Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

Energy Technology Data Exchange (ETDEWEB)

Tsukasaki, Masayuki [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Suzuki, Dai [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Aizawa, Ryo [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyazono, Agasa [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Miyamoto, Yoichi; Suzawa, Tetsuo; Takami, Masamichi; Yoshimura, Kentaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan); Morimura, Naoko [Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta, Tokyo 145-8515 (Japan); Kamijo, Ryutaro [Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555 (Japan)

2011-07-15

Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.

Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

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Lin, Hui-Hsuan [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Tsai, Chia-Wen [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Chou, Fen-Pi [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Wang, Chau-Jong [Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Hsuan, Shu-Wen [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China); Wang, Cheng-Kun [E-Chyun Dermatology Clinic, No.70, Sec. 3, Jhonghua E. Rd., East District, Tainan, Taiwan (China); Chen, Jing-Hsien [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China)

2011-02-01

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

Alpha particles induce expression of immunogenic markers on tumour cells

International Nuclear Information System (INIS)

Gorin, J.B.; Gouard, S.; Cherel, M.; Davodeau, F.; Gaschet, J.; Morgenstern, A.; Bruchertseifer, F.

2013-01-01

The full text of the publication follows. Radioimmunotherapy (RIT) is an approach aiming at targeting the radioelements to tumours, usually through the use of antibodies specific for tumour antigens. The radiations emitted by the radioelements then induce direct killing of the targeted cells as well as indirect killing through bystander effect. Interestingly, it has been shown that ionizing radiations, in some settings of external radiotherapy, can foster an immune response directed against tumour cells. Our research team is dedicated to the development of alpha RIT, i.e RIT using alpha particle emitters, we therefore decided to study the effects of such particles on tumour cells in regards to their immunogenicity. First, we studied the effects of bismuth 213, an alpha emitter, on cellular death and autophagy in six different tumour cell lines. Then, we measured the expression of 'danger' signals and MHC molecules at the cell surface to determine whether irradiation with 213 Bi could cause the tumour cells to be recognized by the immune system. Finally a co-culture of dendritic cells with irradiated tumour cells was performed to test whether it would induce dendritic cells to mature. No apoptosis was detected within 48 hours after irradiation in any cell line, however half of them exhibited signs of autophagy. No increase in membrane expression of 'danger' signals was observed after treatment with 213 Bi, but we showed an increase in expression of MHC class I and II for some cell lines. Moreover, the co-culture experiment indicated that the immunogenicity of a human adenocarcinoma cell line (LS 174T) was enhanced in vitro after irradiation with alpha rays. These preliminary data suggest that alpha particles could be of interest in raising an immune response associated to RIT. (authors)

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

Science.gov (United States)

Clausell, N.; de Lima, V. C.; Molossi, S.; Liu, P.; Turley, E.; Gotlieb, A. I.; Adelman, A. G.; Rabinovitch, M.

1995-01-01

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury. Images PMID:7626352

Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells.

Directory of Open Access Journals (Sweden)

Mahendran Botlagunta

2011-03-01

Full Text Available DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

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Chung, Eric; Jakinovich, Paul; Bae, Aekyung [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States); Rebecchi, Mario, E-mail: Mario.rebecchi@SBUmed.org [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States)

2012-10-01

Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

Modulation of tumor necrosis factor {alpha} expression in mouse brain after exposure to aluminum in drinking water

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Tsunoda, M.; Sharma, R.P. [Georgia Univ., Athens (Greece). College of Veterinary Medicine

1999-11-01

Aluminum, a known neurotoxic substance and a ground-water pollutant, is a possible contributing factor in various nervous disorders including Alzheimer's disease. It has been hypothesized that cytokines are involved in aluminum neurotoxicity. We investigated the alterations in mRNA expression of tumor necrosis factor {alpha} (TNF{alpha}), interleukin-1{beta} (IL-1{beta}), and interferon {gamma} (IFN{gamma}), cytokines related to neuronal damage, in cerebrum and peripheral immune cells of mice after exposure to aluminum through drinking water. Groups of male BALB/c mice were administered aluminum ammonium sulfate in drinking water ad libitum at 0, 5, 25, and 125 ppm aluminum for 1 month. An additional group received 250 ppm ammonium as ammonium sulfate. After treatment, the cerebrum, splenic macrophages and lymphocytes were collected. The expression of TNF{alpha} mRNA in cerebrum was significantly increased among aluminum-treated groups compared with the control, in a dose-dependent manner. Other cytokines did not show any aluminum-related effects. In peripheral cells, there were no significant differences of cytokine mRNA expressions among treatment groups. Increased expression of TNF{alpha} mRNA by aluminum in cerebrum may reflect activation of microglia, a major source of TNF{alpha} in this brain region. Because the aluminum-induced alteration in cytokine message occurred at aluminum concentrations similar to those noted in contaminated water, these results may be relevant in considering the risk of aluminum neurotoxicity in drinking water. (orig.)

Estrogen increases smooth muscle expression of alpha2C-adrenoceptors and cold-induced constriction of cutaneous arteries.

Science.gov (United States)

Eid, A H; Maiti, K; Mitra, S; Chotani, M A; Flavahan, S; Bailey, S R; Thompson-Torgerson, C S; Flavahan, N A

2007-09-01

Raynaud's phenomenon, which is characterized by intense cold-induced constriction of cutaneous arteries, is more common in women compared with men. Cold-induced constriction is mediated in part by enhanced activity of alpha(2C)-adrenoceptors (alpha(2C)-ARs) located on vascular smooth muscle cells (VSMs). Experiments were therefore performed to determine whether 17beta-estradiol regulates alpha(2C)-AR expression and function in cutaneous VSMs. 17beta-Estradiol (0.01-10 nmol/l) increased expression of the alpha(2C)-AR protein and the activity of the alpha(2C)-AR gene promoter in human cultured dermal VSMs, which was assessed following transient transfection of the cells with a promoter-reporter construct. The effect of 17beta-estradiol was associated with increased accumulation of cAMP and activation of the cAMP-responsive Rap2 GTP-binding protein. Transient transfection of VSMs with a dominant-negative mutant of Rap2 inhibited the 17beta-estradiol-induced activation of the alpha(2C)-AR gene promoter, whereas a constitutively active mutant of Rap2 increased alpha(2C)-AR promoter activity. The effects of 17beta-estradiol were inhibited by the estrogen receptor (ER) antagonist, ICI-182780 (1 micromol/l), and were mimicked by a cell-impermeable form of the hormone (estrogen:BSA) or by the selective ER-alpha receptor agonist 4,4',4'''-(4-propyl-[(1)H]-pyrazole-1,3,5-triyl)tris-phenol (PPT; 10 nmol/l) or the selective ER-beta receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nmol/l). Therefore, 17beta-estradiol increased expression of alpha(2C)-ARs by interacting with cell surface receptors to cause a cAMP/Rap2-dependent increase in alpha(2C)-AR transcription. In mouse tail arteries, 17beta-estradiol (10 nmol/l) increased alpha(2C)-AR expression and selectively increased the cold-induced amplification of alpha(2)-AR constriction, which is mediated by alpha(2C)-ARs. An estrogen-dependent increase in expression of cold-sensitive alpha(2C)-ARs may contribute

Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor.

Science.gov (United States)

Kim, So Young; Choi, Yong Jun; Joung, Sun Myung; Lee, Byung Ho; Jung, Yi-Sook; Lee, Joo Young

2010-04-01

Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

DEFF Research Database (Denmark)

Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette

2005-01-01

and 2 (HIFs) are clearly related heterodimeric transcription factors that consist of an oxygen-depended alpha-subunit and a constitutive beta-subunit. With hypoxic exposure, HIF-1alpha and HIF-2alpha protein are stabilized. Upon heterodimerization, HIFs induce the transcription of a variety of genes......Regular exercise induces a variety of adaptive responses that enhance the oxidative and metabolic capacity of human skeletal muscle. Although the physiological adjustments of regular exercise have been known for decades, the underlying mechanisms are still unclear. The hypoxia inducible factors 1...... including erythropoietin (EPO), transferrin and its receptor, as well as vascular endothelial growth factor (VEGF) and its receptor. Considering that several of these genes are also induced with exercise, we tested the hypothesis that the mRNA level of HIF-1alpha and HIF-2alpha subunits increases...

The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1{alpha} targeted gene expression

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Miyake, Kotaro, E-mail: hif.panc@gmail.com [Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Nishioka, Masanori; Imura, Satoru; Batmunkh, Erdenebulgan [Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Uto, Yoshihiro [Department of Biological Science and Technology, Institute of Socio Technosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Nagasawa, Hideko [Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Hori, Hitoshi [Department of Biological Science and Technology, Institute of Socio Technosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Shimada, Mitsuo [Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan)

2012-08-01

Tumor hypoxia has been considered to be a potential therapeutic target, because hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. In the present study, we investigated the antitumor effect of a novel hypoxic cytotoxin, 3-[2-hydroxyethyl(methyl)amino]-2-quinoxalinecarbonitrile 1,4-dioxide (TX-2098) in inhibiting the expression of hypoxia inducible factor-1{alpha} (HIF-1{alpha}), and consequently vascular endothelial cell growth factor (VEGF) expression in pancreatic cancer. The antitumor effects of TX-2098 under hypoxia were tested against various human pancreatic cancer cell lines using WST-8 assay. VEGF protein induced pancreatic cancer was determined on cell-free supernatant by ELISA. Moreover, nude mice bearing subcutaneously (s.c.) or orthotopically implanted human SUIT-2 were treated with TX-2098. Tumor volume, survival and expression of HIF-1 and associated molecules were evaluated in treatment versus control groups. In vitro, TX-2098 inhibited the proliferation of various pancreatic cancer cell lines. In s.c model, tumors from nude mice injected with pancreatic cancer cells and treated with TX-2098 showed significant reductions in volume (P < 0.01 versus control). Quantitative real-time reverse transcription-PCR analysis revealed that TX-2098 significantly inhibited mRNA expression of the HIF-1 associated molecules, VEGF, glucose transporter 1 and Aldolase A (P < 0.01 versus control). These treatments also prolong the survival in orthotopic models. These results suggest that the effect of TX-2098 in pancreatic cancer might be correlated with the expression of VEGF and HIF-1 targeted molecules. -- Highlights: Black-Right-Pointing-Pointer We designed and synthesized novel hypoxic cytoxin, TX-2098. Black-Right-Pointing-Pointer TX-2098 inhibited the proliferation of human pancreatic cancer cells than TPZ. Black-Right-Pointing-Pointer TX-2098 reduced VEGF protein level than TPZ. Black-Right-Pointing-Pointer TX-2098

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

Science.gov (United States)

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.

Science.gov (United States)

Knowlton, K U; Baracchini, E; Ross, R S; Harris, A N; Henderson, S A; Evans, S M; Glembotski, C C; Chien, K R

1991-04-25

To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.

17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

Science.gov (United States)

Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

2010-03-01

The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

MicroRNA-195 induced apoptosis in hypoxic chondrocytes by targeting hypoxia-inducible factor 1 alpha.

Science.gov (United States)

Bai, R; Zhao, A-Q; Zhao, Z-Q; Liu, W-L; Jian, D-M

2015-02-01

The chondrocytes, the resident cells of cartilage, are maintained and take effects in the whole life upon chronic hypoxic exposure, which hypoxia-inducible factor 1 alpha (HIF-1α) play pivotal roles in response to. Dysregulation of some microRNA (miRNAs) have also been identified to be involved in hypoxia-related physiologic and pathophysiologic responses in some tissues or cell lines. However, the mechanism of miRNAs reponse to hypoxia remain largely unknown in chondrocytes, including the microRNA-195 (miR-195). AIM To investigate the effects of microRNAs (miRNAs) and hypoxia-inducible factor 1 alpha (HIF-1α) on chondrocytes in physiologic environment. We compared the expression of miR-195 and HIF-1α mRNA on hypoxia with that on normoxia in ATDC 5 cells by qRT-PCR. Further experiments was performed to confirmed the relationships of miR-195 and HIF-1α by bioinformatics analysis and dual reporter gene assay. we also assessed the effect of miR-195 on apoptosis in hypoxic ATDC 5 cells by transfect with miR-195 mimics. It was found the downregulated miR-195 and upregulated HIF-1α were present in hypoxic ATDC 5 cells. miR-195 negatively regulated HIF-1α by targeting its 3'-untranslated region. Moreover, the founding indicated miR-195 greatly increased apoptosis and downregulated HIF-1α mRNA occurred simultaneously in hypoxic chondrocytes. We concluded that miR-195 induced apoptosis in hypoxic chondrocytes by directly targeting HIF-1α.

Hypoxia inducible factor 1-alpha (HIF-1 alpha is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

Directory of Open Access Journals (Sweden)

Elisa Conde

Full Text Available Acute tubular necrosis (ATN caused by ischemia/reperfusion (I/R during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α, using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

Prolactin, TNF alpha and nitric oxide expression in nitroso-N-methylurea-induced-mammary tumours

Directory of Open Access Journals (Sweden)

Vegh Irene

2007-11-01

Full Text Available Abstract Background The N-Nitrosomethylurea breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease. Methods The present experimental study was designed to assess whether metoclopramide administration has any effect on development of MNU-induced tumours, and evaluate the treatment of goserelin acetate on PRL, TNF alpha and NO expression. NMU was administered to female Wistar rats on 2 occasions (5 mg/100 g body w/rat. PRL and TNF alpha were performed by immune-assay. Nitric Oxide by semi automated-assay and ploidy analyses by flow cytometry. Results The administration of metoclopramide made the induction time shorter and increased the incidence and average of tumours per rat. Tumours development was inhibited by a goserelin chronic administration. The ploidy of adenocarcinoma was polyploid-aneuploid type (average S = 60%. It was higher basal PRL plasma levels in rats with NMU induced tumours than in basal controls without tumour (p Conclusion The increase of blood PRL levels in NMU-induced rats may be an indicator of a poor prognosis of mammary cancer evolution. The metoclopramide administration accelerates tumour growth. However goserelin administration achieves regression in tumour development associated to inhibition PRL, TNF alpha and NO expression.

Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

Science.gov (United States)

Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

2004-10-01

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells.

Science.gov (United States)

Shin, K Y; Moon, H S; Park, H Y; Lee, T Y; Woo, Y N; Kim, H J; Lee, S J; Kong, G

2000-10-31

We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines. MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner. However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2. HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9. These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific. Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs.

Down-regulation of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor by HEXIM1 attenuates myocardial angiogenesis in hypoxic mice.

Science.gov (United States)

Yoshikawa, Noritada; Shimizu, Noriaki; Ojima, Hidenori; Kobayashi, Hiroshi; Hosono, Osamu; Tanaka, Hirotoshi

2014-10-24

Pulmonary hypertension (PH) sustains elevation of pulmonary vascular resistance and ultimately leads to right ventricular (RV) hypertrophy and failure and death. Recently, proangiogenic factors hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) have been known to promote left ventricular myocardial angiogenesis and lead to cardiac hypertrophy, and this would be involved in RV hypertrophy of PH patients. Previously, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced cardiomyocyte hypertrophy and hypertrophic genes expression, and that cardiomyocyte-specific HEXIM1 transgenic mice ameliorates RV hypertrophy in hypoxia-induced PH model. Given these results, here we analyzed the effect of HEXIM1 on the expression of HIF-1α and VEGF and on myocardial angiogenesis of RV in PH. We revealed that overexpression of HEXIM1 prevented hypoxia-induced expression of HIF-1α protein and its target genes including VEGF in the cultured cardiac myocytes and fibroblasts, and that cardiomyocyte-specific HEXIM1 transgenic mice repressed RV myocardial angiogenesis in hypoxia-induced PH model. Thus, we conclude that HEXIM1 could prevent RV hypertrophy, at least in part, via suppression of myocardial angiogenesis through down-regulation of HIF-1α and VEGF in the myocardium under hypoxic condition. Copyright © 2014 Elsevier Inc. All rights reserved.

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

Science.gov (United States)

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

Science.gov (United States)

Williams, C M; Coleman, J W

1995-10-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris

DEFF Research Database (Denmark)

Micheelsen, Pernille Ollendorff; Ostergaard, Peter Rahbek; Lange, Lene

2008-01-01

An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed...... and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants...

Enhanced actions of insulin-like growth factor-I and interferon-alpha co-administration in experimental cirrhosis.

Science.gov (United States)

Tutau, Federico; Rodríguez-Ortigosa, Carlos; Puche, Juan Enrique; Juanarena, Nerea; Monreal, Iñigo; García Fernández, María; Clavijo, Encarna; Castilla, Alberto; Castilla-Cortázar, Inma

2009-01-01

Cirrhosis is a diffuse process of hepatic fibrosis and regenerative nodule formation. The liver is the major source of circulating insulin-like growth factor-I (IGF-I) whose plasma levels are diminished in cirrhosis. IGF-I supplementation has been shown to induce beneficial effects in cirrhosis, including antifibrogenic and hepatoprotective effects. On other hand, interferon-alpha (IFN-alpha) therapy seems to suppress the progression of hepatic fibrosis. The aim of this study was to investigate the effect of the co-administration of IGF-I+IFN-alpha to Wistar rats with CCl(4)-induced cirrhosis, exploring liver function tests, hepatic lipid peroxidation and histopathology. The mechanisms underlying the effects of these agents were studied by reverse transcription-polymerase chain reaction, determining the expression of some factors [hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), alpha-smooth muscle actin, collagen, tissular inhibitor of metalloproteinases-1 and pregnane X receptor (PXR)] involved in fibrogenesis, fibrolysis and/or hepatoprotection. Both IGF-I and IFN-alpha exerted significant effects on fibrogenesis. IGF-I significantly increased serum albumin and HGF whereas IFN-alpha-therapy did not. The inhibition of TGF-beta expression was only observed by the effect of IFN-alpha-therapy. In addition, only the co-administration of IGF-I and IFN-alpha was able to increase the PXR. The combined therapy with both factors improved liver function tests, hepatic lipid peroxidation and reduced fibrosis, inducing a relevant histological improvement, reducing fibrosis and recovering hepatic architecture. The co-administration IGF-I+IFN enhanced all the beneficial effects observed with each factor separately, showing an additive action on histopathology and PXR expression, which is involved in the inhibition of fibrogenesis.

