treatment also induces closure of nonunion fractures, at a success rate (85% of cases) similar to that of surgical intervention (68-96%) while avoiding the complications associated with surgery. The regulation of cell adhesion necessary for wound healing depends on cooperative engagement of the extracellular matrix receptors, integrin and syndecan, as exemplified by the wound healing defects observed in syndecan- and integrin-knock-out mice. This report distinguishes the influence of ultrasound on signals downstream of the prototypic fibronectin receptors, α5β1 integrin and syndecan-4, which cooperate to regulate Rac1 and RhoA. Ultrasonic stimulation fails to activate integrins or induce cell spreading on poor, electrostatic ligands. By contrast, ultrasound treatment overcomes the necessity of engagement or expression of syndecan-4 during the process of focal adhesion formation, which normally requires simultaneous engagement of both receptors. Ultrasound exerts an influence downstream of syndecan-4 and PKCα to specifically activate Rac1, itself a critical regulator of tissue repair, and to a lesser extent RhoA. The ability of ultrasound to bypass syndecan-4 signaling, which is known to facilitate efficient tissue repair, explains the reduction in healing times observed in ultrasound-treated patients. By substituting for one of the key axes of adhesion-dependent signaling, ultrasound therapy has considerable potential as a clinical technique. PMID:19147498

Acute Podocyte Vascular Endothelial Growth Factor (VEGF-A) Knockdown Disrupts alphaVbeta3 Integrin Signaling in the Glomerulus

Science.gov (United States)

Veron, Delma; Villegas, Guillermo; Aggarwal, Pardeep Kumar; Bertuccio, Claudia; Jimenez, Juan; Velazquez, Heino; Reidy, Kimberly; Abrahamson, Dale R.; Moeckel, Gilbert; Kashgarian, Michael; Tufro, Alda

2012-01-01

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGFKD) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ∼20% of non-induced controls and urine VEGF-A to ∼30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alphaVbeta3 integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta3 integrin and neuropilin-1 in the kidney in vivo and in VEGFKD podocytes. Podocyte VEGF knockdown disrupts alphaVbeta3 integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGFKD podocytes downregulates fibronectin and disrupts alphaVbeta3 integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alphaVbeta3 integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alphaVbeta3 integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure. PMID:22808199

Polyvalent integrin antagonist-decorated superparamagnetic iron oxide nanoparticles for triggering apoptosis in human leukemia cancer cells

Energy Technology Data Exchange (ETDEWEB)

Say, R Latin-Small-Letter-Dotless-I dvan, E-mail: rsay@anadolu.edu.tr [Anadolu Universitesi, Kimya Boeluemue, Fen Fakueltesi (Turkey); Yazar, Suzan [Sanovel Pharmaceutical Company (Turkey); Ugur, Alper; Huer, Deniz [Anadolu Universitesi, Kimya Boeluemue, Fen Fakueltesi (Turkey); Denizli, Adil [Hacettepe University, Department of Chemistry (Turkey); Ersoez, Arzu [Anadolu Universitesi, Kimya Boeluemue, Fen Fakueltesi (Turkey)

2013-01-15

Integrin family members are the main mediators of cell adhesion to the extracellular matrix and active as intra- and extracellular signaling molecules in a variety of processes. They bind to their ligands by interacting with short amino acid sequences, that is, RGD (arginine-glycine-aspartic acid) sequence. RGD sequences have been used to enhance cell binding to artificial surfaces, so RGD mimics have been used to block integrin binding to its ligand. Integrin-ligand interactions are dependent on divalent cations, and Mg{sup 2+} provide higher-affinity binding to ligand for many integrins. In this study, we have designed new integrin antagonists using methacryloyl amidoaspartic acid (MAASP) monomer-conjugated silanized super paramagnetic iron oxide nanoparticles (SPIONs, the size of the nanoparticles was verified with an average size of 32.6 nm) and poly(MAASP-co-EDMA) shell-decorated silanized SPIONs. Several mechanisms have been proposed to describe uptake of modified SPIONs into the cells, including receptor-mediated endocytosis. Our aim is to bind these modified SPIONs to the integrin-mediated aspartic acid ends of MAASP monomers and block integrin binding to their ligand.

Truncated Bovine Integrin Alpha-v/Beta-6 as a Universal Capture Ligand for FMD Diagnosis.

Directory of Open Access Journals (Sweden)

Gareth Shimmon

Full Text Available Foot-and-mouth disease (FMD is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV serotype-specific antibodies in small animals (rabbits and guinea pigs that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvβ6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvβ6 and FMDV empty capsids in FMD diagnostic assays.

Intrinsic folding of small peptide chains: spectroscopic evidence for the formation of beta-turns in the gas phase.