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ER{alpha} in the liver of male lizard Podarcis sicula

Energy Technology Data Exchange (ETDEWEB)

Verderame, Mariailaria; Prisco, Marina; Andreuccetti, Piero [Department of Biological Sciences, Evolutionary and Comparative Biology Division, University Federico II of Naples, Via Mezzocannone 8, 80134 Naples (Italy); Aniello, Francesco [Department of Biological Sciences, Genetic and Molecular Biology Division, University Federico II of Naples, Via Mezzocannone 8, 80134 Naples (Italy); Limatola, Ermelinda, E-mail: limatola@unina.it [Department of Biological Sciences, Evolutionary and Comparative Biology Division, University Federico II of Naples, Via Mezzocannone 8, 80134 Naples (Italy)

2011-05-15

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic specie. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E{sub 2} treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 {mu}g/{mu}l in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ER{alpha} in the liver which is known to be related to VTG synthesis in Podarcis. - Highlights: > Nonylphenol (NP) polluted diet induces VTG synthesis in a terrestrial vertebrate. > VTG and ER{alpha} genes are unexpressed in the liver of untreated male lizards Podarcis. > In the liver cells of NP-treated males the expression of both VTG and ER{alpha} occurs. > In treated males VTG synthesis is coupled with ER{alpha} expression as in breeding females. - NP-polluted diet induces the expression of ER{alpha} and VTG in the liver.

Expression of transcription factors Slug in the lens epithelial cells undergoing epithelial-mesenchymal transition induced by connective tissue growth factor

Directory of Open Access Journals (Sweden)

Ying-Na Wang

2015-10-01

Full Text Available AIM:To investigate the expression of transcription factors Slug in human lens epithelial cells (HLECs undergoing epithelial-mesenchymal transition (EMT induced by connective tissue growth factor (CTGF.METHODS: HLECs were treated with CTGF of different concentrations (20, 50 and 100 ng/mL or without CTGF (control for 24h. The morphological changes of HLECs were analysed by microscopy. The expression and cellular localization of Slug was evaluated by immumo-fluorescence. Expressions of Slug, E-cadherin and alpha smooth muscle actin (α-SMA were further determined by Western blot analysis. RESULTS: HLECs showed spidle fibrolasts-like characteristics and loosely connected each other after CTGF treatment. The immuno-fluorescence staining indicated that Slug was localized in the nuclei and its expression was induced by CTGF. The relative expressions of Slug protein were 1.64±0.11, 1.96 ±0.03, 3.12 ±0.10, and 4.08±0.14, respectively, in response to control group and treatment with CTGF of 20, 50 and 100 ng/mL (F=443.86, PCONCLUSION: Transcription factor Slug may be involved in EMT of HLECs induced by CTGF in vitro.

Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

Science.gov (United States)

Garcia, J A; Ferreira, H L; Vieira, F V; Gameiro, R; Andrade, A L; Eugênio, F R; Flores, E F; Cardoso, T C

2017-06-01

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy. © 2015 John Wiley & Sons Ltd.

Inhibition of HIF-1{alpha} activity by BP-1 ameliorates adjuvant induced arthritis in rats

Energy Technology Data Exchange (ETDEWEB)

Shankar, J. [Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago (United States); Thippegowda, P.B., E-mail: btprabha@uic.edu [Department of Pharmacology, (M/C 868), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612 (United States); Kanum, S.A. [Department of Chemistry, Yuvaraj' s College, University of Mysore, Mysore (India)

2009-09-18

Rheumatoid arthritis (RA) is a chronic inflammatory, angiogenic disease. Inflamed synovitis is a hallmark of RA which is hypoxic in nature. Vascular endothelial growth factor (VEGF), one of the key regulators of angiogenesis, is overexpressed in the pathogenesis of RA. VEGF expression is regulated by hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), a master regulator of homeostasis which plays a pivotal role in hypoxia-induced angiogenesis. In this study we show that synthetic benzophenone analogue, 2-benzoyl-phenoxy acetamide (BP-1) can act as a novel anti-arthritic agent in an experimental adjuvant induced arthritis (AIA) rat model by targeting VEGF and HIF-1{alpha}. BP-1 administered hypoxic endothelial cells and arthritic animals clearly showed down regulation of VEGF expression. Further, BP-1 inhibits nuclear translocation of HIF-1{alpha}, which in turn suppresses transcription of the VEGF gene. These results suggest a further possible clinical application of the BP-1 derivative as an anti-arthritic agent in association with conventional chemotherapeutic agents.

Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells.

Directory of Open Access Journals (Sweden)

Ilker Kudret Sariyer

Full Text Available PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV, which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs. We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF. SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen.Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins.Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to

Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-alpha in monocyte-derived macrophages.

Science.gov (United States)

Kim, Sung-Jo

2010-06-01

In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production in differentiated THP-1 cells, a human monocytic cell line. LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-alpha and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Leptin enhanced P. intermedia LPS-induced TNF-alpha production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-alpha expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-alpha production was not mediated by the leptin receptor. The ability of leptin to enhance P. intermedia LPS-induced TNF-alpha production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

Extract of corn silk (stigma of Zea mays) inhibits the tumour necrosis factor-alpha- and bacterial lipopolysaccharide-induced cell adhesion and ICAM-1 expression.

Science.gov (United States)

Habtemariam, S

1998-05-01

Treatment of human endothelial cells with cytokines such as tumour necrosis factor-alpha (TNF) or E. coli lipopolysaccharide (LPS) induces the expression of several adhesion molecules and enhances leukocyte adhesion to endothelial cell surface. Interfering with this leukocyte adhesion or adhesion molecules upregulation is an important therapeutic target for the treatment of bacterial sepsis and various inflammatory diseases. In the course of screening marketed European anti-inflammatory herbal drugs for TNF antagonistic activity, a crude ethanolic extract of corn silk (stigma of Zea mays) exhibited significant activity. The extract at concentrations of 9-250 micrograms/ml effectively inhibited the TNF- and LPS-induced adhesiveness of EAhy 926 endothelial cells to monocytic U937 cells. Similar concentration ranges of corn silk extract did also block the TNF and LPS but not the phorbol 12-myristate 13-acetate-induced ICAM-1 expression on EAhy 926 endothelial cell surface. The extract did not alter the production of TNF by LPS-activated macrophages and failed to inhibit the cytotoxic activity of TNF. It is concluded that corn silk possesses important therapeutic potential for TNF- and LPS-mediated leukocyte adhesion and trafficking.

Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells

DEFF Research Database (Denmark)

Ortis, Fernanda; Naamane, Najib; Flamez, Daisy

2010-01-01

by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells. RESEARCH DESIGN AND METHODS: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h......-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression...... of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine...

Muscle-specific expression of hypoxia-inducible factor in human skeletal muscle

DEFF Research Database (Denmark)

Mounier, Rémi; Pedersen, Bente Klarlund; Plomgaard, Peter

2010-01-01

fibres that possess unique patterns of protein and gene expression, producing different capillarization and energy metabolism systems. In this work, we analysed HIF-1alpha mRNA and protein expression related to the fibre-type composition in untrained human skeletal muscle by obtaining muscle biopsies...... from triceps brachii (characterized by a high proportion of type II fibres), from soleus (characterized by a high proportion of type I fibres) and from vastus lateralis (characterized by an equal proportion of type I and II fibres). The hypothesis was that type I muscle fibres would have lower HIF-1......alpha protein level. Interestingly, none of the HIF-1alpha target genes, like the most studied angiogenic factor involved in muscle angiogenesis, vascular endothelial growth factor (VEGF), exhibited a muscle fibre-specific-related mRNA expression at rest in normoxia. However, soleus presented...

Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

Energy Technology Data Exchange (ETDEWEB)

Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Takeuchi, Kentaro; Inada, Hirohiko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yamasaki, Daisuke [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Doi, Takefumi [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

2009-11-20

Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

Energy Technology Data Exchange (ETDEWEB)

Miettinen, Johanna A., E-mail: johanna.miettinen@oulu.fi [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Pietilae, Mika [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Salonen, Riikka J. [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Ohlmeier, Steffen [Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 Oulu (Finland); Ylitalo, Kari; Huikuri, Heikki V. [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland)

2011-04-01

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-{alpha}) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-{alpha} exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-{alpha} exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-{alpha} exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-{alpha} exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-{alpha} exposure, which might influence MSC differentiation stage and capacity.

Benfotiamine alleviates diabetes-induced cerebral oxidative damage independent of advanced glycation end-product, tissue factor and TNF-alpha.

Science.gov (United States)

Wu, Shan; Ren, Jun

2006-02-13

Diabetes mellitus leads to thiamine deficiency and multiple organ damage including diabetic neuropathy. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cerebral oxidative stress. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg, i.p.). Fourteen days later, control and diabetic (fasting blood glucose >13.9 mM) mice received benfotiamine (100 mg/kg/day, i.p.) for 14 days. Oxidative stress and protein damage were evaluated by glutathione/glutathione disulfide (GSH/GSSG) assay and protein carbonyl formation, respectively. Pro-oxidative or pro-inflammatory factors including advanced glycation end-product (AGE), tissue factor and tumor necrosis factor-alpha (TNF-alpha) were evaluated by immunoblot analysis. Four weeks STZ treatment led to hyperglycemia, enhanced cerebral oxidative stress (reduced GSH/GSSG ratio), elevated TNF-alpha and AGE levels without changes in protein carbonyl or tissue factor. Benfotiamine alleviated diabetes-induced cerebral oxidative stress without affecting levels of AGE, protein carbonyl, tissue factor and TNF-alpha. Collectively, our results indicated benfotiamine may antagonize diabetes-induced cerebral oxidative stress through a mechanism unrelated to AGE, tissue factor and TNF-alpha.

Hypoxia-inducible factor-1α expression requires PI 3-kinase activity and correlates with Akt1 phosphorylation in invasive breast carcinomas

NARCIS (Netherlands)

Gort, E.H.; Groot, A.J.; Derks van de Ven, T.L.P.; Groep, P. van der; Verlaan, I.; Laar, T. van; Diest, P.J. van; Wall, E. van der; Shvarts, A.

2006-01-01

Hypoxia-inducible factor-1 alpha (HIF-1a) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1a protein is associated with poor patient survival and therapy resistance in many types of solid

PGC-1{alpha} is required for AICAR induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle

DEFF Research Database (Denmark)

Leick, Lotte; Fentz, Joachim; Biensø, Rasmus S

2010-01-01

We tested the hypothesis that repeated activation of AMPK induces mitochondrial and glucose membrane transporter gene/protein expression via a peroxisome proliferator activated receptor Upsilon co-activator (PGC)-1alpha dependent mechanism. Whole body PGC-1alpha knockout (KO) and littermate wild...... GLUT4, cytochrome c oxidase (COX)I and cytochrome (cyt) c protein expression ~10-40% relative to saline in white muscles of the WT mice, but not of the PGC-1alpha KO mice. In line, GLUT4 and cyt c mRNA content increased 30-60% 4h after a single AICAR injection relative to saline only in WT mice. One...... and PGC-1alpha KO mice. In conclusion, we here provide genetic evidence for a major role of PGC-1alpha in AMPK mediated regulation of mitochondrial and glucose membrane transport protein expression in skeletal muscle....

Prognostic impact of HIF-1{alpha} expression in patients with definitive radiotherapy for cervical cancer

Energy Technology Data Exchange (ETDEWEB)

Dellas, K.; Bache, M.; Kappler, M.; Haensgen, G. [Halle-Wittenberg Univ., Halle (Germany). Dept. of Radiotherapy; Pigorsch, S.U. [Technische Univ. Muenchen (Germany). Dept. of Radiotherapy and Radiation Oncology; Taubert, H.; Holzhausen, H.J. [Halle-Wittenberg Univ., Halle (Germany). Inst. of Pathology; Holzapfel, D.; Zorn, E. [Halle-Wittenberg Univ., Halle (Germany). Dept. of Radiotherapy; Halle-Wittenberg Univ., Halle (Germany). Inst. of Pathology

2008-03-15

Purpose: To investigate the relationship between the hypoxia-inducible factor-(HIF-)1{alpha} expression in tumor tissue, tumor oxygenation and hemoglobin levels in patients with advanced cervical cancers prior to radiotherapy and the effect on clinical outcome. Patients and Methods: The investigation included 44 patients who underwent definitive radiotherapy for advanced cervical cancers between May 1995 and March 1999. Tumor biopsies were taken prior to treatment, and HIF-1{alpha} expression was determined by immunohistochemistry. In the same tumor area, tumor tissue oxygenation (pO{sub 2}) was measured using the Eppendorf device. Results: The 5-year cancer-specific survival of all patients was 60%. Twelve of 44 tumor specimens were HIF-1{alpha}-negative with a significantly better 5-year survival (92 {+-} 8%) versus 32 patients who were HIF-1{alpha}-positive (45 {+-} 10%; p < 0.02). There was no correlation between HIF-1{alpha} expression and tumor oxygenation (p = 0.57 both for pO{sub 2} median and hypoxic fraction < 5 mmHg vs. HIF-1{alpha} expression). However, patients with hemoglobin levels < 11 g/dl showed elevated HIF-1{alpha} expression compared to patients with hemoglobin levels > 12.5 g/dl (p = 0.04). Furthermore, HIF-1{alpha} correlated with vascular endothelial growth factor expression (p = 0.002). In a multivariate Cox regression model, HIF-1{alpha} expression (relative risk [RR] = 7.5; p = 0.05) revealed an increased risk of tumor-related death. Conclusion: The study indicates, that endogenous tumor markers such as HIF-1{alpha} may serve as prognostic markers of clinical outcome concerning cervical cancer after primary radiotherapy. (orig.)

The role of hepatocyte nuclear factor 4-alpha in perfluorooctanoic acid- and perfluorooctanesulfonic acid-induced hepatocellular dysfunction

Energy Technology Data Exchange (ETDEWEB)

Beggs, Kevin M., E-mail: kbeggs2@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, 4052 HLSIC, Kansas City, KS 66160 (United States); McGreal, Steven R., E-mail: smcgreal@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, 4052 HLSIC, Kansas City, KS 66160 (United States); McCarthy, Alex [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, 4052 HLSIC, Kansas City, KS 66160 (United States); Gunewardena, Sumedha, E-mail: sgunewardena@kumc.edu [Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd, 2027 HLSIC, Kansas City, KS 66160 (United States); Lampe, Jed N., E-mail: jlampe@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, 4052 HLSIC, Kansas City, KS 66160 (United States); Lau, Christoper, E-mail: lau.christopher@epa.gov [Developmental Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Apte, Udayan, E-mail: uapte@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, 4052 HLSIC, Kansas City, KS 66160 (United States)

2016-08-01

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers. - Highlights: • PFOA and PFOS cause decreased HNF4α protein expression in human hepatocytes. • PFOA and PFOS promote changes associated with lipid metabolism and carcinogenesis. • PFOA and PFOS induced changes in gene expression associated with cellular dedifferentiation. • PFOA and PFOS induce expression of Nanog, a transcription factor involved in stem cell development.

Intermitted pharmacologic pretreatment by xenon, isoflurane, nitrous oxide, and the opioid morphine prevents tumor necrosis factor alpha-induced adhesion molecule expression in human umbilical vein endothelial cells

NARCIS (Netherlands)

Weber, Nina C.; Kandler, Jennis; Schlack, Wolfgang; Grueber, Yvonne; Frädorf, Jan; Preckel, Benedikt

2008-01-01

BACKGROUND: The barrier properties of the endothelium are of critical importance during pathophysiologic processes. These barrier properties depend on an intact cytoskeleton and are regulated by cell adhesion molecules. Tumor necrosis factor alpha (TNF-alpha) is known to induce cell adhesion

The tumor necrosis factor-alpha-induced protein 8 family in immune homeostasis and inflammatory cancer diseases.

Science.gov (United States)

Luan, Y Y; Yao, Y M; Sheng, Z Y

2013-01-01

Within the immune system homeostasis is maintained by a myriad of mechanisms that include the regulation of immune cell activation and programmed cell death. The breakdown of immune homeostasis may lead to fatal inflammatory diseases. We set out to identify genes of tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family that has a functional role in the process of immune homeostasis. Tumor necrosis factor-alpha-induced protein 8 (TNFAIP8), which functions as an oncogenic molecule, is also associated with enhanced cell survival and inhibition of apoptosis. Tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) governs immune homeostasis in both the innate and adaptive immune system and prevents hyper-responsiveness by negatively regulating signaling via T cell receptors and Toll-like receptors (TLRs). There also exist two highly homologous but uncharacterized proteins, TIPE1 and TIPE3. This review is an attempt to provide a summary of TNFAIP8 family associated with immune homeostasis and inflammatory cancer diseases.

Effect of hypoxia-inducible factor 1-alpha (HIF-1α) on proliferation ...

African Journals Online (AJOL)

Jane

2011-07-25

Jul 25, 2011 ... Full Length Research Paper. Effect of hypoxia-inducible factor 1-alpha ... 1Department of Neurosurgery, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025,. China. 2Department of Neurosurgery, Chaoyang Hospital, Huainan, Anhui, China. 3Department of Neurosurgery ...

[Genetic cloning and expression of hypoxia inducible factor 1 alpha in high altitude hypoxic adaptation species Tibetan antelope (Pantholops hodgsonii)].