Science.gov (United States)

Chin, Wutharath; Dognon, Jean-Pierre; Piuzzi, François; Tardivel, Benjamin; Dimicoli, Iliana; Mons, Michel

2005-01-19

Laser desorption of model peptides coupled to laser spectroscopic techniques enables the gas-phase observation of genuine secondary structures of biology. Spectroscopic evidence for the formation of beta-turns in gas-phase peptide chains containing glycine and phenylalanine residues establishes the intrinsic stability of these forms and their ability to compete with other stable structures. The precise characterization of local minima on the potential energy surface from IR spectroscopy constitutes an acute assessment for the state-of-the-art quantum mechanical calculations also presented. The observation of different types of beta-turns depending upon the residue order within the sequence is found to be consistent with the residue propensities in beta-turns of proteins, which suggests that the prevalence of glycine in type II and II' turns stems essentially from an energetic origin, already at play under isolated conditions.

Activation of p53 pathway by Nutlin-3a inhibits the expression of the therapeutic target alpha 5 integrin in colon cancer cells

Czech Academy of Sciences Publication Activity Database

Janoušková, Hana; Ray, A.M.; Noulet, F.; Lelong-Rebel, I.; Choulier, L.; Schaffner, F.; Lehmann, M.; Martin, S.; Teisinger, Jan; Dontenwill, M.

2013-01-01

Roč. 336, č. 2 (2013), s. 307-318 ISSN 0304-3835 Institutional support: RVO:67985823 Keywords : colon cancer * integrin alpha 5 beta 1 * p53 * Nutlin-3a Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.016, year: 2013

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

International Nuclear Information System (INIS)

Hasan, Nazarul; Hu, Chuan

2010-01-01

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

Integrin αβ1, αvβ, α6β effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

International Nuclear Information System (INIS)

Sansing, Hope A.; Sarkeshik, Ali; Yates, John R.; Patel, Vyomesh; Gutkind, J. Silvio; Yamada, Kenneth M.; Berrier, Allison L.

2011-01-01

Research highlights: → Proteomics of clustered integrin αβ1, α v β, α 6 β receptors in oral carcinoma. → p130Cas, Dek, Src and talin regulate oral carcinoma invasion. → p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin. -- Abstract: Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin αβ1, α v β or α 6 β receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.

Differential effects of ongoing EEG beta and theta power on memory formation.

Science.gov (United States)

Scholz, Sebastian; Schneider, Signe Luisa; Rose, Michael

2017-01-01

Recently, elevated ongoing pre-stimulus beta power (13-17 Hz) at encoding has been associated with subsequent memory formation for visual stimulus material. It is unclear whether this activity is merely specific to visual processing or whether it reflects a state facilitating general memory formation, independent of stimulus modality. To answer that question, the present study investigated the relationship between neural pre-stimulus oscillations and verbal memory formation in different sensory modalities. For that purpose, a within-subject design was employed to explore differences between successful and failed memory formation in the visual and auditory modality. Furthermore, associative memory was addressed by presenting the stimuli in combination with background images. Results revealed that similar EEG activity in the low beta frequency range (13-17 Hz) is associated with subsequent memory success, independent of stimulus modality. Elevated power prior to stimulus onset differentiated successful from failed memory formation. In contrast, differential effects between modalities were found in the theta band (3-7 Hz), with an increased oscillatory activity before the onset of later remembered visually presented words. In addition, pre-stimulus theta power dissociated between successful and failed encoding of associated context, independent of the stimulus modality of the item itself. We therefore suggest that increased ongoing low beta activity reflects a memory promoting state, which is likely to be moderated by modality-independent attentional or inhibitory processes, whereas high ongoing theta power is suggested as an indicator of the enhanced binding of incoming interlinked information.

CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

Science.gov (United States)

Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

2017-11-09

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

Inhibition of Alzheimer amyloid {beta} aggregation by polyvalent trehalose

Energy Technology Data Exchange (ETDEWEB)

Miura, Yoshiko; You, Chouga [School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan); Ohnishi, Reiko [Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)], E-mail: miuray@jaist.ac.jp

2008-04-15

A glycopolymer carrying trehalose was found to suppress the formation of amyloid fibrils from the amyloid {beta} peptide (1-42) (A{beta}), as evaluated by thioflavin T assay and atomic force microscopy. Glycopolymers carrying sugar alcohols also changed the aggregation properties of A{beta}, and the inhibitory effect depended on the type of sugar and alkyl side chain. Neutralization activity was confirmed by in vitro assay using HeLa cells. The glycopolymer carrying trehalose strongly inhibited amyloid formation and neutralized cytotoxicity.

Mechanism of Integrim-Mediated Growth Control in Normal, Transformed, and Neoplastic Breast Cells

National Research Council Canada - National Science Library

Wayner, Elizabeth

1999-01-01

.... The primary cell adhesion receptors that mediate binding to extracellular matrix proteins are integrins Our data suggest that alpha 3 beta 1 and alpha 6 beta 4 are the primary integrins responsible...

Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor.

Science.gov (United States)

Orgovan, Norbert; Peter, Beatrix; Bősze, Szilvia; Ramsden, Jeremy J; Szabó, Bálint; Horvath, Robert

2014-02-07

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (v(RGD)) of integrin ligand RGD-motifs. v(RGD) was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 μm(-2) (corresponding to a 3D dissociation constant of ~30 μM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels.

Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

Science.gov (United States)

Golla, R; Philp, N; Safer, D; Chintapalli, J; Hoffman, R; Collins, L; Nachmias, V T

1997-01-01

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of

Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta(2)-microglobulin

DEFF Research Database (Denmark)

Heegaard, N.H.H.; Jørgensen, T.J.D.; Rozlosnik, N.

2005-01-01

. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that Delta K58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is I order of magnitude faster in Delta K58-beta(2)M than in wt-beta(2)m...... in the circulation of dialysis patients. This beta(2)M variant, Delta K58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)M...

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

Science.gov (United States)

Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

1997-01-01

Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

Differential effects of ongoing EEG beta and theta power on memory formation.

Directory of Open Access Journals (Sweden)

Sebastian Scholz

Full Text Available Recently, elevated ongoing pre-stimulus beta power (13-17 Hz at encoding has been associated with subsequent memory formation for visual stimulus material. It is unclear whether this activity is merely specific to visual processing or whether it reflects a state facilitating general memory formation, independent of stimulus modality. To answer that question, the present study investigated the relationship between neural pre-stimulus oscillations and verbal memory formation in different sensory modalities. For that purpose, a within-subject design was employed to explore differences between successful and failed memory formation in the visual and auditory modality. Furthermore, associative memory was addressed by presenting the stimuli in combination with background images. Results revealed that similar EEG activity in the low beta frequency range (13-17 Hz is associated with subsequent memory success, independent of stimulus modality. Elevated power prior to stimulus onset differentiated successful from failed memory formation. In contrast, differential effects between modalities were found in the theta band (3-7 Hz, with an increased oscillatory activity before the onset of later remembered visually presented words. In addition, pre-stimulus theta power dissociated between successful and failed encoding of associated context, independent of the stimulus modality of the item itself. We therefore suggest that increased ongoing low beta activity reflects a memory promoting state, which is likely to be moderated by modality-independent attentional or inhibitory processes, whereas high ongoing theta power is suggested as an indicator of the enhanced binding of incoming interlinked information.

Time-temperature dependent variations in beta-carotene contents in carrot using different spectrophotometric techniques

Science.gov (United States)

Ullah, Rahat; Khan, Saranjam; Shah, Attaullah; Ali, Hina; Bilal, Muhammad

2018-05-01

The current study presents time dependent variations in the concentration of beta-carotene in carrot under different storage-temperature conditions using UV–VIS and Raman spectrophotometric techniques. The UV–VIS absorption spectra of beta-carotene extracted from carrot shows three distinct absorption peaks at 442, 467, and 500 nm with maximum absorption at 467 nm. These absorption peaks are very much reproducible and are assigned to β-carotene. Similarly, Raman spectra of carrot samples also confirmed the three main Raman peaks of beta-carotene at shift positions 1003, 1150, and 1515 cm‑1. An overall decrease in beta-carotene content has been observed for time-temperature conditions. These results depict a decrease of about 40% in the content of beta-carotene when carrot samples were stored in a refrigerator (4 °C) for the first 20 d, whereas a decrease of about 25% was observed when carrot samples were stored in a freezer (‑16 °C) for the same period. The objective of this study is to investigate the possible use of Raman spectroscopy and UV–VIS spectroscopy for quick and detailed analysis of changes (degradation) in beta-carotene content associated with time and temperature in storage (frozen foods) in order to promote quality foods for consumers. Future study with a greater focus on the concentration/content of beta-carotene in other fruits/vegetables is also desirable.

Connective tissue growth factor inhibits gastric cancer peritoneal metastasis by blocking integrin α3β1-dependent adhesion.

Science.gov (United States)

Chen, Chiung-Nien; Chang, Cheng-Chi; Lai, Hong-Shiee; Jeng, Yung-Ming; Chen, Chia-I; Chang, King-Jeng; Lee, Po-Huang; Lee, Hsinyu

2015-07-01

Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential. CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3β1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3β1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3β1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3β1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.

Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin β4.

Science.gov (United States)

Niu, Gengming; Ye, Taiyang; Qin, Liuliang; Bourbon, Pierre M; Chang, Cheng; Zhao, Shengqiang; Li, Yan; Zhou, Lei; Cui, Pengfei; Rabinovitz, Issac; Mercurio, Arthur M; Zhao, Dezheng; Zeng, Huiyan

2015-01-01

Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin β4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin β4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin β4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing. © FASEB.

Polyvalent integrin antagonist-decorated superparamagnetic iron oxide nanoparticles for triggering apoptosis in human leukemia cancer cells

International Nuclear Information System (INIS)

Say, Rıdvan; Yazar, Suzan; Uğur, Alper; Hür, Deniz; Denizli, Adil; Ersöz, Arzu

2013-01-01

Integrin family members are the main mediators of cell adhesion to the extracellular matrix and active as intra- and extracellular signaling molecules in a variety of processes. They bind to their ligands by interacting with short amino acid sequences, that is, RGD (arginine-glycine-aspartic acid) sequence. RGD sequences have been used to enhance cell binding to artificial surfaces, so RGD mimics have been used to block integrin binding to its ligand. Integrin–ligand interactions are dependent on divalent cations, and Mg 2+ provide higher-affinity binding to ligand for many integrins. In this study, we have designed new integrin antagonists using methacryloyl amidoaspartic acid (MAASP) monomer-conjugated silanized super paramagnetic iron oxide nanoparticles (SPIONs, the size of the nanoparticles was verified with an average size of 32.6 nm) and poly(MAASP-co-EDMA) shell-decorated silanized SPIONs. Several mechanisms have been proposed to describe uptake of modified SPIONs into the cells, including receptor-mediated endocytosis. Our aim is to bind these modified SPIONs to the integrin-mediated aspartic acid ends of MAASP monomers and block integrin binding to their ligand.