Science.gov (United States)

Liu, Fang; Wuren, Tana; Ma, Lan; Yang, Ying-Zhong; Ge, Ri-Li

2011-12-25

In order to investigate the role of the hypoxia inducible factor 1 alpha (HIF-1α) in the adaptation mechanism to high altitude hypoxia, the cloning of the HIF-1α gene cDNA of Tibetan antelope (Pantholops hodgsonii), using RT-PCR and RACE, was applied, and the comparative analysis of the tissue-specific expressions of HIF-1α among Tibetan antelope, Tibetan sheep and plain sheep was performed using real-time PCR and Western blot. The sequence analysis indicated that the cDNA sequences acquired by cloning from the HIF-1α gene of Tibetan antelope comprised a 2 471-bp open reading frame (ORF) and a 1 911-bp 3'UTR. The similarity between its coding sequence, predicted amino acid sequence and HIF-1α of other mammals exceeded 87%, in which the similarity with cow was up to more than 98%, which showed that this sequence was the cDNA of HIF-1α of Tibetan antelope. The results of real-time PCR and Western blot showed that expressions of HIF-1α mRNA and protein appeared in Tibetan antelope's lung, cardiac muscle and skeletal muscle, with the highest expression in lung. HIF-1α mRNA and protein had obvious differential expression in these tissues. Further research showed that Tibetan antelope and Tibetan sheep possessed higher expressions of HIF-1α protein in the three tissues above-mentioned compared with plain sheep, and the expressions of HIF-1α mRNA and protein in Tibetan antelope's lung, cardiac muscle and skeletal muscle were higher than those of Tibetan sheep. It illustrates that the hypoxic HIF-1α-specific expression is one of the molecular bases of high altitude hypoxia adaptation in Tibetan antelope.

Quantitative immuno-electron microscopic analysis of depolarization-induced expression of PGC-1alpha in cultured rat visual cortical neurons.

Science.gov (United States)

Meng, Hui; Liang, Huan Ling; Wong-Riley, Margaret

2007-10-17

Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC- 1alpha) is a coactivator of nuclear receptors and other transcription factors that regulate several metabolic processes, including mitochondrial biogenesis, energy homeostasis, respiration, and gluconeogenesis. PGC-1alpha plays a vital role in stimulating genes that are important to oxidative metabolism and other mitochondrial functions in brown adipose tissue and skeleton muscles, but the significance of PGC-1alpha in the brain remains elusive. The goal of our present study was to determine by means of quantitative immuno-electron microscopy the expression of PGC-1alpha in cultured rat visual cortical neurons under normal conditions as well as after depolarizing stimulation for varying periods of time. Our results showed that: (a) PGC-1alpha was normally located in both the nucleus and the cytoplasm. In the nucleus, PGC-1alpha was associated mainly with euchromatin rather than heterochromatin, consistent with active involvement in transcription. In the cytoplasm, it was associated mainly with free ribosomes. (b) Neuronal depolarization by KCl for 0.5 h induced a significant increase in PGC-1alpha labeling density in both the nucleus and the cytoplasm (Pneuronal activity by synthesizing more proteins in the cytoplasm and translocating them to the nucleus for gene activation. PGC-1alpha level in neurons is, therefore, tightly regulated by neuronal activity.

A correlation study between high resolution CT appearances and expression of transforming growth factor-β, tumor necrosis factor-α in radiation-induced lung injury of rats

International Nuclear Information System (INIS)

Guo Lili; Cheng Guangjun; Li Shaodong; Xu Kai

2008-01-01

Objective: To study the correlation between high resolution computed tomography manifestations and expression of transforming growth factor beta, tumor necrosis factor alpha in radiation- induced lung injury of rats, and to investigate the values of cytokine detection and HRCT scanning for the prediction and early diagnosis of radiation-induced lung injury. Methods: Forty-eight Sprague-Dawley (SD) rats were randomly divided into eight groups, group A was normal control group, and group B- H were irradiated with a single dose of 15 Gy to the lungs. HRCT scanning was performed before and 1 week, 2, 4, 8, 12, 16, 24 weeks after radiation in group A-H respectively. The expression of TGF-beta and TNF-alpha were detected with ELISA. All the rats were killed to observe pathological changes of their lungs. HRCT signs, levels of cytokine were simultaneously compared and analyzed. The t-test and Spearman rank correlation were used for the statistics. Results: Four HRCT signs were observed during the 24 weeks after radiation, including ground-glass opacity (1 case), patchy consolidation (8 cases), massive consolidation (7 cases) and fibrosis (3 cases). The average levels of TGF-beta in group B-H [(3.33± 0.47), (3.20±0.65), (3.12±0.45), (3.54±0.80), (3.30±1.13), (2.49±0.67), (4.19± 0.22) μg/L, respectively] were higher than the control group [(0.45±0.14) μg/L, P 0.05). There were no rank correlations between HRCT manifestations and expression of TGF-beta and TNF-alpha (r s = 0.5570 and 0.1013,P>0.05). HRCT signs were correlated with pathological changes. Conclusions: The monitoring of TGF-beta and TNF-alpha in the serum after irradiation can predict the development of radiation-induced lung injury. There are no rank correlations between HRCT manifestations and expression of TGF-beta and TNF-alpha. (authors)

HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

Science.gov (United States)

Niehof, Monika; Borlak, Jürgen

2011-01-27

Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS). In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII) remain elusive. Notably, angiotensinogen (AGT) is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1), angiotensin I converting enzyme (ACE), and angiotensin I converting enzyme 2 (ACE2) as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition of nuclear

HNF4alpha dysfunction as a molecular rational for cyclosporine induced hypertension.

Directory of Open Access Journals (Sweden)

Monika Niehof

Full Text Available Induction of tolerance against grafted organs is achieved by the immunosuppressive agent cyclosporine, a prominent member of the calcineurin inhibitors. Unfortunately, its lifetime use is associated with hypertension and nephrotoxicity. Several mechanism for cyclosporine induced hypertension have been proposed, i.e. activation of the sympathetic nervous system, endothelin-mediated systemic vasoconstriction, impaired vasodilatation secondary to reduction in prostaglandin and nitric oxide, altered cytosolic calcium translocation, and activation of the renin-angiotensin system (RAS. In this regard the molecular basis for undue RAS activation and an increased signaling of the vasoactive oligopeptide angiotensin II (AngII remain elusive. Notably, angiotensinogen (AGT is the precursor of AngII and transcriptional regulation of AGT is controlled by the hepatic nuclear factor HNF4alpha. To better understand the molecular events associated with cyclosporine induced hypertension, we investigated the effect of cyclosporine on HNF4alpha expression and activity and searched for novel HNF4alpha target genes among members of the RAS cascade. Using bioinformatic algorithm and EMSA bandshift assays we identified angiotensin II receptor type 1 (AGTR1, angiotensin I converting enzyme (ACE, and angiotensin I converting enzyme 2 (ACE2 as genes targeted by HNF4alpha. Notably, cyclosporine represses HNF4alpha gene and protein expression and its DNA-binding activity at consensus sequences to AGT, AGTR1, ACE, and ACE2. Consequently, the gene expression of AGT, AGTR1, and ACE2 was significantly reduced as evidenced by quantitative real-time RT-PCR. While RAS is composed of a sophisticated interplay between multiple factors we propose a decrease of ACE2 to enforce AngII signaling via AGTR1 to ultimately result in vasoconstriction and hypertension. Taken collectively we demonstrate cyclosporine to repress HNF4alpha activity through calcineurin inhibitor mediated inhibition

Role of tumor necrosis factor-alpha and platelet-activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis.

Science.gov (United States)

Lupia, E; Montrucchio, G; Battaglia, E; Modena, V; Camussi, G

1996-08-01

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF-alpha-like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti-TNF-alpha neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti-TNF-alpha mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF-alpha and PAF.

[Effects of feixin decoction on the contents of hypoxia-inducible factor-1alpha and vascular endothelial growth factor in the rat model of hypoxic pulmonary hypertension].

Science.gov (United States)

He, Hong-Jun; Dai, Ai-Guo

2012-05-01

To explore the effects of Feixin Decoction (FXD) on the hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in the rat model of hypoxic pulmonary hypertension (HPH), and to study its mechanisms for treating HPH. Forty healthy male SD rats were randomly divided into four groups, i. e., the normal control group, the HPH model group, the FXD group, and the Nifedipine group, 10 rats in each group. The HPH rat model was prepared using normal pressure intermittent hypoxia method. Except the normal control group, rats in the rest groups were fed in a self-made hypoxic plexiglass cabin, with the poor oxygen condition for 8 h daily for 14 successive days. Then the distilled water (at 30 mL/kg) was given by gastrogavage to rats in the normal control group and the HPH model group. FXD (at 28 g/kg) and Nifedipine (at 20 mg/kg) were given by gastrogavage to rats in the FXD group and the Nifedipine group respectively, once daily, for 14 successive days. Besides, hypoxia was continued for 14 days while medicating. The mean pulmonary artery pressure (mPAP) was detected on the second day after the last medication. The morphology of the pulmonary arteriole was detected. The ratio of pulmonary artery wall area and tube area (WA%) was determined. The protein and mRNA expressions of HIF-1alpha and VEGF were detected using immunohistochemistry and in situ hybridization technique. Compared with the normal control group, mPAP, WA%, and the protein and mRNA expressions of HIF-1alpha and VEGF significantly increased in the model group (P < 0.01, P < 0.05). Compared with the HPH model group, mPAP, WA%, and the protein and mRNA expressions of HIF-1alpha and VEGF significantly decreased in the FXD group (P < 0.01, P < 0.05). FXD down-regulated the expression of VEGF through decreasing the expression of HIF-1alpha. One of its mechanisms for treating HPH might be partially due to reversing the remodeling of pulmonary vascular smooth muscle.

A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

Directory of Open Access Journals (Sweden)

Anna H. Y. Law

2013-04-01

Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

Interaction of a gibberellin-induced factor with the upstream region of an alpha-amylase gene in rice aleurone tissue.

OpenAIRE

Ou-Lee, T M; Turgeon, R; Wu, R

1988-01-01

The interaction between the DNA sequences of an alpha-amylase (EC 3.2.1.1) gene and a tissue-specific factor induced in rice (Oryza sativa L.) aleurone tissue by gibberellin was studied. DNA mobility-shift during electrophoresis indicated that a 500-base-pair sequence (HS500) of a rice alpha-amylase genomic clone (OSamy-a) specifically interacted with a factor from gibberellin-induced rice aleurone tissue. The amount of complex formed between the HS500 DNA fragment and the gibberellin-induced...

Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

Science.gov (United States)

Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

2005-03-01

In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of

Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation☆

OpenAIRE

Cui, Hong; Han, Weijuan; Yang, Lijun; Chang, Yanzhong

2013-01-01

Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor 1α, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage. There is little evidence of direct regulatory effects of hypoxia-inducible factor 1α on oligodendrocyte lineage gene-1. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxy...

Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds.

Science.gov (United States)

Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko

2004-06-01

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

Hantaan Virus Nucleocapsid Protein Binds to Importin alpha Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B

Science.gov (United States)

2008-11-19

Microbiology . All Rights Reserved. Hantaan Virus Nucleocapsid Protein Binds to Importin Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced...Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702,1 and Department of Microbiology , Mount Sinai...34–36. 32. Prescott , J., C. Ye, G. Sen, and B. Hjelle. 2005. Induction of innate immune response genes by Sin Nombre hantavirus does not require

TNF-alpha, produced by feline infectious peritonitis virus (FIPV)-infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages.

Science.gov (United States)

Takano, Tomomi; Hohdatsu, Tsutomu; Toda, Ayako; Tanabe, Maki; Koyama, Hiroyuki

2007-07-20

The pathogenicity of feline infectious peritonitis virus (FIPV) is known to depend on macrophage tropism, and this macrophage infection is enhanced by mediation via anti-S antibody (antibody-dependent enhancement, ADE). In this study, we found that TNF-alpha production was increased with viral replication in macrophages inoculated with a mixture of FIPV and anti-S antibody, and demonstrated that this culture supernatant had feline PBMC apoptosis-inducing activity. We also demonstrated that the expression level of the FIPV virus receptor, feline aminopeptidase N (fAPN), was increased in macrophages of FIP cats. For upregulation of TNF-alpha and fAPN in macrophages, viral replication in macrophages is necessary, and their expressions were increased by ADE of FIPV infection. It was demonstrated that a heat-resistant fAPN-inducing factor was present in the culture supernatant of FIPV-infected macrophages, and this factor was TNF-alpha: fAPN expression was upregulated in recombinant feline TNF-alpha-treated macrophages, and FIPV infectivity was increased in these macrophages. These findings suggested that FIPV replication in macrophages increases TNF-alpha production in macrophages, and the produced TNF-alpha acts and upregulates fAPN expression, increasing FIPV sensitivity.

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...

The histone deacetylase inhibitor, Vorinostat, represses hypoxia inducible factor 1 alpha expression through translational inhibition.

Directory of Open Access Journals (Sweden)

Darren M Hutt

Full Text Available Hypoxia inducible factor 1α (HIF-1α is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor--eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.

Specific inhibition of hypoxia-inducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids.

Science.gov (United States)

Hasebe, Yuki; Egawa, Kiyoshi; Yamazaki, Yoko; Kunimoto, Setsuko; Hirai, Yasuaki; Ida, Yoshiteru; Nose, Kiyoshi

2003-10-01

Screening using a reporter under the control of the hypoxia-response element (HRE) identified several flavonoids and homoisoflavonoids that inhibit the activation of HRE under hypoxic conditions. Among various compounds, isorhamnetin, luteolin, quercetin, and methyl ophiopogonanone B (MOB) were effective at 3 to 9 microg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by MOB in HepG2 cells, but the effective doses were 10 to 20 microg/ml. MOB caused destabilization of hypoxia-inducible factor (HIF)-1alpha, as revealed by Western blotting, that was dependent on proteasome activity and the tumor suppressor, p53. The tubular formation and migration of human umbilical vein endothelial cells was also inhibited by MOB. MOB is expected to act as an inhibitor of angiogenesis.

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway.

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Lim, Jung Hwa; Jung, Cho-Rok; Lee, Chan-Hee; Im, Dong-Soo

2008-11-01

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.

SDF-1 alpha expression during wound healing in the aged is HIF dependent.

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Loh, Shang A; Chang, Edward I; Galvez, Michael G; Thangarajah, Hariharan; El-ftesi, Samyra; Vial, Ivan N; Lin, Darius A; Gurtner, Geoffrey C

2009-02-01

Age-related impairments in wound healing are associated with decreased neovascularization, a process that is regulated by hypoxia-responsive cytokines, including stromal cell-derived factor (SDF)-1 alpha. Interleukin-1 beta is an important inflammatory cytokine involved in wound healing and is believed to regulate SDF-1 alpha expression independent of hypoxia signaling. Thus, the authors examined the relative importance of interleukin (IL)-1 beta and hypoxia-inducible factor (HIF)-1 alpha on SDF-1 alpha expression in aged wound healing. Young and aged mice (n = 4 per group) were examined for wound healing using a murine excisional wound model. Wounds were harvested at days 0, 1, 3, 5, and 7 for histologic analysis, immunohistochemistry, enzyme-linked immunosorbent assay, and Western blot. An engineered wild-type and mutated SDF luciferase reporter construct were used to determine HIF transactivation. Aged mice demonstrated significantly impaired wound healing, reduced granulation tissue, and increased epithelial gap compared with young controls. Real-time polymerase chain reaction demonstrated reduced SDF-1 alpha levels in aged wounds that correlated with reduced CD31+ neovessels. Western blots revealed decreased HIF-1 alpha protein in aged wounds. However, both IL-1 beta and macrophage infiltrate were unchanged between young and aged animals. Using the wild-type and mutated SDF luciferase reporter construct in which the hypoxia response element was deleted, only young fibroblasts were able to respond to IL-1 beta stimulation, and this response was abrogated by mutating the HIF-binding sites. This suggests that HIF binding is essential for SDF-1 transactivation in response to both inflammatory and hypoxic stimuli. SDF-1 alpha deficiency observed during aged wound healing is attributable predominantly to decreased HIF-1 alpha levels rather than impaired IL-1 beta expression.

[Cellular adhesion signal transduction network of tumor necrosis factor-alpha induced hepatocellular carcinoma cells].

Science.gov (United States)

Zheng, Yongchang; Du, Shunda; Xu, Haifeng; Xu, Yiyao; Zhao, Haitao; Chi, Tianyi; Lu, Xin; Sang, Xinting; Mao, Yilei

2014-11-18

To systemically explore the cellular adhesion signal transduction network of tumor necrosis factor-alpha (TNF-α)-induced hepatocellular carcinoma cells with bioinformatics tools. Published microarray dataset of TNF-α-induced HepG2, human transcription factor database HTRI and human protein-protein interaction database HPRD were used to construct and analyze the signal transduction network. In the signal transduction network, MYC and SP1 were the key nodes of signaling transduction. Several genes from the network were closely related with cellular adhesion.Epidermal growth factor receptor (EGFR) is a possible key gene of effectively regulating cellular adhesion during the induction of TNF-α. EGFR is a possible key gene for TNF-α-induced metastasis of hepatocellular carcinoma.

Increased expression of protein kinase A inhibitor alpha (PKI-alpha) and decreased PKA-regulated genes in chronic intermittent alcohol exposure.

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Repunte-Canonigo, Vez; Lutjens, Robert; van der Stap, Lena D; Sanna, Pietro Paolo

2007-03-23

Intermittent models of alcohol exposure that mimic human patterns of alcohol consumption produce profound physiological and biochemical changes and induce rapid increases in alcohol self-administration. We used high-density oligonucleotide microarrays to investigate gene expression changes during chronic intermittent alcohol exposure in three brain regions that receive mesocorticolimbic dopaminergic projections and that are believed to be involved in alcohol's reinforcing actions: the medial prefrontal cortex, the nucleus accumbens and the amygdala. An independent replication of the experiment was used for RT-PCR validation of the microarray results. The protein kinase A inhibitor alpha (PKI-alpha, Pkia), a member of the endogenous PKI family implicated in reducing nuclear PKA activity, was found to be increased in all three regions tested. Conversely, we observed a downregulation of the expression of several PKA-regulated transcripts in one or more of the brain regions studied, including the activity and neurotransmitter-regulated early gene (Ania) - 1, -3, -7, -8, the transcription factors Egr1 and NGFI-B (Nr4a1) and the neuropeptide NPY. Reduced expression of PKA-regulated genes in mesocorticolimbic projection areas may have motivational significance in the rapid increase in alcohol self-administration induced by intermittent alcohol exposure.

HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

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Freya M Mowat

2010-06-01

Full Text Available Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Expression and regulation of HIF-1 alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor

NARCIS (Netherlands)

Westra, Johanna; Brouwer, Elisabeth; van Roosmalen, Ingrid A. M.; Doornbos-van der Meer, Berber; van Leeuwen, Miek A.; Posthumus, Marcel D.; Kallenberg, Cees G. M.

2010-01-01

Background: Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory

PPAR-alpha agonist treatment increases trefoil factor family-3 expression and attenuates apoptosis in the liver tissue of bile duct-ligated rats.

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Karakan, Tarkan; Kerem, Mustafa; Cindoruk, Mehmet; Engin, Doruk; Alper, Murat; Akın, Okan

2013-01-01

Peroxisome proliferators-activated receptor alpha activation modulates cholesterol metabolism and suppresses bile acid synthesis. The trefoil factor family comprises mucin-associated proteins that increase the viscosity of mucins and help protect epithelial linings from insults. We evaluated the effect of short-term administration of fenofibrate, a peroxisome proliferators activated receptor alpha agonist, on trefoil factor family-3 expression, degree of apoptosis, generation of free radicals, and levels of proinflammatory cytokines in the liver tissue of bile duct-ligated rats. Forty male Wistar rats were randomly divided into four groups: 1 = sham operated, 2 = bile duct ligation, 3 = bile duct-ligated + vehicle (gum Arabic), and 4 = bile duct-ligated + fenofibrate (100 mg/kg/day). All rats were sacrificed on the 7 th day after obtaining blood samples and liver tissue. Liver function tests, tumor necrosis factor-alpha and interleukin 1 beta in serum, and trefoil factor family-3 mRNA expression, degree of apoptosis (TUNEL) and tissue malondialdehyde (malondialdehyde, end-product of lipid peroxidation by reactive oxygen species) in liver tissue were evaluated. Fenofibrate administration significantly reduced serum total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and tumor necrosis factor-alpha and interleukin-1β levels. Apoptosis and malondialdehyde were significantly reduced in the fenofibrate group. Trefoil factor family-3 expression increased with fenofibrate treatment in bile duct-ligated rats. The peroxisome proliferators-activated receptor alpha agonist fenofibrate significantly increased trefoil factor family-3 expression and decreased apoptosis and lipid peroxidation in the liver and attenuated serum levels of proinflammatory cytokines in bile duct-ligated rats. Further studies are needed to determine the protective role of fenofibrate in human cholestatic disorders.

Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation

International Nuclear Information System (INIS)

Wong, G.H.; McHugh, T.; Weber, R.; Goeddel, D.V.

1991-01-01

We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha.

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Dietrich, Christoph G; Martin, Ina V; Porn, Anne C; Voigt, Sebastian; Gartung, Carsten; Trautwein, Christian; Geier, Andreas

2007-09-01

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.

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Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

2010-07-16

Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1alpha and HIF-2alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice

DEFF Research Database (Denmark)

Clausen, Bettina H; Lambertsen, Kate L; Babcock, Alicia A

2008-01-01

BACKGROUND: Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1beta is primarily neurotoxic in ischemic stroke, TNF-alpha may have neurotoxic and/or neuroprotective effects. We inv...

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

Science.gov (United States)

Matsumoto, M; Imagawa, M; Aoki, Y

2000-07-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors.

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Aprelikova, Olga; Chandramouli, Gadisetti V R; Wood, Matthew; Vasselli, James R; Riss, Joseph; Maranchie, Jodi K; Linehan, W Marston; Barrett, J Carl

2004-06-01

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Copyright 2004 Wiley-Liss, Inc.

Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

Science.gov (United States)

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

2003-04-01

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Effect of peroxisome proliferator-activated receptor alpha activators on tumor necrosis factor expression in mice during endotoxemia.

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Hill, M R; Clarke, S; Rodgers, K; Thornhill, B; Peters, J M; Gonzalez, F J; Gimble, J M

1999-07-01

Inflammatory mediators orchestrate the host immune and metabolic response to acute bacterial infections and mediate the events leading to septic shock. Tumor necrosis factor (TNF) has long been identified as one of the proximal mediators of endotoxin action. Recent studies have implicated peroxisome proliferator-activated receptor alpha (PPARalpha) as a potential target to modulate regulation of the immune response. Since PPARalpha activators, which are hypolipidemic drugs, are being prescribed for a significant population of older patients, it is important to determine the impact of these drugs on the host response to acute inflammation. Therefore, we examined the role of PPARalpha activators on the regulation of TNF expression in a mouse model of endotoxemia. CD-1 mice treated with dietary fenofibrate or Wy-14,643 had fivefold-higher lipopolysaccharide (LPS)-induced TNF plasma levels than LPS-treated control-fed animals. Higher LPS-induced TNF levels in drug-fed animals were reflected physiologically in significantly lower glucose levels in plasma and a significantly lower 50% lethal dose than those in LPS-treated control-fed animals. Utilizing PPARalpha wild-type (WT) and knockout (KO) mice, we showed that the effect of fenofibrate on LPS-induced TNF expression was indeed mediated by PPARalpha. PPARalpha WT mice fed fenofibrate also had a fivefold increase in LPS-induced TNF levels in plasma compared to control-fed animals. However, LPS-induced TNF levels were significantly decreased and glucose levels in plasma were significantly increased in PPARalpha KO mice fed fenofibrate compared to those in control-fed animals. Data from peritoneal macrophage studies indicate that Wy-14,643 modestly decreased TNF expression in vitro. Similarly, overexpression of PPARalpha in 293T cells decreased activity of a human TNF promoter-luciferase construct. The results from these studies suggest that any anti-inflammatory activity of PPARalpha in vivo can be masked by other

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression.

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Shannon, Diane B; McKeown, Scott T W; Lundy, Fionnuala T; Irwin, Chris R

2006-01-01

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

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Montrucchio, G.; Lupia, E.; de Martino, A.; Battaglia, E.; Arese, M.; Tizzani, A.; Bussolino, F.; Camussi, G.

1997-01-01

We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent. Images Figure 3 Figure 4 PMID:9250168

Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

DEFF Research Database (Denmark)

Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

2010-01-01

-alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

Polymorphisms in the hypoxia-inducible factor 1 alpha gene in Mexican patients with preeclampsia: A case-control study

Directory of Open Access Journals (Sweden)

Nava-Salazar Sonia

2011-03-01

Full Text Available Abstract Background Although the etiology of preeclampsia is still unclear, recent work suggests that changes in circulating angiogenic factors play a key role in its pathogenesis. In the trophoblast of women with preeclampsia, hypoxia-inducible factor 1 alpha (HIF-1α is over-expressed, and induces the expression of non-angiogenic factors and inhibitors of trophoblast differentiation. This observation prompted the study of HIF-1α and its relation to preeclampsia. It has been described that the C1772T (P582S and G1790A (A588T polymorphisms of the HIF1A gene have significantly greater transcriptional activity, correlated with an increased expression of their proteins, than the wild-type sequence. In this work, we studied whether either or both HIF1A variants contribute to preeclampsia susceptibility. Results Genomic DNA was isolated from 150 preeclamptic and 105 healthy pregnant women. Exon 12 of the HIF1A gene was amplified by PCR, and the genotypes of HIF1A were determined by DNA sequencing. In preeclamptic women and controls, the frequencies of the T allele for C1772T were 4.3 vs. 4.8%, and the frequencies of the A allele for G1790A were 0.0 vs. 0.5%, respectively. No significant differences were found between groups. Conclusion The frequency of the C1772T and G1790A polymorphisms of the HIF1A gene is very low, and neither polymorphism is associated with the development of preeclampsia in the Mexican population.

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

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Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

1999-03-15

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

Science.gov (United States)

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

[Effects of intermittent hypoxic exposure on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha and erythropoietin levels].

Science.gov (United States)

Zhang, Cheng-yan; Zhang, Ji-xin; Lü, Xiao-tao; Li, Bao-yu

2009-10-01

To investigate the effects of intermittent hypoxic exposure and normoxic convalescence on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) levels. Rat models of intermittent hypoxic exposure were established, combined with the clinical research on volunteers experiencing the intermittent plateau work. Blood samples for red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (HCT) were collected, serum HIF-1alpha and EPO levels were measured using enzyme linked immunosorbent assay. RBC counts, Hb concentration and HCT were significantly higher than the normoxic group (P hypoxic exposure can enhance serum hypoxia inducible factor-1 alpha and erythropointin levels and the generation of red blood cells, which leads to an increase in hemoglobin concentration and hematocrit. The results have changed with the hypoxic exposure period prolonged. Normoxic convalescence after intermittent hypoxic exposure can make the related indexes reduced, and contribute to the organism recovery.

D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of); Baek, Won-Ki, E-mail: wonki@dsmc.or.kr [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of)

2009-04-24

D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

Science.gov (United States)

Koo, Hyun-Na; Hong, Seung-Heon; Song, Bong-Keun; Kim, Cheorl-Ho; Yoo, Young-Hyun; Kim, Hyung-Min

2004-01-16

Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

International Nuclear Information System (INIS)

Choi, Hyun-Jeung; Kim, Tae Yong; Ruiz-Llorente, Sergio; Jeon, Min Ji; Han, Ji Min; Kim, Won Gu; Shong, Young Kee; Kim, Won Bae

2012-01-01

Introduction: Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Methods: Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Results: Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. Conclusions: ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

OpenAIRE

Matsumoto, M; Imagawa, M; Aoki, Y

2000-01-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did no...

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

Science.gov (United States)

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Immunoreactive transforming growth factor alpha and epidermal growth factor in oral squamous cell carcinomas

DEFF Research Database (Denmark)

Therkildsen, M H; Poulsen, Steen Seier; Bretlau, P

1993-01-01

Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected...... previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases......., the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus...

Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures

DEFF Research Database (Denmark)

Bernardino, Liliana; Xapelli, Sara; Silva, Ana P

2005-01-01

The inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects...... of mouse recombinant TNF-alpha (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-alpha to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol....... By using TNF-alpha receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-alpha involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed...

Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

Science.gov (United States)

Schulmeister, Ulrike; Hochwallner, Heidrun; Swoboda, Ines; Focke-Tejkl, Margarete; Geller, Beate; Nystrand, Mats; Härlin, Annika; Thalhamer, Josef; Scheiblhofer, Sandra; Keller, Walter; Niggemann, Bodo; Quirce, Santiago; Ebner, Christoph; Mari, Adriano; Pauli, Gabrielle; Herz, Udo; Valenta, Rudolf; Spitzauer, Susanne

2009-06-01

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

The MC160 Protein Expressed by the Dermatotropic Poxvirus Molluscum Contagiosum Virus Prevents Tumor Necrosis Factor Alpha-Induced NF-κB Activation via Inhibition of I Kappa Kinase Complex Formation

Science.gov (United States)

Nichols, Daniel Brian; Shisler, Joanna L.

2006-01-01

The pluripotent cytokine tumor necrosis factor alpha (TNF-α) binds to its cognate TNF receptor I (TNF-RI) to stimulate inflammation via activation of the NF-κB transcription factor. To prevent the detrimental effects of TNF-α in keratinocytes infected with the molluscum contagiosum virus (MCV), this poxvirus is expected to produce proteins that block at least one step of the TNF-RI signal transduction pathway. One such product, the MC160 protein, is predicted to interfere with this cellular response because of its homology to other proteins that regulate TNF-RI-mediated signaling. We report here that expression of MC160 molecules did significantly reduce TNF-α-mediated NF-κB activation in 293T cells, as measured by gene reporter and gel mobility shift assays. Since we observed that MC160 decreased other NF-κB activation pathways, namely those activated by receptor-interacting protein, TNF receptor-associated factor 2, NF-κB-inducing kinase, or MyD88, we hypothesized that the MC160 product interfered with I kappa kinase (IKK) activation, an event common to multiple signal transduction pathways. Indeed, MC160 protein expression was associated with a reduction in in vitro IKK kinase activity and IKK subunit phosphorylation. Further, IKK1-IKK2 interactions were not detected in MC160-expressing cells, under conditions demonstrated to induce IKK complex formation, but interactions between the MC160 protein and the major IKK subunits were undetectable. Surprisingly, MC160 expression correlated with a decrease in IKK1, but not IKK2 levels, suggesting a mechanism for MC160 disruption of IKK1-IKK2 interactions. MCV has probably retained its MC160 gene to inhibit NF-κB activation by interfering with signaling via multiple biological mediators. In the context of an MCV infection in vivo, MC160 protein expression may dampen the cellular production of proinflammatory molecules and enhance persistent infections in host keratinocytes. PMID:16378960

Transforming growth factor alpha and epidermal growth factor in laryngeal carcinomas demonstrated by immunohistochemistry

DEFF Research Database (Denmark)

Christensen, M E; Therkildsen, M H; Poulsen, Steen Seier

1993-01-01

the basal cell layer. The present investigation and our previous results confirm the existence of EGF receptors, TGF-alpha and EGF in laryngeal carcinomas. In addition, we conclude that the conditions do exist for growth factors to act through an autocrine system in poorly differentiated tumours and through......Fifteen laryngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) using immunohistochemical methods. In a recent study the same material was characterized for epidermal growth factor receptors (EGF...... receptors) which were confined predominantly to the undifferentiated cells. The expression of this growth factor system in malignant cells may play a role in carcinogenesis and/or tumour growth. All carcinomas were positive for TGF-alpha and 12 were positive for EGF. In moderately-to-well differentiated...

Suppressor of cytokine signalling-3 inhibits Tumor necrosis factor-alpha induced apoptosis and signalling in beta cells

DEFF Research Database (Denmark)

Bruun, Christine; Heding, Peter E; Rønn, Sif G

2009-01-01

Tumor necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine involved in the pathogenesis of several diseases including type 1 diabetes mellitus (T1DM). TNFalpha in combination with interleukin-1-beta (IL-1beta) and/or interferon-gamma (IFNgamma) induces specific destruction...

Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat

NARCIS (Netherlands)

Buttini, M; Appel, K; Sauter, A; GebickeHaerter, PJ; Boddeke, HWGM

Induction of tumor necrosis factor alpha was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for in situ hybridization histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

Science.gov (United States)

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Liver alpha-amylase gene expression as an early obesity biomarker.

Science.gov (United States)

Mojbafan, Marzieh; Afsartala, Zohreh; Amoli, Mahsa M; Mahmoudi, Mahdi; Yaghmaei, Parichehreh; Larijani, Bagher; Ebrahim-Habibi, Azadeh

2017-04-01

Obesity is a major health problem worldwide, for which preventive and therapeutic means are still needed. Alpha-amylase is a digestive enzyme whose inhibition has been targeted as a potential anti-obesity strategy. However, alpha-amylase gene expression has not been particularly attended to, and in contrast with pancreatic and salivary amylases, fewer studies have focused on liver alpha-amylase. The present study aimed at investigating the expression of alpha-amylase gene in obese and normal mice at RNA and protein level as well as acarbose effect on this gene expression in hepatocyte cell culture. Control and case groups were fed by normal mouse pellet and high-fat diet respectively, during 8 weeks. After this period, serum biochemical parameters including glucose, cholesterol, triglycerides, AST, ALT and alpha-amylase were assayed. Liver alpha-amylase gene was analyzed by real time PCR, and liver enzyme was assayed with Bernfeld and ELISA methods Hepatocyte cell culture derived from both group were also treated by acarbose and alpha-amylase activity and gene expression was analyzed by above mentioned methods. All biochemical factors showed an increase in obese mice, but the increase in ALT and AST were not statistically significant. Alpha-amylase levels were also increased in obese mice, both at RNA and protein level, while a decrease was seen in obese mice derived hepatocytes after acarbose treatment. Elevated liver alpha-amylase levels may be indicative of initial stages of obesity and the use of acarbose could be considered as a treatment of obesity which could be potentially effective at multiple levels. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Reoxygenation of human coronary smooth muscle cells suppresses HIF-1{alpha} gene expression and augments radiation-induced growth delay and apoptosis

Energy Technology Data Exchange (ETDEWEB)

Grumann, T.; Arab, A.; Bode, C.; Hehrlein, C. [Dept. of Cardiology, Univ. Clinic of Freiburg (Germany); Guttenberger, R. [Dept. of Radiotherapy, Univ. Clinic of Freiburg (Germany)

2006-01-01

Background and Purpose: Catheter-based coronary brachytherapy with {beta}- and {gamma}-radiation is an evidence-based method to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) and stent implantation, but the outcome may be PTCA are hypoxic. A lack of oxygen decreases the effect of low LET (linear energy transfer) irradiation. The authors assumed that reoxygenation of hypoxic human coronary smooth muscle cells (HCSMCs) improves the results of coronary brachytherapy. The expression of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) gene, and the rates of growth and apoptosis of hypoxic and reoxygenated HCSMCs after {gamma}-iradiation were therefore analyzed. Material and Methods: An in vitro model of megacolonies of HCSMCs was developed. After exposure to chronic hypoxia the HCSMCs were irradiated with graded doses of 2, 4, 8, and 16 Gy using a {sup 60}Co source either under hypoxia (pO{sub 2}<3 mmHg) or after reoxygenation (pO{sub 2}{approx}150 mmHg). RT-PCR (reverse transcription-polymerase chain reaction) analysis was used to quantify HIF-1{alpha} gene expression and the growth of HCSMC megacolonies was measured serially. The oxygen enhancement ratio (OER) was calculate from the specific growth delay. Apoptosis of HCSMCs was quantified by counting cells with specific DNA strand breaks using the TUNEL assy. Results: HIF-1{alpha} gene expression was markedly suppressed in reoxygenated cells versus hypoxic cells 30 min after {gamma}-irradiation at all radiation doses (158{+-}46% vs. 1,675{+-}1,211%; p<0.01). Apoptosis was markedly increased in reoxygenated HCSMCs. The OER was 1.8(95% CI[confidence interval]1.3-2.4). Therefore, reoxygenated HCSMCs require 44% less radiation dose to achieve the equivalent biological radiation effect compared to hypoxic HCSMCs. Conclusion: Reoxygenation of coronary smooth muscle cells should be considered an option to increase efficacy of coronary brachytherapy. This could be used to reduce radiation dose

Exercise preconditioning reduces brain damage and inhibits TNF-alpha receptor expression after hypoxia/reoxygenation: an in vivo and in vitro study.