Inhibition of Focal Adhesion Kinase Signaling by Integrin α6β1 Supports Human Pluripotent Stem Cell Self-Renewal.

Science.gov (United States)

Villa-Diaz, Luis G; Kim, Jin Koo; Laperle, Alex; Palecek, Sean P; Krebsbach, Paul H

2016-07-01

Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin β1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6β1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6β1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753-1764. © 2016 AlphaMed Press.

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

Science.gov (United States)

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Role of beta-adrenoceptors in memory consolidation: beta3-adrenoceptors act on glucose uptake and beta2-adrenoceptors on glycogenolysis.

Science.gov (United States)

Gibbs, Marie E; Hutchinson, Dana S; Summers, Roger J

2008-09-01

Noradrenaline, acting via beta(2)- and beta(3)-adrenoceptors (AR), enhances memory formation in single trial-discriminated avoidance learning in day-old chicks by mechanisms involving changes in metabolism of glucose and/or glycogen. Earlier studies of memory consolidation in chicks implicated beta(3)- rather than beta(2)-ARs in enhancement of memory consolidation by glucose, but did not elucidate whether stimulation of glucose uptake or of glycolysis was responsible. This study examines the role of glucose transport in memory formation using central injection of the nonselective facilitative glucose transporter (GLUT) inhibitor cytochalasin B, the endothelial/astrocytic GLUT-1 inhibitor phloretin and the Na(+)/energy-dependent endothelial glucose transporter (SGLT) inhibitor phlorizin. Cytochalasin B inhibited memory when injected into the mesopallium (avian cortex) either close to or between 25 and 45 min after training, whereas phloretin and phlorizin only inhibited memory at 30 min. This suggested that astrocytic/endothelial (GLUT-1) transport is critical at the time of consolidation, whereas a different transporter, probably the neuronal glucose transporter (GLUT-3), is important at the time of training. Inhibition of glucose transport by cytochalasin B, phloretin, or phlorizin also interfered with beta(3)-AR-mediated memory enhancement 20 min posttraining, whereas inhibition of glycogenolysis interfered with beta(2)-AR agonist enhancement of memory. We conclude that in astrocytes (1) activities of both GLUT-1 and SGLT are essential for memory consolidation 30 min posttraining; (2) neuronal GLUT-3 is essential at the time of training; and (3) beta(2)- and beta(3)-ARs consolidate memory by different mechanisms; beta(3)-ARs stimulate central glucose transport, whereas beta(2)-ARs stimulate central glycogenolysis.

Clonorchis sinensis excretory-secretory products promote the migration and invasion of cholangiocarcinoma cells by activating the integrin β4-FAK/Src signaling pathway.

Science.gov (United States)

Pak, Jhang Ho; Bashir, Qudsia; Kim, In Ki; Hong, Sung-Jong; Maeng, Sejung; Bahk, Young Yil; Kim, Tong-Soo

2017-06-01

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4-FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasma pre beta-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus : Role of phospholipid transfer protein

NARCIS (Netherlands)

Dallinga-Thie, G. M.; van Tol, A.; Dullaart, R. P. F.

Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-beta-HDL, generation in vitro. We determined the effect of atorvastatin on pre-beta-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-beta-HDL formation as well as

{alpha}{sub v}{beta}{sub 3} imaging can accurately distinguish between mature teratoma and necrosis in {sup 18}F-FDG-negative residual masses after treatment of non-seminomatous testicular cancer: a preclinical study

Energy Technology Data Exchange (ETDEWEB)

Aide, Nicolas [Francois Baclesse Cancer Centre and Caen University, Bioticla Team, EA1772, IFR 146 ICORE, GRECAN, Caen (France); Caen University Hospital and Francois Baclesse Cancer Centre, PET Unit, Caen (France); Centre Francois Baclesse, Service de Medecine Nucleaire, Caen (France); Briand, Melanie; Dutoit, Soizic; Deslandes, Edwiges; Poulain, Laurent [Francois Baclesse Cancer Centre and Caen University, Bioticla Team, EA1772, IFR 146 ICORE, GRECAN, Caen (France); Bohn, Pierre; Rouvet, Jean; Modzelewski, Romain; Vera, Pierre [Henri Becquerel Cancer Center and Rouen University Hospital and QuantIF- LITIS (EA4108), Department of Nuclear Medicine, Rouen (France); Lasnon, Charline [Caen University Hospital and Francois Baclesse Cancer Centre, PET Unit, Caen (France); Chasle, Jacques [Francois Baclesse Cancer Centre and Caen University, Pathology Department, Caen (France); Vela, Antony [Francois Baclesse Cancer Centre and Caen University, Radiophysics Unit, Caen (France); Carreiras, Franck [Universite de Cergy Pontoise, UFR Sciences et Techniques, ERRMECe, EA 1391, Institut des materiaux, Cergy-Pontoise (France)