Science.gov (United States)

Ding, Yun-Hong; Mrizek, Michael; Lai, Qin; Wu, Yimin; Reyes, Raul; Li, Jie; Davis, William W; Ding, Yuchuan

2006-11-01

Exercise reduces ischemia and reperfusion injury in rat stroke models. We investigated whether gradual increases in tumor necrosis factor-alpha (TNF-alpha) reported during exercise down-regulates expression of TNF-alpha receptors I and II (TNFRI and II) in stroke, leading to reduced brain damage. Adult male Sprague Dawley rats were subjected to 30 minutes of exercise on a treadmill each day for 3 weeks. Then, stroke was induced by a 2-hour middle cerebral artery (MCA) occlusion using an intra-luminal filament. Expressions of TNFRI and II mRNA in the brain were detected using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Protein expressions of TNFRI and II were determined by enzyme-linked immunoabsorbant assay (ELISA) in serum and brain homogenates. Spatial distribution of TNF-alpha receptors in brain regions was determined with immunocytochemistry. In human umbilical vein endothelial cells (HUVEC), we addressed the causal effect of TNF-alpha pretreatment on TNF I and II expression using ELISA and real-time PCR. In exercised rats after stroke, brain infarct was significantly (p<0.01) reduced in the entire MCA supplied regions, associated with a mild expression of TNFRI and II mRNA and protein. The TNF-alpha receptors were restricted to the ischemic core. In contrast, a robust expression of TNFRI and II molecules was found in non-exercised rats subjected to similar ischemia/reperfusion insults. An in vitro study revealed a causal link between TNF-alpha pretreatment and reduced cellular expression of TNF-alpha receptors under hypoxic/reoxygenated conditions. Our results suggest that reduced-brain damage in ischemic rats after exercise preconditioning may be attributable to the reduced expression of TNF-alpha receptors. Chronically increased TNF-alpha expression was also found to reduce TNFI and II responding to acute ischemia/reperfusion insult.

Prognostic role of hypoxia-inducible factor-1 alpha expression in osteosarcoma: a meta-analysis

Directory of Open Access Journals (Sweden)

Ren HY

2016-03-01

Full Text Available Hai-Yong Ren,1 Yin-Hua Zhang,1,2 Heng-Yuan Li,1 Tao Xie,1 Ling-Ling Sun,1 Ting Zhu,1 Sheng-Dong Wang,1 Zhao-Ming Ye1 1Department of Orthopaedics, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 2The First Department of Orthopaedics, Hospital of Zhejiang General Corps of Armed Police Forces, Jiaxing, People’s Republic of China Background: Hypoxia-inducible factor-1α (HIF-1α plays an important role in tumor progression and metastasis. A number of studies have investigated the association of HIF-1α with prognosis and clinicopathological characteristics of osteosarcoma but yielded inconsistent results.  Method: Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs and odds ratios (ORs with corresponding confidence intervals (CIs were calculated to assess the prognostic value of HIF-1α expression. The standard mean difference was used to analyze the continuous variable.  Results: Finally, nine studies comprising 486 patients were subjected to final analysis. Protein expression level of HIF-1α was found to be significantly related to overall survival (HR =3.0; 95% CI: 1.46–6.15, disease-free survival (HR =2.23; 95% CI: 1.26–3.92, pathologic grade (OR =21.33; 95% CI: 4.60–98.88, tumor stage (OR =10.29; 95% CI: 3.55–29.82, chemotherapy response (OR =9.68; 95% CI: 1.87–50.18, metastasis (OR =5.06; 95% CI: 2.87–8.92, and microvessel density (standard mean difference =2.83; 95% CI: 2.28–3.39.  Conclusion: This meta-analysis revealed that overexpression of HIF-1α is a predictive factor of poor outcomes for osteosarcoma. HIF-1α appeared to play an important role in prognostic evaluation and may be a potential target in antitumoral therapy. Keywords: HIF-1α, osteosarcoma, prognosis, meta-analysis

Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

Directory of Open Access Journals (Sweden)

Bernhard F Gibbs

Full Text Available Stem cell factor (SCF is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1 in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

Science.gov (United States)

Xia, Tian; Cheng, Hao; Zhu, Yu

2014-01-01

Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

Molecular cloning and functional characterization of the human platelet-derived growth factor alpha receptor gene promoter

NARCIS (Netherlands)

Afink, G. B.; Nistér, M.; Stassen, B. H.; Joosten, P. H.; Rademakers, P. J.; Bongcam-Rudloff, E.; van Zoelen, E. J.; Mosselman, S.

1995-01-01

Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R

Alpha Power Modulates Perception Independently of Endogenous Factors

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Sasskia Brüers

2018-04-01

Full Text Available Oscillations are ubiquitous in the brain. Alpha oscillations in particular have been proposed to play an important role in sensory perception. Past studies have shown that the power of ongoing EEG oscillations in the alpha band is negatively correlated with visual outcome. Moreover, it also co-varies with other endogenous factors such as attention, vigilance, or alertness. In turn, these endogenous factors influence visual perception. Therefore, it remains unclear how much of the relation between alpha and perception is indirectly mediated by such endogenous factors, and how much reflects a direct causal influence of alpha rhythms on sensory neural processing. We propose to disentangle the direct from the indirect causal routes by introducing modulations of alpha power, independently of any fluctuations in endogenous factors. To this end, we use white-noise sequences to constrain the brain activity of 20 participants. The cross-correlation between the white-noise sequences and the concurrently recorded EEG reveals the impulse response function (IRF, a model of the systematic relationship between stimulation and brain response. These IRFs are then used to reconstruct rather than record the brain activity linked with new random sequences (by convolution. Interestingly, this reconstructed EEG only contains information about oscillations directly linked to the white-noise stimulation; fluctuations in attention and other endogenous factors may still modulate brain alpha rhythms during the task, but our reconstructed EEG is immune to these factors. We found that the detection of near-perceptual threshold targets embedded within these new white-noise sequences depended on the power of the ~10 Hz reconstructed EEG over parieto-occipital channels. Around the time of presentation, higher power led to poorer performance. Thus, fluctuations in alpha power, induced here by random luminance sequences, can directly influence perception: the relation between

Enhancement of alpha particles-induced cell transformation by oxygen free radicals and tumor necrosis factor released from phagocytes

International Nuclear Information System (INIS)

Gong Yifen; Guo Renfeng; Zhu Maoxiang; Shou Jiang; Ge Guixiu; Yang Zhihua; Hieber, L.; Peters, K.; Schippel, C.

1997-01-01

To illustrate the role of several endogenous factors released from phagocytes under chronic inflammation in radiation-induced cancer. C 3 T 10 T 1/2 and SHE cells were used as targets, and 238 Pu alpha source was used in alpha irradiation. The enhancement of TF in alpha particles-induced cell transformation by PMA-stimulated human blood and zymosan-stimulated U-937 cells was studied using formation of transformed foci. Transformation frequency (TF) of C 3 H 10 T 1/2 cells exposed to alpha particles of 0.5 Gy increased 2.1 and 2.8 fold by PMA-and PMA-stimulated neutrophils, respectively. TF of irradiated SHE cells at a dose of 0.5 Gy increased 12 fold by the addition of the supernatant of macrophage-like U-937 cell line. It was shown that TF of irradiated SHE cells at above dose increased 8 fold by the supernatant treated with anti-TNF-α could be subcultured continuously in vitro. The cells at 40 th passage and two lines of monoclone cells have the ability to develop malignant tumors in nude mice. The overdose of free radicals and TNF-α released from neutrophils and macrophages have played an important role in low dose radiation-induced cancer

Tamoxifen induces the expression of maspin through estrogen receptor-alpha.

Science.gov (United States)

Liu, Zesheng; Shi, Heidi Y; Nawaz, Zafar; Zhang, Ming

2004-06-08

Maspin (mammary serine protease inhibitor) is a tumor suppressor gene that plays an important role in inhibiting tumor growth, invasion and metastasis. Maspin expression is down regulated at transcription level in primary and metastatic breast tumor cells. Previous studies on hormonal regulation of maspin prompt us to test whether an estrogen antagonist tamoxifen (TAM) can exert its anti-tumor function by up regulating maspin gene expression. For this purpose, we first tested whether maspin promoter could be activated in normal and several breast tumor cells. We then carried out a series of promoter analysis in which estrogen receptors and TAM were reconstituted in an in vitro cell culture system. Here we report our new finding that tumor suppresser gene maspin is one of the TAM target genes. TAM induces a maspin/luciferase reporter in cell culture and this induction requires the presence of (estrogen receptor alpha) ERalpha but not estrogen receptor-beta (ERbeta). Maspin promoter deletion and mutation analysis showed that the cis element(s) within a region between -90and+87 bp but not the HRE site (-272 bp) was involved in TAM induction of maspin expression. TAM bound ERalpha may directly control maspin gene expression through the interaction with cofactor (s). Analysis using several ERalpha mutants showed that the N-terminal A/B motif (AF-1) was critical for maspin basal level transcription activation. An ERalpha mutant with point mutations at DNA binding domain abolished estrogen induction of an ERE-luciferase reporter but was still active in activating maspin promoter by TAM. LBD-AF2 domain was required for ERalpha-dependent TAM induction. Deletion of LBD-AF2 or a point mutation in the ERalpha LBD-AF2 region (LBDmtL539A) completely abolished the activation of maspin promoter, suggesting that TAM induction of maspin involves the recruitment of cofactor(s) by ERalpha to the maspin promoter region. This finding indicates that one of the pathways for cancer

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

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Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

2012-01-06

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

Energy Technology Data Exchange (ETDEWEB)

Raman, Dayanidhi; Milatovic, Snjezana-Zaja [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Milatovic, Dejan [Department of Pediatrics/Pediatric Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Splittgerber, Ryan [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Fan, Guo-Huang [Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37221 (United States); Richmond, Ann, E-mail: ann.richmond@vanderbilt.edu [VA Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

2011-11-15

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxia-inducible factor-1alpha expression via poly(ADP-ribose) polymerase-1.

Science.gov (United States)

Pan, Rong; Chen, Chen; Liu, Wen-Lan; Liu, Ke-Jian

2013-07-01

Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study tests the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-h hypoxic treatment. Although 3-h hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc-concentration-dependent manner. Exposure of astrocytes to hypoxia for 3 h remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably, HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. © 2013 John Wiley & Sons Ltd.

Regulation of hypoxia-inducible factor-1α (HIF-1α expression by interleukin-1β (IL-1 β, insulin-like growth factors I (IGF-I and II (IGF-II in human osteoarthritic chondrocytes

Directory of Open Access Journals (Sweden)

Angelica Rossi Sartori-Cintra

2012-01-01

Full Text Available OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α can regulate cytokines (catabolic action and/or growth factors (anabolic action in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β and insulin-like growth factors I (IGF-I and II (IGF-II and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.

Interferon beta 1, an intermediate in the tumor necrosis factor alpha- induced increased MHC class I expression and an autocrine regulator of the constitutive MHC class I expression

OpenAIRE

1987-01-01

In conclusion, our observations indicate that the constitutive MHC class I expression is regulated by autocrine production of IFN-beta 1. TNF-alpha acts as an enhancer of the autocrine production of IFN-beta 1, and consequently as an enhancer of the MHC class I expression and viral protection.

Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

Directory of Open Access Journals (Sweden)

Xiaodong Pan

Full Text Available Transforming growth factor-beta (TGF-β, a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1 expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

Molecular evidence for the existence of lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus).

Science.gov (United States)

De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Whang, Ilson; Lee, Jae-Seong; Jung, Sung-Ju; Choi, Cheol Young; Lee, Jehee

2010-01-01

The lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn(+2)) binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-alpha expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-alpha, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca. 2010 Elsevier Ltd. All rights reserved.

Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

International Nuclear Information System (INIS)

Tachibana, Keisuke; Takeuchi, Kentaro; Inada, Hirohiko; Yamasaki, Daisuke; Ishimoto, Kenji; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Doi, Takefumi

2009-01-01

Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial β-oxidation. The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that plays an important role in the regulation of β-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPARα and found that PPARα induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPARα regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

Development of a chromosomally integrated metabolite-inducible Leu3p-alpha-IPM "off-on" gene switch.

Directory of Open Access Journals (Sweden)

Maria Poulou

2010-08-01

Full Text Available Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression.Here, we show that a chromosomally integrated yeast 'Leu3p-alpha-IotaRhoMu' system constitutes a ligand-inducible regulatory "off-on" genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of alpha-isopropylmalate (alpha-IotaRhoMu in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable alpha-IPM presents an EC(50 equal to 0.8837 mM and fast "OFF-ON" kinetics (t(50ON = 43 min, t(50OFF = 2.18 h, it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo.Our results demonstrate that the 'Leu3p-alpha-IotaRhoMu' constitutes a simpler and safer system for inducible gene expression in biomedical applications.

Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

Science.gov (United States)

Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

2004-11-01

GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

Irradiation induces a biphasic expression of pro-inflammatory cytokines in the lung

Energy Technology Data Exchange (ETDEWEB)

Ruebe, C.E.; Wilfert, F.; Palm, J.; Burdak-Rothkamm, S.; Ruebe, C. [Dept. of Radiotherapy - Radiooncology, Saarland Univ., Homburg/Saar (Germany); Koenig, J. [Inst. of Medical Biometrics, Epidemiology and Medical Informatics, Saarland Univ., Homburg/Saar (Germany); Liu Li [Dept. of Radiotherapy - Radiooncology, Saarland Univ., Homburg/Saar (Germany); Cancer Center, Union Hospital Tongji Medical Coll., Huazhong Univ. of Science and Technology, Wuhan (China); Schuck, A.; Willich, N. [Dept. of Radiotherapy - Radiooncology, Univ. of Muenster (Germany)

2004-07-01

Background and purpose: the precise pathophysiological mechanisms of radiation-induced lung injury are poorly understood, but have been shown to correlate with dysregulation of different cytokines. The purpose of this study was to evaluate the time course of the pro-inflammatory cytokines tumor necrosis factor-(TNF-){alpha}, interleukin-(IL)-1{alpha} and IL-6 after whole-lung irradiation. Material and methods: the thoraces of C57BL/6J mice were irradiated with 12 Gy. Treated and control mice were sacrificed at 0.5, 1, 3, 6, 12, 24, 48, 72 h, 1, 2, 4, 8, 16, and 24 weeks post irradiation (p.i.). Real-time multiplex RT-PCR (reverse transcriptase polmyerase chain reaction) was established to evaluate the expression of TNF-{alpha}, IL-1{alpha} and IL-6 in the lung tissue of the mice. For histological analysis, lung tissue sections were stained by hematoxylin and eosin. Results: multiplex RT-PCR analysis revealed a biphasic expression of these pro-inflammatory cytokines in the lung tissue after irradiation. After an initial increase at 1 h p.i. for TNF-{alpha} and at 6 h p.i. for IL-1{alpha} and IL-6, the mRNA expression of these pro-inflammatory cytokines returned to basal levels (48 h, 72 h, 1 week, 2 weeks p.i.). During the pneumonic phase, TNF-{alpha}, IL-1{alpha} and IL-6 were significantly elevated and revealed their maximum at 8 weeks p.i. Histopathologic evaluation of the lung sections obtained within 4 weeks p.i. revealed only minor lung damage in 5-30% of the lung tissue. By contrast, at 8, 16, and 24 weeks p.i., 70-90% of the lung tissue revealed histopathologically detectable organizing alveolitis. Conclusion: irradiation induces a biphasic expression of pro-inflammatory cytokines in the lung. The initial transitory cytokine response occurred within the first hours after lung irradiation with no detectable histopathologic alterations. The second, more persistent cytokine elevation coincided with the onset of histologically discernible organizing acute

The potential role of Brachyury in inducing epithelial-to-mesenchymal transition (EMT) and HIF-1α expression in breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Shao, Chao [Department of Mammary Surgery, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China); Zhang, Jingjing, E-mail: jingjingzhangzs@163.com [Department of Cancer Radiotherapy, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China); Fu, Jianhua [Department of Thoracic Surgery, Cancer Center, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China); Ling, Feihai, E-mail: feihailingfhl@163.com [Department of Mammary Surgery, Zhongshan Hospital of Sun Yat-sen University, Zhongshan, 528403 (China)

2015-11-27

One of transcription factors of the T-box family, Brachyury has been implicated in tumorigenesis of many types of cancers, regulating cancer cell proliferation, metastasis, invasion and epithelial-to-mesenchymal transition (EMT). However, the role of Brachyury in breast cancer cells has been scarcely reported. The present study aimed to investigate the expression and role of Brachyury in breast cancer. Brachyury expression was analyzed by qRT-PCR and Western blot. The correlations between Brachyury expression and clinicopathological factors of breast cancer were determined. Involvement of EMT stimulation and hypoxia-inducible factor-1α (HIF-1α) expression induction by Brachyury was also evaluated. Moreover, the effect of Brachyury on tumor growth and metastasis in vivo was examined in a breast tumor xenograft model. Brachyury expression was enhanced in primary breast cancer tissues and Brachyury expression was correlated with tumor stage and lymph node metastasis. Hypoxia enhanced Brachyury expression, the silencing of which blocked the modulation effect of hypoxia on E-cadherin and vimentin expression. Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling as well as accelerated cell proliferation and migration in vitro. Additionally, Brachyury accelerated breast tumor xenograft growth and increased lung metastasis in nude mice. In summary, our data confirmed that Brachyury might contribute to hypoxia-induced EMT of breast cancer and trigger HIF-1alpha expression via PTEN/Akt signaling. - Highlights: • Brachyury expression was correlated with tumor stage and lymph node metastasis. • Hypoxia enhanced Brachyury expression, which contributes to hypoxia-induced EMT. • Brachyury significantly augmented HIF-1alpha expression via PTEN/Akt signaling. • Brachyury accelerated tumor xenograft growth and increased lung metastasis.

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha

OpenAIRE

Camenisch, G; Tini, M; Chilov, D; Kvietikova, I; Srinivas, V; Caro, J; Spielmann, P; Wenger, R H; Gassmann, M

1999-01-01

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the alpha subunit of the human hypoxia-inducible factor 1 (HIF-1alpha). Anti-HIF-1alpha IgY antibodies were affinity purified and...

Induction of VEGF expression by alpha-tocopherol and alpha-tocopheryl phosphate via PI3Kgamma/PKB and hTAP1/SEC14L2-mediated lipid exchange

Science.gov (United States)

In several studies, vitamin E has been observed to influence angiogenesis and vasculogenesis. We recently showed that the phosphorylated form of alpha-tocopherol (alphaT), alpha-tocopheryl phosphate (alphaTP), increases the expression of the vascular endothelial growth factor (VEGF). Thus, alphaTP m...

Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor necrosis factor alpha and tumor necrosis factor beta

DEFF Research Database (Denmark)

Issazadeh-Navikas, Shohreh; Ljungdahl, A; Höjeberg, B

1995-01-01

in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-beta (= lymphotoxin-alpha) and cytolysin appeared early and before onset of clinical...... signs of EAE; (ii) TNF-alpha peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-gamma (IFN-gamma) mRNA shown previously is consistent with a role of IL-12 in promoting...... proliferation and activation of T helper 1 (Th1) type cells producing IFN-gamma. The TNF-beta mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-alpha was suggested from these observations that TNF-alpha mRNA expression...

SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

International Nuclear Information System (INIS)

Campos, D; Peeters, W; Nickel, K; Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M

2015-01-01

Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response

SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

Energy Technology Data Exchange (ETDEWEB)

Campos, D [University of Wisconsin Madison, Madison, WI (United States); Peeters, W [Radboud University Medical Center, Nijmegen, GA (United States); Nickel, K [University of Wisconsin, Madison, WI (United States); Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M [University of Wisconsin, Madison, Wisconsin (United States)

2015-06-15

Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

Energy Technology Data Exchange (ETDEWEB)

Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Xiao, Shaobo, E-mail: shaoboxiao@yahoo.com [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)

2010-08-13

Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

Science.gov (United States)

Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

1993-01-15

In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

OpenAIRE

Southgate, V J; Steyn, A J; Pretorius, I S; Van Vuuren, H J

1993-01-01

Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

Interleukin-4 inhibits both paracrine and autocrine tumor necrosis factor-alpha-induced proliferation of B chronic lymphocytic leukemia cells

NARCIS (Netherlands)

van Kooten, C.; Rensink, I.; Aarden, L.; van Oers, R.

1992-01-01

The proliferative response of purified malignant B cells from 26 patients with chronic lymphocytic leukemia (CLL) was investigated in vitro. In the majority of these patients, a proliferative response could be induced by the combination of tumor necrosis factor (TNF)-alpha and PMA. The concentration

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

Science.gov (United States)

Tran, An Hue Vy; Han, Se Hee; Kim, Joon; Grasso, Francesca; Kim, In San; Han, Ye Sun

2017-07-01

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Developmental expression of the alpha-skeletal actin gene

Directory of Open Access Journals (Sweden)

Vonk Freek J

2008-06-01

Full Text Available Abstract Background Actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. In higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. Expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. All members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins. Results We present here a model for the evolution of the genomic organization of alpha-skeletal actin and by molecular modeling, illustrate the structural differences of actin proteins of different phyla. We further describe and compare alpha-skeletal actin expression in two developmental stages of five vertebrate species (mouse, chicken, snake, salamander and fish. Our findings confirm that alpha-skeletal actin is expressed in skeletal muscle and in the heart of all five species. In addition, we identify many novel non-muscular expression domains including several in the central nervous system. Conclusion Our results show that the high sequence homology of alpha-skeletal actins is reflected by similarities of their 3 dimensional protein structures, as well as by conserved gene expression patterns during vertebrate development. Nonetheless, we find here important differences in 3D structures, in gene architectures and identify novel expression domains for this structural and functional important gene.

Protection against dexamethasone-induced muscle atrophy is related to modulation by testosterone of FOXO1 and PGC-1{alpha}

Energy Technology Data Exchange (ETDEWEB)

Qin, Weiping, E-mail: weiping.qin@mssm.edu [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Department of Medicine, Mount Sinai School of Medicine, NY (United States); Pan, Jiangping; Wu, Yong [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Bauman, William A. [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Department of Medicine, Mount Sinai School of Medicine, NY (United States); Department of Rehabilitation Medicine, Mount Sinai School of Medicine, NY (United States); Cardozo, Christopher, E-mail: Chris.Cardozo@mssm.edu [Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peters VA Medical Center, Bronx, NY (United States); Department of Medicine, Mount Sinai School of Medicine, NY (United States); Department of Rehabilitation Medicine, Mount Sinai School of Medicine, NY (United States)

2010-12-17

Research highlights: {yields} In rat gastrocnemius muscle, dexamethasone reduced PGC-1{alpha} cellular and nuclear levels without altering mRNA levels for this factor. {yields} Dexamethasone reduced phosphorylating of p38 MAPK, which stabilizes PGC-1{alpha} and promotes its nuclear entry. {yields} Co-administration of testosterone with dexamethasone increased cellular and nuclear levels of PGC-1{alpha} protein without changing its mRNA levels. {yields} Co-administration of testosterone restored p38 MAPK levels to those of controls. -- Abstract: Glucocorticoid-induced muscle atrophy results from muscle protein catabolism and reduced protein synthesis, associated with increased expression of two muscle-specific ubiquitin ligases (MAFbx and MuRF1), and of two inhibitors of protein synthesis, REDD1 and 4EBP1. MAFbx, MuRF1, REDD1 and 4EBP1 are up-regulated by the transcription factors FOXO1 and FOXO3A. The transcriptional co-activator PGC-1{alpha} has been shown to attenuate many forms of muscle atrophy and to repress FOXO3A-mediated transcription of atrophy-specific genes. Dexamethasone-induced muscle atrophy can be prevented by testosterone, which blocks up-regulation by dexamethasone of FOXO1. Here, an animal model of dexamethasone-induced muscle atrophy was used to further characterize effects of testosterone to abrogate adverse actions of dexamethasone on FOXO1 levels and nuclear localization, and to determine how these agents affect PGC-1{alpha}, and its upstream activators, p38 MAPK and AMPK. In rat gastrocnemius muscle, testosterone blunted the dexamethasone-mediated increase in levels of FOXO1 mRNA, and FOXO1 total and nuclear protein. Dexamethasone reduced total and nuclear PGC-1{alpha} protein levels in the gastrocnemius; co-administration of testosterone with dexamethasone increased total and nuclear PGC-1{alpha} levels above those present in untreated controls. Testosterone blocked dexamethasone-induced decreases in activity of p38 MAPK in the gastrocnemius

Proposal of a weight factor for alpha radiation aiming biota radioprotection

International Nuclear Information System (INIS)

Pereira, Wagner de S.; Py Junior, Delcy de A.; Kelecom, Alphonse; Goncalves, Simone

2009-01-01

Several proposals based on the environmental radioprotection of calculating the absorbed dose in biota have been suggested. The absorbed dose expresses the deposition of energy per mass unit. The differences in biological effects of the absorbed dose can be quantified by applying a correction factor to the absorbed dose. The correction factor for radiation is easier to establish, because radiations exist in smaller number (alpha, beta, neutrons and photons) and can be set for groups of organisms. This work aims to propose a correction factor for radiation, in order to adequate the concept of absorbed dose currently used to the concept of equivalent dose. A survey of the literature on correction factors proposed for alpha radiation was carried out and, when possible, the biological endpoint was identified, as well as the radionuclide and the biological target. A variation of the weight factor for alpha radiation from 1 to 377 was observed and a number of biological endpoints, biological target and alpha emitter radionuclide were identified. Finally we propose a weight value for alpha radiation of 40, and we propose also the name of correction factor for radiation alpha as being ecological radiation weighting factor (WRE) the name 'equivalent dose for flora and fauna' (HTFF) to name of the new dose. (author)

The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes

DEFF Research Database (Denmark)

Benfield, T L; Lundgren, Bettina; Levine, S J

1997-01-01

Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting...... the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0.......01). In a similar fashion, MSG elicited release of TNF-alpha. Initial increases were also seen at 4 h, and at 20 h, TNF-alpha levels reached 6.4 +/- 1.1 ng/ml, compared to 0.08 +/- 0.01 ng/ml for control cultures (P alpha secretion was observed at 20 h...

Fano factor evaluation of diamond detectors for alpha particles

Energy Technology Data Exchange (ETDEWEB)

Shimaoka, Takehiro; Kaneko, Junichi H.; Tsubota, Masakatsu; Shimmyo, Hiroaki [Graduate School of Engineering, Hokkaido University, Kita 13, Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-8628 (Japan); Sato, Yuki [Naraha Remote Technology Development Center, Japan Atomic Energy Agency, Naraha-machi, Futaba-gun, Fukushima, 979-0513 (Japan); Chayahara, Akiyoshi; Umezawa, Hitoshi; Mokuno, Yoshiaki [Advanced Power Electronics Research Center, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577 (Japan); Watanabe, Hideyuki [Research Institute for Electronics and Photonics, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, 305-8565 (Japan)

2016-10-15

This report is the first describing experimental evaluation of Fano factor for diamond detectors. High-quality self-standing chemical vapor deposited diamond samples were produced using lift-off method. Alpha-particle induced charge measurements were taken for three samples. A 13.1 ±0.07 eV of the average electron-hole pair creation energy and excellent energy resolution of approximately 0.3% were found for 5.486 MeV alpha particles from an {sup 241}Am radioactive source. The best Fano factor for 5.486 MeV alpha particles, calculated from experimentally obtained epsilon values and the detector intrinsic energy resolution, was 0.382 ± 0.007. (copyright 2016 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Alpha-driven magnetohydrodynamics (MHD) and MHD-induced alpha loss in the Tokamak Fusion Test Reactor

International Nuclear Information System (INIS)

Chang, Z.; Nazikian, R.; Fu, G.Y.

1997-02-01

Alpha-driven toroidal Alfven eigenmodes (TAEs) are observed as predicted by theory in the post neutral beam phase in high central q (safety factor) deuterium-tritium (D-T) plasmas in the Tokamak Fusion Test Reactor (TFTR). The mode location, poloidal structure and the importance of q profile for TAE instability are discussed. So far no alpha particle loss due to these modes was detected due to the small mode amplitude. However, alpha loss induced by kinetic ballooning modes (KBMs) was observed in high confinement D-T discharges. Particle orbit simulation demonstrates that the wave-particle resonant interaction can explain the observed correlation between the increase in alpha loss and appearance of multiple high-n (n ≥ 6, n is the toroidal mode number) modes

Polychlorinated biphenyls alter expression of alpha-synuclein, synaptophysin and parkin in the rat brain

DEFF Research Database (Denmark)

Malkiewicz, Katarzyna; Mohammed, Roma; Folkesson, Ronnie

2006-01-01

Polychlorinated Biphenyls (PCBs)-induced changes in synaptic transmission are one of the effects of their neurotoxicity but the mechanism remains unknown. We assessed the in vivo effects of the PCBs mixture, Aroclor 1254 on the expression of neuronal proteins that are involved in the synaptic...... function and/or are associated with neurodegeneration. Wistar rats were treated orally with repeated doses of Aroclor 1254 and the levels of soluble alpha-synuclein, parkin, synaptophysin and amyloid precursor protein (APP) in the brain were determined by Western blotting. The results showed that Aroclor...... did not cause changes in the expression and processing of APP but at a dose 100 microg/g/day repeated for 6 days caused a decrease in the expression of alpha-synuclein in the cerebellum, cortex, hippocampus and hypothalamus of the animals sacrificed 2 days after treatment. The decrease in alpha...

Expression of HIF-1{alpha} in irradiated tissue is altered by topical negative-pressure therapy

Energy Technology Data Exchange (ETDEWEB)

Grimm, A.; Stange, S.; Labanaris, A.; Horch, R.E. [Erlangen-Nuernberg Univ., Erlangen (Germany). Dept. of Plastic and Hand Surgery; Dimmler, A. [Erlangen-Nuernberg Univ., Erlangen (Germany). Dept. of Pathology; Sauer, R.; Grabenbauer, G. [Erlangen-Nuernberg Univ. (Germany). Dept. of Radiation Oncology

2007-03-15

Background and Purpose: Despite the enormous therapeutic potential of modern radiotherapy, common side effects such as radiation-induced wound healing disorders remain a well-known clinical phenomenon. Topical negative pressure therapy (TNP) is a novel tool to alleviate intraoperative, percutaneous irradiation or brachytherapy. Since TNP has been shown to positively influence the perfusion of chronic, poorly vascularized wounds, the authors applied this therapeutic method to irradiated wounds and investigated the effect on tissue oxygenation in irradiated tissue in five patients. Material and Methods: With informed patients' consent, samples prior to and 4 and 8 days after continuous TNP with -125 mmHg were obtained during routine wound debridements. Granulation tissue was stained with hematoxylin-eosin, and additionally with CD31, HIF-1{alpha} (hypoxia-inducible factor-1{alpha}), and D2-40 to detect blood vessels, measure indirect signs of hypoxia, and lymph vessel distribution within the pre- and post-TNP samples. Results: In this first series of experiments, a positive influence of TNP onto tissue oxygenation in radiation-induced wounds could be demonstrated. TNP led to a significant decrease of 53% HIF-1{alpha}-positive cell nuclei. At the same time, a slight reduction of CD31-stained capillaries was seen in comparison to samples before TNP. Immunostaining with D2-40 revealed an increased number of lymphatic vessels with distended lumina and an alteration of the parallel orientation within the post-TNP samples. Conclusion: This study is, to the authors' knowledge, the first report on a novel previously not described histological marker to demonstrate the effects of TNP on HIF-1{alpha} expression as an indirect marker of tissue oxygenation in irradiated wounds, as demonstrated by a reduction of HIF-1{alpha} concentration after TNP. Since this observation may be of significant value to develop possible new strategies to treat radiation-induced tissue

Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

Directory of Open Access Journals (Sweden)

Bin Shan

Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Differential expression of largemouth bass (Micropterus salmoides) estrogen receptor isotypes alpha, beta, and gamma by estradiol.

Science.gov (United States)

Sabo-Attwood, Tara; Kroll, Kevin J; Denslow, Nancy D

2004-04-15

The expression levels of three estrogen receptor (ER) isotypes alpha, beta, and gamma were quantified in female largemouth bass (Micropterus salmoides) (LMB) liver, ovary, brain, and pituitary tissues. ER alpha and beta expression predominated in the liver, while ERs beta and gamma predominated in the other tissues. Temporally in females, ER alpha was highly up-regulated, ER gamma was slightly up-regulated, and ER beta levels remained unchanged in the liver when plasma 17-beta estradiol (E2) and vitellogenin (Vtg) levels were elevated in the spring. In ovarian tissue from these same fish, all three ERs were maximally expressed in the fall, during early oocyte development and prior to peak plasma E2 levels. When males were injected with E2, ER alpha was highly inducible, ER gamma was moderately up-regulated, and ER beta levels were not affected. None of the ER isotypes were induced by E2 in gonadal tissues. These results combined suggest that the ERs themselves are not regulated in the same manner by E2, and furthermore, do not contribute equally to the transcriptional regulation of genes involved in fish reproduction such as Vtg.

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

Science.gov (United States)

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

LENUS (Irish Health Repository)

Gupta, Sanjeev

2010-01-01

Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha\\/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats.

Science.gov (United States)

Khamphaya, Tanaporn; Chansela, Piyachat; Piyachaturawat, Pawinee; Suksamrarn, Apichart; Nathanson, Michael H; Weerachayaphorn, Jittima

2016-10-15

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases. Copyright �

Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells.

NARCIS (Netherlands)

Paleolog, E.M.; Delasalle, S.A.; Buurman, W.A.; Feldmann, M.