2011-02-15

We assessed whether imaging {alpha}{sub v}{beta}{sub 3} integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses. Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8 weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles).{sup 18}F-Fluorodeoxyglucose ({sup 18}F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8 weeks with cisplatin+ATRA). Imaging of {alpha}{sub v}{beta}{sub 3} expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [{sup 99m}Tc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse {alpha}{sub v}{beta}{sub 3} expression were performed. Cisplatin+ATRA induced differentiation of the xenografts. After 8 weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for {sup 18}F-FDG [mean standardised uptake value (SUV{sub mean}) = 0.48 {+-} 0.05] compared to untreated embryonal carcinoma (SUV{sub mean} = 0.92 {+-} 0.13) (p = 0.005). {alpha}{sub v}{beta}{sub 3} imaging accurately distinguished mature teratoma (tumour to muscle ratio = 4.29 {+-} 1.57) from necrosis (tumour to muscle ratio = 1.3 {+-} 0.26) (p = 0.0002). Immunohistochemistry studies showed that {alpha}{sub v}{beta}{sub 3} integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma. Imaging {alpha}{sub v}{beta}{sub 3} integrin accurately distinguished mature teratoma from

The β3-Integrin Binding Protein β3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1

Science.gov (United States)

Kračun, Damir; Rieß, Florian; Kanchev, Ivan; Gawaz, Meinrad

2014-01-01

Abstract Aims: Integrins are multifunctional heterodimeric adhesion receptors that mediate the attachment between a cell and the extracellular matrix or other surrounding cells. In endothelial cells, integrins can modulate cell migration and motility. In particular, β3-integrin is expressed in angiogenic vessels. Signal transduction by β3-integrins requires the recruitment of intracellular signaling molecules. β3-endonexin is a highly spliced molecule that has been identified as a β3-integrin binding protein. β3-endonexin isoforms are expressed in endothelial cells and have been suggested to act as shuttle proteins between the membrane and the nucleus. However, their functional role in angiogenesis is unclear. In this study, we investigated whether β3-endonexin isoforms are involved in endothelial angiogenic processes under hypoxia. Results: The overexpression of β3-endonexin isoforms decreased endothelial proliferation and tube formation under hypoxia, while the depletion of β3-endonexin by RNAi promoted angiogenic responses in vitro and in vivo. In hypoxia, β3-endonexin accumulated in the nucleus, and prevention of this response by depletion of β3-endonexin increased hypoxic activation and induction of the hypoxia-inducible factor (HIF)-1 and its target genes VEGF and PAI-1. β3-endonexin diminished nuclear factor kappa B (NFκB) activation and decreased NFκB binding to the HIF-1α promoter under hypoxia, subsequently diminishing NFκB-dependent transcription of HIF-1α under hypoxia. Innovation: Our results indicate for the first time that the overexpression of β3-endonexin can decrease hypoxic induction and activation of HIF-1α and can prevent hypoxic endothelial proliferation and angiogenic responses. Conclusion: β3-endonexin can act as a novel anti-angiogenic factor specifically in the response to hypoxia due to its negative impact on the activation of HIF-1. Antioxid. Redox Signal. 20, 1964–1976. PMID:24386901

The changing integrin expression and a role for integrin β8 in the chondrogenic differentiation of mesenchymal stem cells.

Directory of Open Access Journals (Sweden)

Vanessa L S LaPointe

Full Text Available Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype.

The Changing Integrin Expression and a Role for Integrin β8 in the Chondrogenic Differentiation of Mesenchymal Stem Cells

Science.gov (United States)

LaPointe, Vanessa L. S.; Verpoorte, Amanda; Stevens, Molly M.

2013-01-01

Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs) into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype. PMID:24312400

Oncofetal chondroitin sulfate glycosaminoglycans are key players in integrin signaling and tumor cell motility

DEFF Research Database (Denmark)

Clausen, Thomas Mandel; Bento Ayres Pereira, Marina Maria; Al Nakouzi, Nader

2016-01-01

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2...... revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-β1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes β-1......,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor...

Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis.

Science.gov (United States)

Merwin, J R; Anderson, J M; Kocher, O; Van Itallie, C M; Madri, J A

1990-01-01

Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with

Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents

DEFF Research Database (Denmark)

Christensen, Gitte L.; Jacobsen, Maria L. B.; Wendt, Anna

2015-01-01

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression...

Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

Science.gov (United States)

Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

1994-10-01

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber.

Science.gov (United States)

Papanikolopoulou, Katerina; Schoehn, Guy; Forge, Vincent; Forsyth, V Trevor; Riekel, Christian; Hernandez, Jean-François; Ruigrok, Rob W H; Mitraki, Anna

2005-01-28

Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.

NCBI nr-aa BLAST: CBRC-CJAC-01-0777 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-CJAC-01-0777 ref|YP_001278902.1| Integrin alpha beta-propellor repeat protein [Rose...iflexus sp. RS-1] gb|ABQ92952.1| Integrin alpha beta-propellor repeat protein [Roseiflexus sp. RS-1] YP_001278902.1 4e-23 41% ...

Texture Of Zircaloy-4 Result Of Beta-Quenching, Cold Rolling And Recrystallization

International Nuclear Information System (INIS)

Futichah; Sulistioso

1998-01-01

Differences of crystallographic texture of zircaloy-4 plate depends on cold working and heat treatment.To determine the change of zircaloy-4 textures, the solid solution treatment process at beta phase which was followed by quenching on water was employed for this sample. The next step was cold rolling until deformation epsilon = 1.62. The specimens were recrystallized at 750 o C, for 2 hours. The result of beta-quench gave a spread and different orientations and the main orientation occurred at (0001)[1010] and (0001)[1120]. Result of cold rolling with epsilon = 1.39 and epsilon 1.62 is the deformation texture at the main orientation of (0001)[1010] with the angle of inclination was around 38 o. However, the result of Recrystallization process on 750 o C for 2 hours gave annealing textures with orientations of (0001)[1120]. It means that the recrystallization process of zircaloy-4 plate can not remove the deformation textures, but can change the crystallographic orientation

An α4β1 integrin antagonist decreases airway inflammation in ovalbumin-exposed mice

Science.gov (United States)

Kenyon, Nicholas J.; Liu, Ruiwu; O’Roark, Erin M.; Huang, Wenzhe; Peng, Li; Lam, Kit S.

2008-01-01

Inhibition of the α4 subunit of both the α4β1 and α4β7 integrins has shown promise in decreasing airway inflammation and airway hyperresponsiveness in various animal models. We hypothesized that a novel, high-affinity α4β1 antagonist (LLP2A) would decrease the migration of eosinophils to the lung and ameliorate the airway hyperresponsiveness in a mouse model of ovalbumin-induced airway inflammation. To test this hypothesis, we administered LLP2A, or scrambled LLP2A (a negative control), prior to exposure of sensitized BALB/c mice to ovalbumin aerosol. We can partially prevent, or reverse, the airway inflammatory response, but not airways hyperresponsiveness, by treatment of mice with LLP2A, a synthetic peptidomimetic α4β1 antagonist LLP2A. Specifically engineered, PEGylated (PEG) formulations of this antagonist further reduce the airway inflammatory response to ovalbumin lbumin, presumably by improving the circulating half-life of the drug. PMID:19103195

αMβ2-integrin-intercellular adhesion molecule-1 interactions drive the flow-dependent trafficking of Guillain-Barré syndrome patient derived mononuclear leukocytes at the blood-nerve barrier in vitro

Science.gov (United States)

Yosef, Nejla; Ubogu, Eroboghene E.

2012-01-01

The mechanisms of hematogenous leukocyte trafficking at the human blood-nerve barrier (BNB) are largely unknown. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the pathogenesis of Guillain-Barré syndrome (GBS). We developed a cytokine-activated human in vitro BNB model using primary endoneurial endothelial cells. Endothelial treatment with 10 U/mL tissue necrosis factor-α and 20 U/mL interferon-γ resulted in de novo expression of proinflammatory chemokines CCL2, CXCL9, CXCL11 and CCL20, with increased expression of CXCL2-3, CXCL8 and CXCL10 relative to basal levels. Cytokine treatment induced/ enhanced ICAM-1, E- and P-selectin, vascular cell adhesion molecule-1 and the alternatively spliced pro-adhesive fibronectin variant, fibronectin connecting segment-1 expression in a time-dependent manner, without alterations in junctional adhesion molecule-A expression. Lymphocytes and monocytes from untreated GBS patients express ICAM-1 counterligands, αM- and αL-integrin, with differential regulation of αM-integrin expression compared to healthy controls. Under flow conditions that mimic capillary hemodynamics in vivo, there was a >3-fold increase in total GBS patient and healthy control mononuclear leukocyte adhesion/ migration at the BNB following cytokine treatment relative to the untreated state. Function neutralizing monoclonal antibodies against human αM-integrin (CD11b) and ICAM-1 reduced untreated GBS patient mononuclear leukocyte trafficking at the BNB by 59% and 64.2% respectively. Monoclonal antibodies against αL-integrin (CD11a) and human intravenous immunoglobulin reduced total leukocyte adhesion/migration by 22.8% and 17.6% respectively. This study demonstrates differential regulation of αM-integrin on circulating mononuclear cells in GBS, as well as an important role for αM-integrin-ICAM-1 interactions in pathogenic GBS patient leukocyte trafficking at the human BNB in vitro. PMID:22552879

The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis

DEFF Research Database (Denmark)

Danen, Erik H J; Sonneveld, Petra; Brakebusch, Cord

2002-01-01

We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels ...... receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.......We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels...... of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated...