1994-01-01

Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion

Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

Science.gov (United States)

Proper, Steven P; Saini, Yogesh; Greenwood, Krista K; Bramble, Lori A; Downing, Nathaniel J; Harkema, Jack R; Lapres, John J

2014-02-01

Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1α contrib utes to cobalt toxicity in vitro, loss of HIF1α in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1α and HIF2α show unique tissue expression profiles, and HIF2α is known to be the predominant HIF mRNA isoform in the adult lung. Thus, if HIF2α activation by cobalt contributes to pathophysiology of HMLD, we hypothesized that loss of HIF2α in lung epithelium would provide protection from cobalt-induced inflammation. Mice with HIF2α-deficiency in Club and alveolar type II epithelial cells (ATIIs) (HIF2α(Δ/Δ)) were exposed to cobalt (60 µg/day) or saline using a subacute occupational exposure model. Bronchoalveolar lavage cellularity, cytokines, qRT-PCR, and histopathology were analyzed. Results show that loss of HIF2α leads to enhanced eosinophilic inflammation and increased goblet cell metaplasia. Additionally, control mice demonstrated a mild recovery from cobalt-induced lung injury compared with HIF2α(Δ/Δ) mice, suggesting a role for epithelial HIF2α in repair mechanisms. The expression of important cytokines, such as interleukin (IL)-5 and IL-10, displayed significant differences following cobalt exposure when HIF2α(Δ/Δ) and control mice were compared. In summary, our data suggest that although loss of HIF2α does not afford protection from cobalt-induced lung inflammation, epithelial HIF2α signaling does play an important role in modulating the inflammatory and repair response in the lung.

TNF-{alpha} mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Energy Technology Data Exchange (ETDEWEB)

Kim, Beom-Jun [Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap2-Dong, Seoul (Korea, Republic of); Bae, Sung Jin [Health Promotion Center, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap2-Dong, Seoul (Korea, Republic of); Lee, Sun-Young; Lee, Young-Sun; Baek, Ji-Eun; Park, Sook-Young [Asan Institute for Life Sciences, 388-1 Poongnap2-Dong, Seoul (Korea, Republic of); Lee, Seung Hun [Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap2-Dong, Seoul (Korea, Republic of); Koh, Jung-Min, E-mail: jmkoh@amc.seoul.kr [Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap2-Dong, Seoul (Korea, Republic of); Kim, Ghi Su [Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Poongnap2-Dong, Seoul (Korea, Republic of)

2012-07-20

Highlights: Black-Right-Pointing-Pointer Estrogen deprivation stimulates the bony sclerostin levels with reversal by estrogen. Black-Right-Pointing-Pointer TNF-{alpha} increases the activity and expression of MEF2 in UMR-106 cells. Black-Right-Pointing-Pointer TNF-{alpha} blocker prevents the stimulation of bony sclerostin expression by ovariectomy. Black-Right-Pointing-Pointer No difference in bony sclerostin expression between sham-operated and ovariectomized nude mice. -- Abstract: Although recent clinical studies have suggested a possible role for sclerostin, a secreted Wnt antagonist, in the pathogenesis of postmenopausal osteoporosis, the detailed mechanisms how estrogen deficiency regulates sclerostin expression have not been well-elucidated. Bilateral ovariectomy or a sham operation in female C57BL/6 mice and BALB/c nude mice was performed when they were seven weeks of age. The C57BL/6 mice were intraperitoneally injected with phosphate-buffered serum (PBS), 5 {mu}g/kg {beta}-estradiol five times per week for three weeks, or 10 mg/kg TNF-{alpha} blocker three times per week for three weeks. Bony sclerostin expression was assessed by immunohistochemistry staining in their femurs. The activity and expression of myocyte enhancer factors 2 (MEF2), which is essential for the transcriptional activation of sclerostin, in rat UMR-106 osteosarcoma cells were determined by luciferase reporter assay and western blot analysis, respectively. Bony sclerostin expression was stimulated by estrogen deficiency and it was reversed by estradiol supplementation. When the UMR-106 cells were treated with well-known, estrogen-regulated cytokines, only TNF-{alpha}, but not IL-1 and IL-6, increased the MEF2 activity. Consistently, TNF-{alpha} also increased the nuclear MEF2 expression. Furthermore, the TNF-{alpha} blocker prevented the stimulation of bony sclerostin expression by ovariectomy. We also found that there was no difference in sclerostin expression between ovariectomized

Divergent effects of 17-{beta}-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-{alpha}-induced neointima formation

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Nintasen, Rungrat [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom); Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University (Thailand); Riches, Kirsten; Mughal, Romana S. [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom); Viriyavejakul, Parnpen; Chaisri, Urai; Maneerat, Yaowapa [Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University (Thailand); Turner, Neil A. [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom); Porter, Karen E., E-mail: medkep@leeds.ac.uk [Division of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT (United Kingdom); Multidisciplinary Cardiovascular Research Center (MCRC), University of Leeds, Leeds LS2 9JT (United Kingdom)

2012-04-20

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} augments neointimal hyperplasia in human saphenous vein. Black-Right-Pointing-Pointer TNF-{alpha} induces detrimental effects on endothelial and smooth muscle cell function. Black-Right-Pointing-Pointer Estradiol exerts modulatory effects on TNF-induced vascular cell functions. Black-Right-Pointing-Pointer The modulatory effects of estradiol are discriminatory and cell-type specific. -- Abstract: Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-{alpha} on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-{alpha} (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-{alpha}, an effect that was abolished by co-culture with E2. TNF-{alpha} increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-{alpha} alone. SV-EC migration was significantly impaired by TNF-{alpha} (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-{alpha} increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-{alpha} potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there

Epidermal growth factor and insulin-like growth factor I upregulate the expression of the epidermal growth factor system in rat liver

DEFF Research Database (Denmark)

Bor, M V; Sørensen, B S; Vinter-Jensen, L

2000-01-01

BACKGROUND/AIM: Both epidermal growth factor and insulin-like growth factor I play a role in connection with the liver. In the present study, the possible interaction of these two growth factor systems was studied by investigating the effect of epidermal growth factor or insulin-like growth factor...... I treatment on the expression of the epidermal growth factor receptor, and its activating ligands, transforming growth factor-alpha and epidermal growth factor. METHODS: Fifty-five male rats received no treatment, human recombinant epidermal growth factor or human recombinant insulin-like growth.......8+/-1.6 fmol/mg protein epidermal growth factor and 144+/-22 fmol/mg protein transforming growth factor-alpha. Both epidermal growth factor and insulin-like growth factor I treatment increased the expression of mRNA for transforming growth factor-alpha and epidermal growth factor receptor, as well...

Drug-Induced Endoplasmic Reticulum and Oxidative Stress Responses Independently Sensitize Toward TNF alpha-Mediated Hepatotoxicity

NARCIS (Netherlands)

Fredriksson, Lisa; Wink, Steven; Herpers, Bram; Benedetti, Giulia; Hadi, Mackenzie; de Bont, Hans; Groothuis, Geny; Luijten, Mirjam; Danen, Erik; de Graauw, Marjo; Meerman, John; van de Water, Bob

Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha) to cause cell death of

Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

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Mervi Alanne-Kinnunen

Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

Astrocyte-targeted expression of interleukin-6 protects the central nervous system during neuroglial degeneration induced by 6-aminonicotinamide

DEFF Research Database (Denmark)

Penkowa, Milena; Camats, Jordi; Hadberg, Hanne

2003-01-01

). This study demonstrates that transgenic IL-6 expression significantly increases the 6-AN-induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL-6 induced significant increases in proinflammatory cytokines IL-1, IL-12, and tumor necrosis factor......-alpha as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor-beta, neurotrophin-3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP-IL6 mice relative to controls after 6-AN. Moreover, oxidative...

Expression of tumor necrosis factor-alpha and receptor I(P55in pterygium

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Bing Wu

2014-06-01

Full Text Available AIM:To observe the expression of tumor necrosis factor- alpha(TNF-αand its receptor I(P55in different pterygium and discuss the role of TNF-α and receptor I(P55in pterygium.METHODS: Immunohistochemistical staining method(PVwas adopted to detect the expression of TNF-α and receptor I in pterygium(72 eyesand para-pterygium conjunctival tissue(30 eyes. The relationship between the expression and clinical-pathological parameters was also analyzed. RESULTS: The positive rates of TNF-α were 65.3%(47/72, 26.7%(8/30in pterygium and para-pterygium conjunctival tissue. The positive expression of TNF-α had statistic difference between the two groups(χ2=12.706, Pχ2=13.875, Pχ2=6.547, P=0.011. There had no statistically significance of the expression intensity between the two groups(F=1.288, P=0.393; the positive rate in advanced pterygium group was higher than quiescent pterygium group(χ2=4.082, P=0.043. The expression intensity had no statistically significance between the two groups(F=0.489, P=0.708. The positive rate of P55 in recurrent pterygium group was higher than primary pterygium group(χ2=9.907, P=0.002. There had no statistically significance of the two group's expression intensity(F=1.175, P=0.424; the positive rate in advanced pterygium group was higher than in quiescent pterygium group(χ2=11.140, P=0.001. The expression intensity had no statistically significance between the two groups(F=0.665, P=0.621. CONCLUSION:The expression of TNF-α and P55 are changing according to the development of clinical staging and onset. The expression of TNF-α and P55 may be related to clinical classification, staging and patient's working conditions of pterygium. There has no significant difference expression intensity of TNF-α and P55 in clinical staging and onset of pterygium.

HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

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Sanjeev Gupta

2010-07-01

Full Text Available Endoplasmic reticulum (ER stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR. Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

Altered expression of hypoxia-inducible factor-1α (HIF-1α and its regulatory genes in gastric cancer tissues.

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Jihan Wang

Full Text Available Tissue hypoxia induces reprogramming of cell metabolism and may result in normal cell transformation and cancer progression. Hypoxia-inducible factor 1-alpha (HIF-1α, the key transcription factor, plays an important role in gastric cancer development and progression. This study aimed to investigate the underlying regulatory signaling pathway in gastric cancer using gastric cancer tissue specimens. The integration of gene expression profile and transcriptional regulatory element database (TRED was pursued to identify HIF-1α ↔ NFκB1 → BRCA1 → STAT3 ← STAT1 gene pathways and their regulated genes. The data showed that there were 82 differentially expressed genes that could be regulated by these five transcription factors in gastric cancer tissues and these genes formed 95 regulation modes, among which seven genes (MMP1, TIMP1, TLR2, FCGR3A, IRF1, FAS, and TFF3 were hub molecules that are regulated at least by two of these five transcription factors simultaneously and were associated with hypoxia, inflammation, and immune disorder. Real-Time PCR and western blot showed increasing of HIF-1α in mRNA and protein levels as well as TIMP1, TFF3 in mRNA levels in gastric cancer tissues. The data are the first study to demonstrate HIF-1α-regulated transcription factors and their corresponding network genes in gastric cancer. Further study with a larger sample size and more functional experiments is needed to confirm these data and then translate into clinical biomarker discovery and treatment strategy for gastric cancer.

Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity.

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Hans Rempel

2008-04-01

Full Text Available HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14(+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1, a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.We analyzed sialoadhesin expression on CD14(+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14(+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-alpha and interferon-gamma but not tumor necrosis factor-alpha. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.Increased sialoadhesin expression on CD14(+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14(+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein {alpha}-subunit

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Abdulaev, Najmoutin G. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Zhang Cheng; Dinh, Andy [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States); Ngo, Tony; Bryan, Philip N. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Brabazon, Danielle M. [Loyola College in Maryland, Department of Chemistry (United States); Marino, John P. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States)], E-mail: marino@carb.nist.gov; Ridge, Kevin D. [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States)

2005-05-15

Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein {alpha}-subunit (G{sub {alpha}}) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G{sub {alpha}} chimera ({approx}40 kDa polypeptide) has been tested. The results show that a prodomain fused G{sub {alpha}} chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G{sub {alpha}} isolated from natural sources. To assay for the functional integrity of the purified G{sub {alpha}} chimera at NMR concentrations and probe for changes in the structure and dynamics of G{sub {alpha}} that result from activation, {sup 15}N-HSQC spectra of the GDP/Mg{sup 2+} bound form of G{sub {alpha}} obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the {sup 15}N-HSQC spectra reveals a number of changes in chemical shifts of the {sup 1}HN, {sup 15}N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G{sub {alpha}} activation.

Nicotinic Receptor Alpha7 Expression during Tooth Morphogenesis Reveals Functional Pleiotropy

Science.gov (United States)

Rogers, Scott W.; Gahring, Lorise C.

2012-01-01

The expression of nicotinic acetylcholine receptor (nAChR) subtype, alpha7, was investigated in the developing teeth of mice that were modified through homologous recombination to express a bi-cistronic IRES-driven tau-enhanced green fluorescent protein (GFP); alpha7GFP) or IRES-Cre (alpha7Cre). The expression of alpha7GFP was detected first in cells of the condensing mesenchyme at embryonic (E) day E13.5 where it intensifies through E14.5. This expression ends abruptly at E15.5, but was again observed in ameloblasts of incisors at E16.5 or molar ameloblasts by E17.5–E18.5. This expression remains detectable until molar enamel deposition is completed or throughout life as in the constantly erupting mouse incisors. The expression of alpha7GFP also identifies all stages of innervation of the tooth organ. Ablation of the alpha7-cell lineage using a conditional alpha7Cre×ROSA26-LoxP(diphtheria toxin A) strategy substantially reduced the mesenchyme and this corresponded with excessive epithelium overgrowth consistent with an instructive role by these cells during ectoderm patterning. However, alpha7knock-out (KO) mice exhibited normal tooth size and shape indicating that under normal conditions alpha7 expression is dispensable to this process. The function of ameloblasts in alpha7KO mice is altered relative to controls. High resolution micro-computed tomography analysis of adult mandibular incisors revealed enamel volume of the alpha7KO was significantly reduced and the organization of enamel rods was altered relative to controls. These results demonstrate distinct and varied spatiotemporal expression of alpha7 during tooth development, and they suggest that dysfunction of this receptor would have diverse impacts upon the adult organ. PMID:22666322

Ets-2 and p53 mediate cAMP-induced MMP-2 expression, activity and trophoblast invasion

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Goldman Shlomit

2009-11-01

Full Text Available Abstract Background We have previously shown that Matrix metalloproteinase (MMP -2 is a key-enzyme in early trophoblast invasion and that Protein Kinase A (PKA increases MMP-2 expression and trophoblast invasion. The aim of this study was to examine MMP -2 regulation by PKA in invasive trophoblasts: JAR choriocarcinoma cell-line and 6-8 w first trimester trophoblasts. Methods The effect of Forskolin (PKA on MMP-2 expression was assessed by Northern Blot and RT-PCR. Possible transcription factors binding to consensus MMP-2 promoter sequences in response to Forskolin, were detected by EMSA binding assay and their expression assessed by western blot analysis. Antisense transfection of relevant transcription factors was performed and the inhibitory effect assessed on MMP-2 expression (RT-PCR, secretion (zymography and trophoblast invasiveness (transwell migration assay. Results We found that Forskolin increased MMP-2 mRNA in JAR cells within 24 hours, and induced binding to p53, Ets, C/EBP and AP-2. Transcription factors Ets-2, phospho- p53, C/EBP epsilon, C/EBP lambda and AP-2 alpha bound to their respective binding sequences in response to Forskolin and the expressions of these transcription factors were all elevated in Forskolin- treated cells. Inhibition of Ets-2 and p53 reduced MMP-2 expression, secretion and invasiveness of Forskolin treated cells. Conclusion MMP-2 is regulated by PKA through several binding sites and transcription factors including Ets-2, p53, C/EBP, C/EBP lambda and AP-2 alpha. Ets-2 and p53 mediate cAMP- induced trophoblast invasiveness, through regulation of MMP-2.

Hypoxia-inducible factor 1 alpha is a poor prognostic factor and potential therapeutic target in malignant peripheral nerve sheath tumor.

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Suguru Fukushima

Full Text Available Malignant peripheral nerve sheath tumor (MPNST is a rare soft tissue sarcoma with poor prognosis. Hypoxia-inducible factor 1 (HIF-1 plays a crucial role in the cellular response to hypoxia and regulates the expression of multiple genes involved in tumor progression in various cancers. However, the importance of the expression of HIF-1α in MPNSTs is unclear.The expression of HIF-1α was examined immunohistochemically in 82 MPNST specimens. Cell culture assays of human MPNST cells under normoxic and hypoxic conditions were used to evaluate the impact of anti-HIF-1α-specific siRNA inhibition on cell survival. A screening kit was employed to identify small molecules that inhibited HIF-1α.The nuclear expression of HIF-1α was positive in 75.6% of MPNST samples (62/82 cases. Positivity for HIF-1α was a significant poor prognostic factor both in univariate (P = 0.048 and multivariate (P ≤ 0.0001 analyses. HIF-1α knockdown abrogated MPNST cell growth, inducing apoptosis. Finally, chetomin, an inhibitor of HIF-1α, effectively inhibited the growth of MPNST cells and induced their apoptosis.Inhibition of HIF-1α signaling is a potential treatment option for MPNSTs.

IgE-mediated basophil tumour necrosis factor alpha induces matrix metalloproteinase-9 from monocytes

DEFF Research Database (Denmark)

Falkencrone, Sidsel; Poulsen, Lars K.; Bindslev-Jensen, Carsten

2013-01-01

IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have...

The role of hypoxia inducible factor-1 alpha in bypassing oncogene-induced senescence.

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Mehtap Kilic Eren

Full Text Available Oncogene induced senescence (OIS is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR, senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs. We showed here that hypoxia prevents execution of oncogene induced senescence (OIS, through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α. In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.

High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

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Jiang, Shao-Yun, E-mail: jiangshaoyun@yahoo.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China); Deng, Jia-Yin, E-mail: yazhou2991@126.com [School of Dentistry, Tianjin Medical University, 12 Qi Xiang Tai Street, Heping District, Tianjin 300070 (China)

2012-10-26

Highlights: Black-Right-Pointing-Pointer High glucose significantly induced TLR2 expression in gingival fibroblasts. Black-Right-Pointing-Pointer High glucose increased NF-{kappa}B p65 nuclear activity, IL-1{beta} and TNF-{alpha} levels. Black-Right-Pointing-Pointer PKC-{alpha}/{delta}-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-{kappa}B) p65 nuclear activity, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-l{beta} (IL-1{beta}) levels. Protein kinase C (PKC)-{alpha} and {delta} knockdown with siRNA significantly decreased TLR2 and NF-{kappa}B p65 expression (p < 0.05), whereas inhibition of PKC-{beta} had no effect on TLR2 and NF-{kappa}B p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-{kappa}B expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-{alpha} and IL-1{beta} secretion via inducing TLR2 through PKC-{alpha} and PKC-{delta} in human gingival fibroblasts.