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

Science.gov (United States)

Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

2005-02-01

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Thyroid status affects the rat cardiac beta-adrenoceptor system transiently and time-dependently

NARCIS (Netherlands)

Zwaveling, J.; Batink, H. D.; Taguchi, K.; de Jong, J.; Michel, M. C.; Pfaffendorf, M.; van Zwieten, A.

1998-01-01

1. The aim of this study was to investigate the time-dependency of the influence of dysthyroid states on the beta-adrenoceptor system in rat heart left ventricle. Therefore, the influence of acute and chronic hyper- and hypothyroidism on beta-adrenoceptor-induced left ventricular responses,

Beta limits for ETF

International Nuclear Information System (INIS)

Helton, F.J.; Miller, R.L.

1982-01-01

ETF (Engineering Test Facility) one-dimensional transport simulations indicate that a volume-average beta of 4% is required for ignition. It is therefore important that theoretical beta limits, determined by requiring equilibria to be stable to all ideal modes, exceed 4%. This paper documents an ideal MHD analysis wherein it is shown that, with appropriate plasma cross-sectional shape and current profile optimization, operation near 5% is possible. The critical beta value, however, depends on the functional form used for ff', which suggests that higher critical betas could be achieved by directly optimizing the safety factor profile. (author)

Neutrophil adhesion and chemotaxis depend on substrate mechanics

Energy Technology Data Exchange (ETDEWEB)

Jannat, Risat A; Hammer, Daniel A [Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 South 33rd Street, Philadelphia, PA 19104 (United States); Robbins, Gregory P; Ricart, Brendon G [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 311A Towne Building, 220 South 33rd Street, Philadelphia, PA 19104 (United States); Dembo, Micah, E-mail: hammer@seas.upenn.ed [Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215 (United States)

2010-05-19

Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K{sub D} of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against {beta}{sub 2}-integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production

DEFF Research Database (Denmark)

Thykær, Jette; Kildegaard, Kanchana Rueksomtawin; Noorman, H.

2008-01-01

was detected in either of the Delta gdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed......The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA...... continued beta-lactam production for the reference strains whereas the Delta gdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results...

Mixed Fibronectin-Derived Peptides Conjugated to a Chitosan Matrix Effectively Promotes Biological Activities through Integrins, α4β1, α5β1, αvβ3, and Syndecan

Directory of Open Access Journals (Sweden)

Hozumi Kentaro

2016-11-01

Full Text Available Mimicking the biological function of the extracellular matrix is an approach to developing cell adhesive biomaterials. The RGD peptide, derived from fibronectin (Fn, mainly binds to integrin αvβ3 and has been widely used as a cell adhesive peptide on various biomaterials. However, cell adhesion to Fn is thought to be mediated by several integrin subtypes and syndecans. In this study, we synthesized an RGD-containing peptide (FIB1 and four integrin α4β1-binding-related motif-containing peptides (LDV, IDAPS, KLDAPT, and PRARI and constructed peptide-chitosan matrices. The FIB1-chitosan matrix promoted human dermal fibroblast (HDF attachment, and the C-terminal elongated PRARI (ePRARI-C-conjugated chitosan matrix significantly promoted HDF attachment through integrin α4β1 and syndecan binding. Next, we constructed a mixed ePRARI-C- and FIB1-chitosan matrix to develop a Fn mimetic biomaterial. The mixed ePRARI-C/FIB1-chitosan matrix promoted significantly better cell attachment and neurite outgrowth compared to those of either ePRARI-C- or FIB1-chitosan matrices. HDF adhesion to the ePRARI-C/FIB1-chitosan matrix was mediated by integrin, α4β1, α5β1, and αvβ3, similar to HDF adhesion to Fn. These data suggest that an ePRARI-C/FIB1-chitosan matrix can be used as a tool to analyze the multiple functions of Fn and can serve as a Fn-mimetic biomaterial.

Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

International Nuclear Information System (INIS)

Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

2010-01-01

Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

Thermoluminescent response of CaSO{sub 4}: Dy + PTFE to beta particles; Respuesta termoluminiscente de CaSO{sub 4}: Dy + PTFE a particulas beta

Energy Technology Data Exchange (ETDEWEB)

Aguirre C, A.; Azorin N, J. [Colegio de Bachilleres No. 13, Xochimilco-Tepepan, 16000 Mexico D.F. (Mexico)

2000-07-01

In this work the results of studying the thermoluminescent properties of CaSO{sub 4}: Dy + PTFE are presented when it is irradiated with beta particles. The conclusion was the obtention of the Tl response curve in function of dose is that to desexcite the dosemeters at temperature 300 C during 30 minutes and after that were irradiated at different times in groups and to do the reading of dosemeter, it can be observed that a greater irradiation time major is the Tl response and this depends of the material has been used. (Author)

Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

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Cheng-Tao Yang

2009-07-01

Full Text Available The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels.To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm.These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

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Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

The colocalization potential of HIV-specific CD8+ and CD4+ T-cells is mediated by integrin β7 but not CCR6 and regulated by retinoic acid.

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Vanessa Sue Wacleche

Full Text Available CD4(+ T-cells from gut-associated lymphoid tissues (GALT are major targets for HIV-1 infection. Recruitment of excess effector CD8(+ T-cells in the proximity of target cells is critical for the control of viral replication. Here, we investigated the colocalization potential of HIV-specific CD8(+ and CD4(+ T-cells into the GALT and explored the role of retinoic acid (RA in regulating this process in a cohort of HIV-infected subjects with slow disease progression. The expression of the gut-homing molecules integrin β7, CCR6, and CXCR3 was identified as a "signature" for HIV-specific but not CMV-specific CD4(+ T-cells thus providing a new explanation for their enhanced permissiveness to infection in vivo. HIV-specific CD8(+ T-cells also expressed high levels of integrin β7 and CXCR3; however CCR6 was detected at superior levels on HIV-specific CD4(+ versus CD8(+ T-cells. All trans RA (ATRA upregulated the expression of integrin β7 but not CCR6 on HIV-specific T-cells. Together, these results suggest that HIV-specific CD8(+ T-cells may colocalize in excess with CD4(+ T-cells into the GALT via integrin β7 and CXCR3, but not via CCR6. Considering our previous findings that CCR6(+CD4(+ T-cells are major cellular targets for HIV-DNA integration in vivo, a limited ability of CD8(+ T-cells to migrate in the vicinity of CCR6(+CD4(+ T-cells may facilitate HIV replication and dissemination at mucosal sites.

Beyond the Matrix: The Many Non-ECM Ligands for Integrins

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Bryce LaFoya

2018-02-01

Full Text Available The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM, and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins.

Beta2- and beta3-adrenoceptors activate glucose uptake in chick astrocytes by distinct mechanisms: a mechanism for memory enhancement?

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Hutchinson, Dana S; Summers, Roger J; Gibbs, Marie E

2007-11-01

Isoprenaline, acting at beta-adrenoceptors (ARs), enhances memory formation in single trial discriminated avoidance learning in day-old chicks by mechanisms involving alterations in glucose and glycogen metabolism. Earlier studies of memory consolidation in chicks indicated that beta3-ARs enhanced memory by increasing glucose uptake, whereas beta2-ARs enhance memory by increasing glycogenolysis. This study examines the ability of beta-ARs to increase glucose uptake in chick forebrain astrocytes. The beta-AR agonist isoprenaline increased glucose uptake in a concentration-dependent manner, as did insulin. Glucose uptake was increased by the beta2-AR agonist zinterol and the beta3-AR agonist CL316243, but not by the beta1-AR agonist RO363. In chick astrocytes, reverse transcription-polymerase chain reaction studies showed that beta1-, beta2-, and beta3-AR mRNA were present, whereas radioligand-binding studies showed the presence of only beta2- and beta3-ARs. beta-AR or insulin-mediated glucose uptake was inhibited by phosphatidylinositol-3 kinase and protein kinase C inhibitors, suggesting a possible interaction between the beta-AR and insulin pathways. However beta2- and beta3-ARs increase glucose uptake by two different mechanisms: beta2-ARs via a Gs-cAMP-protein kinase A-dependent pathway, while beta3-ARs via interactions with Gi. These results indicate that activation of beta2- and beta3-ARs causes glucose uptake in chick astrocytes by distinct mechanisms, which may be relevant for memory enhancement.

The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

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Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

2011-05-27

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

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Ana E. González Wusener

2016-01-01

Full Text Available Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO cells and PTP1B reconstituted (WT cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

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González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

2016-01-01

ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

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Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

2008-07-01

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

Depletion of 4-hydroxynonenal in hGSTA4-transfected HLE B-3 cells results in profound changes in gene expression

International Nuclear Information System (INIS)

Patrick, Brad; Li Jie; Jeyabal, Prince V.S.; Reddy, Prasada M.R.V.; Yang Yusong; Sharma, Rajendra; Sinha, Mala; Luxon, Bruce; Zimniak, Piotr; Awasthi, Sanjay; Awasthi, Yogesh C.

2005-01-01

Previously, we have shown that overexpression of 4-hydroxy-2-nonenal (HNE)-detoxifying enzyme glutathione S-transferase A4-4 (hGSTA4-4) in human lens epithelial cells (HLE B-3) leads to pro-carcinogenic phenotypic transformation of these cells [R. Sharma, et al. Eur. J. Biochem. 271 (2004) 1960-1701]. We now demonstrate that hGSTA4-transfection also causes a profound change in the expression of genes involved in cell adhesion, cell cycle control, proliferation, cell growth, and apoptosis, which is consistent with phenotypic changes of the transformed cells. The expression of p53, p21, p16, fibronectin 1, laminin γ1, connexin 43, Fas, integrin α6, TGFα, and c-jun was down-regulated, while the expression of protein kinase C beta II (PKCβII), c-myc, cyclin-dependent kinase 2 (CDK2), and TGFβ was up-regulated in transfected cells. These results demonstrate that HNE serves as a crucial signaling molecule and, by modulating the expression of genes, can influence cellular functions