Effect of particle size on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages.

Science.gov (United States)

Nadra, Imad; Boccaccini, Aldo R; Philippidis, Pandelis; Whelan, Linda C; McCarthy, Geraldine M; Haskard, Dorian O; Landis, R Clive

2008-01-01

Macrophages may promote a vicious cycle of inflammation and calcification in the vessel wall by ingesting neointimal calcific deposits (predominantly hydroxyapatite) and secreting tumor necrosis factor (TNF)alpha, itself a vascular calcifying agent. Here we have investigated whether particle size affects the proinflammatory potential of hydroxyapatite crystals in vitro and whether the nuclear factor (NF)-kappaB pathway plays a role in the macrophage TNFalpha response. The particle size and nano-topography of nine different crystal preparations was analyzed by X-ray diffraction, Raman spectroscopy, scanning electron microscopy and gas sorbtion analysis. Macrophage TNFalpha secretion was inversely related to hydroxyapatite particle size (P=0.011, Spearman rank correlation test) and surface pore size (P=0.014). A necessary role for the NF-kappaB pathway was demonstrated by time-dependent I kappaB alpha degradation and sensitivity to inhibitors of I kappaB alpha degradation. To test whether smaller particles were intrinsically more bioactive, their mitogenic activity on fibroblast proliferation was examined. This showed close correlation between TNFalpha secretion and crystal-induced fibroblast proliferation (P=0.007). In conclusion, the ability of hydroxyapatite crystals to stimulate macrophage TNFalpha secretion depends on NF-kappaB activation and is inversely related to particle and pore size, with crystals of 1-2 microm diameter and pore size of 10-50 A the most bioactive. Microscopic calcific deposits in early stages of atherosclerosis may therefore pose a greater inflammatory risk to the plaque than macroscopically or radiologically visible deposits in more advanced lesions.

Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma.

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Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

2016-07-21

Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

Cellular localization of transforming growth factor-alpha mRNA in rat forebrain.

Science.gov (United States)

Seroogy, K B; Lundgren, K H; Lee, D C; Guthrie, K M; Gall, C M

1993-05-01

The cellular localization of transforming growth factor-alpha (TGF alpha) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a 35S-labeled cRNA probe. TGF alpha cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocampal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGF alpha cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGF alpha in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGF alpha in the development of several forebrain systems. Our results demonstrating the prominent and wide-spread expression of TGF alpha mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGF alpha is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.

Epigenetic control of hypoxia inducible factor-1α-dependent expression of placental growth factor in hypoxic conditions.

Science.gov (United States)

Tudisco, Laura; Della Ragione, Floriana; Tarallo, Valeria; Apicella, Ivana; D'Esposito, Maurizio; Matarazzo, Maria Rosaria; De Falco, Sandro

2014-04-01

Hypoxia plays a crucial role in the angiogenic switch, modulating a large set of genes mainly through the activation of hypoxia-inducible factor (HIF) transcriptional complex. Endothelial cells play a central role in new vessels formation and express placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, mainly involved in pathological angiogenesis. Despite several observations suggest a hypoxia-mediated positive modulation of PlGF, the molecular mechanism governing this regulation has not been fully elucidated. We decided to investigate if epigenetic modifications are involved in hypoxia-induced PlGF expression. We report that PlGF expression was induced in cultured human and mouse endothelial cells exposed to hypoxia (1% O 2), although DNA methylation at the Plgf CpG-island remains unchanged. Remarkably, robust hyperacetylation of histones H3 and H4 was observed in the second intron of Plgf, where hypoxia responsive elements (HREs), never described before, are located. HIF-1α, but not HIF-2α, binds to identified HREs. Noteworthy, only HIF-1α silencing fully inhibited PlGF upregulation. These results formally demonstrate a direct involvement of HIF-1α in the upregulation of PlGF expression in hypoxia through chromatin remodeling of HREs sites. Therefore, PlGF may be considered one of the putative targets of anti-HIF therapeutic applications.

Obesity-induced endoplasmic reticulum stress suppresses nuclear factor-Y expression.

Science.gov (United States)

Liu, Yulan; Zhang, Yuwei; Zhang, Yanjie; Zhang, Jinlong; Liu, Yin; Feng, Peiqun; Su, Zhiguang

2017-02-01

Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor composed of three subunits, NF-YA, NF-YB, and NF-YC. NF-Y plays crucial roles in pre-adipocyte maintenance and/or commitment to adipogenesis. NF-YA dysfunction in adipocyte resulted in an age-dependent progressive loss of adipose tissue associated with metabolic complications. Endoplasmic reticulum (ER) stress has emerged as an important mediator in the pathogenesis of obesity. However, it is not known if NF-YA is involved in the ER stress-mediated pathogenesis of obesity. We first examined the effects of ER stress on the NF-YA expression in cultured 3T3-L1 adipocytes; then in ob/ob genetic obesity mice, we tested the effect of chemical chaperones alleviating ER stress on the expression levels of NF-YA. Subsequently, we inhibited the new mRNA synthesis using actinomycin D in 3T3-L1 cells to explore the mechanism modulating NF-YA expression. Finally, we evaluated the involvement of PPARg in the regulation of NF-YA expression by ER stress. We demonstrated that both obesity- and chemical chaperone -induced ER stress suppressed NF-YA expression and alleviation of ER stress by chemical chaperone could recover NF-YA expression in ob/ob mice. Moreover, we showed that ER stress suppressed NF-YA mRNA transcription through the involvement of peroxisome proliferator-activated receptor gamma (PPARg). Activation of PPARg ameliorates the ER stress-induced NF-YA suppression. Our findings may point to a possible role of NF-YA in stress conditions that occur in chronic obesity, ER stress might be involved in the pathogenesis of obesity through NF-YA depletion.

Involvement of growth factors and their receptors in radon-induced rat lung tumors

International Nuclear Information System (INIS)

Leung, F.C.; Dagle, G.E.; Cross, F.T.

1992-01-01

In this paper we examine the role of growth factors (GF) and their receptors (GFR) in radon-induced rat lung tumors. Inhalation exposure of radon and its daughters induced lung tumors in rats, but the molecule/cellular mechanisms are not known. Recent evidence suggests that GF/GFR play a critical role in the growth and development of lung cancer in humans and animals. We have developed immunocytochemical methods for identifying sites of production and action of GF/GFR at the cellular level; for example, the avidin-biotin horseradish peroxidase technique. In radon-induced rat epidermoid carcinomas, epidermal growth factor (EGF), EGF-receptors (EGF-R), transforming growth factor alpha (TGF-α), and bombesin were found to be abnormally expressed. These abnormal expressions, mainly associated with epidermoid carcinomas of the lung, were not found in any other lung tumor types. Our data suggest that EGF, EGF-R, TGF-α, and bombesin are involved in radon oncogenesis in rat lungs, especially in epidermoid carcinomas, possibly through the autocrine/paracrine pathway

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...

Arsenite and its metabolites, MMAIII and DMAIII, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

International Nuclear Information System (INIS)

Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.; Ramirez, P.; Vega, L.; Elizondo, G.

2009-01-01

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA III ) and dimethylarsinous acid (DMA III ) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA III induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA III increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA III induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

Science.gov (United States)

Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

1999-01-01

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Adverse cutaneous reactions induced by TNF-alpha antagonist therapy.

Science.gov (United States)

Borrás-Blasco, Joaquín; Navarro-Ruiz, Andrés; Borrás, Consuelo; Casterá, Elvira

2009-11-01

To review adverse cutaneous drug reactions induced by tumor necrosis factor alpha (TNF-alpha) antagonist therapy. A literature search was performed using PubMed (1996-March 2009), EMBASE, and selected MEDLINE Ovid bibliography searches. All language clinical trial data, case reports, letters, and review articles identified from the data sources were used. Since the introduction of TNF-alpha antagonist, the incidence of adverse cutaneous drug reactions has increased significantly. A wide range of different skin lesions might occur during TNF-alpha antagonist treatment. New onset or exacerbation of psoriasis has been reported in patients treated with TNF-alpha antagonists for a variety of rheumatologic conditions. TNF-alpha antagonist therapy has been associated with a lupus-like syndrome; most of these case reports occurred in patients receiving either etanercept or infliximab. Serious skin reactions such as erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis have been reported rarely with the use of TNF-alpha antagonists. As the use of TNF-alpha antagonists continues to increase, the diagnosis and management of cutaneous side effects will become an increasingly important challenge. In patients receiving TNF-alpha antagonist treatment, skin disease should be considered, and clinicians need to be aware of the adverse reactions of these drugs.

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

Science.gov (United States)

Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

1991-04-01

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Yang, Bin [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Li, Wei [Department of Gerontology, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Zheng, Qichang [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Qin, Tao [Department of Hepatobiliary Pancreatic Surgery, People' s Hospital of Zhengzhou University, School of Medicine, Zhengzhou University, Zhengzhou 450003 (China); Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China); Liu, Sanguang, E-mail: sanguang1998@sina.com [Department of Hepatobiliary Surgery, The Second Hospital, Hebei Medical University, Shijiazhuang 050000 (China); Song, Zifang, E-mail: zsong@hust.edu.cn [Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430022 (China)

2015-07-17

Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation.

Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

International Nuclear Information System (INIS)

Yang, Bin; Li, Wei; Zheng, Qichang; Qin, Tao; Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen; Liu, Sanguang; Song, Zifang

2015-01-01

Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation

Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression.

Science.gov (United States)

Layé, S; Gheusi, G; Cremona, S; Combe, C; Kelley, K; Dantzer, R; Parnet, P

2000-07-01

The present study was designed to determine the role of endogenous brain interleukin (IL)-1 in the anorexic response to lipopolysaccharide (LPS). Intraperitoneal administration of LPS (5-10 microgram/mouse) induced a dramatic, but transient, decrease in food intake, associated with an enhanced expression of proinflammatory cytokine mRNA (IL-1beta, IL-6, and tumor necrosis factor-alpha) in the hypothalamus. This dose of LPS also increased plasma levels of IL-1beta. Intracerebroventricular pretreatment with IL-1 receptor antagonist (4 microgram/mouse) attenuated LPS-induced depression of food intake and totally blocked the LPS-induced enhanced expression of proinflammatory cytokine mRNA measured in the hypothalamus 1 h after treatment. In contrast, LPS-induced increases in plasma levels of IL-1beta were not altered. These findings indicate that endogenous brain IL-1 plays a pivotal role in the development of the hypothalamic cytokine response to a systemic inflammatory stimulus.

Phaleria macrocarpa reduces glomerular growth factor expression in alloxan-induced diabetic rats

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Evy Sulistyoningrum

2013-08-01

Full Text Available Background Diabetic nephropathy (DN is the most serious complication of diabetes, causing end-stage renal disease throughout the world. Recent studies have reported a direct role of vascular endothelial growth factor (VEGF and transforming growth factor-â (TGF-â in DN pathogenesis. VEGF and TGF-â are expressed early in glomeruli in response to hyperglycemia. Active substances of Phaleria macrocarpa (PM pericarp are known to have nephroprotective effects. This study aimed to evaluate the effects of Phaleria macrocarpa (Scheff. Boerl pericarp extract on VEGF and TGF-â expression in alloxan-induced diabetic rats. Methods An experimental study was conducted on twenty five male albino (Sprague Dawley rats divided into five groups (of five each: normal control; diabetic; diabetic + metformin 100 mg/kgBW; diabetic + methanolic PM extract 250 mg/kgBW; and diabetic + aqueous PM extract 250 mg/kgBW. Diabetes was induced by alloxan monohydrate 150 mg/BW intraperitoneally. Treatment was given for 3 weeks. VEGF and TGF-â expression analysis was performed by means of immunohistochemical technique. Differences between groups were assessed by one-way ANOVA. Results VEGF expression in the PM extract group was significantly lower than that in the diabetic group and even metformin group (p<0.01. TGF-â expression in methanolic PM extract group was significantly lower than in diabetic and metformin group (p<0.01, but aqueous PM extract group only showed significancy when compared with diabetic group (p< 0.01. Conclusions Phaleria macrocarpa pericarp extract reduces glomerular expression of TGF-â and VEGF in alloxan-induced diabetic rats.

Increased Expression of the Na,K-ATPase alpha4 Isoform Enhances Sperm Motility in Transgenic Mice1

Science.gov (United States)

Jimenez, Tamara; Sanchez, Gladis; McDermott, Jeffrey P.; Nguyen, Anh-Nguyet; Kumar, T. Rajendra; Blanco, Gustavo

2010-01-01

The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions. PMID:20826726

Lactobacillus casei secreting alpha-MSH induces the therapeutic effect on DSS-induced acute colitis in Balb/c Mice.

Science.gov (United States)

Yoon, Sun-Woo; Lee, Chul-Ho; Kim, Jeong-Yoon; Kim, Jie-Youn; Sung, Moon-Hee; Poo, Haryoung

2008-12-01

The neuropeptide alpha-melanocyte-stimulating hormone (alpha- MSH) has anti-inflammatory property by downregulating the expressions of proinflammatory cytokines. Because alpha-MSH elicits the anti-inflammatory effect in various inflammatory disease models, we examined the therapeutic effect of oral administration of recombinant Lactobacillus casei, which secretes alpha-MSH (L. casei-alpha-MSH), on dextran sulfate sodium (DSS)-induced colitis in Balb/c mice. Thus, we constructed the alpha-MSH-secreting Lactobacillus casei by the basic plasmid, pLUAT-ss, which was composed of a PldhUTLS promoter and alpha-amylase signal sequence from Streptococcus bovis strain. Acute colitis was induced by oral administration of 5% DSS in drinking water for 7 days. To investigate the effect of L. casei-alpha-MSH on the colitis, L. casei or L. casei-alpha-MSH was orally administered for 7 days and their effects on body weight, mortality rate, cytokine production, and tissue myeloperoxidase (MPO) activity were observed. Administration of L. casei-alpha-MSH reduced the symptom of acute colitis as assessed by body weight loss (DSS alone: 14.45+/-0. 2 g; L. casei-alpha- MSH: 18.2+/-0.12 g), colitis score (DSS alone: 3.6+/-0.4; L. casei-alpha-MSH: 1.4+/-0.6), MPO activity (DSS alone: 42.7+/-4.5 U/g; L. casei-alpha-MSH: 10.25+/-0.5 U/g), survival rate, and histological damage compared with the DSS alone mice. L. casei-alpha-MSH-administered entire colon showed reduced in vitro production of proinflammatory cytokines and NF-kappaB activation. The alpha-MSH-secreting recombinant L. casei showed significant anti-inflammatory effects in the murine model of acute colitis and suggests a potential therapeutic role for this agent in clinical inflammatory bowel diseases.

The MAPKERK-1,2 pathway integrates distinct and antagonistic signals from TGF alpha and FGF7 in morphogenesis of mouse mammary epithelium

Energy Technology Data Exchange (ETDEWEB)

Fata, Jimmie E; Mori, Hidetoshi; Ewald, Andrew J; Zhang, Hui; Yao, Evelyn; Werb, Zena; Bissell, Mina J

2006-10-03

Transforming growth factor-{alpha} (TGF{alpha}) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK{sup ERK1,2}); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK{sup ERK1,2} for 1 h, induced by TGF{alpha}, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPK{sup ERK1,2}, induced by FGF7, led to growth without branching. Unlike TGF{alpha}, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGF{alpha} indicated that the FGF7-induced MAPK{sup ERK1,2} signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGF{alpha}-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK{sup ERK1,2} activation.

Tumour Necrosis Factor-alpha and Nuclear Factor-kappa B Gene Variants in Sepsis.

Science.gov (United States)

Acar, Leyla; Atalan, Nazan; Karagedik, E Hande; Ergen, Arzu

2018-01-20

The humoral system is activated and various cytokines are released due to infections in tissues and traumatic damage. Nuclear factor-kappa B dimers are encoded by nuclear factor-kappa B genes and regulate transcription of several crucial proteins of inflammation such as tumour necrosis factor-alpha. To investigate the possible effect of polymorphisms on tumour necrosis factor-alpha serum levels with clinical and prognostic parameters of sepsis by determining the nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A) gene polymorphisms and tumour necrosis factor-alpha serum levels. Case-control study. Seventy-two patients with sepsis and 104 healthy controls were included in the study. In order to determine the polymorphisms of nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A), polymerase chain reaction-restriction fragment length polymorphism analysis was performed and serum tumour necrosis factor-alpha levels were determined using an enzyme-linked immunosorbent assay. We observed no significant differences in tumour necrosis factor-alpha serum levels between the study groups. In the patient group, an increase in the tumour necrosis factor-alpha serum levels in patients carrying the tumour necrosis factor-alpha (-308 G/A) A allele compared to those without the A allele was found to be statistically significant. Additionally, an increase in the tumour necrosis factor-alpha serum levels in patients carrying tumour necrosis factor-alpha (-308 G/A) AA genotype compared with patients carrying the AG or GG genotypes was statistically significant. No significant differences were found in these 2 polymorphisms between the patient and control groups (p>0.05). Our results showed the AA genotype and the A allele of the tumour necrosis factor-alpha (-308 G/A) polymorphism may be used as a predictor of elevated tumour necrosis factor-alpha levels in patients with sepsis.

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line.

Science.gov (United States)

Baker, Olga J; Camden, Jean M; Redman, Robert S; Jones, Jonathan E; Seye, Cheikh I; Erb, Laurie; Weisman, Gary A

2008-11-01

Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

alpha-MSH and its receptors in regulation of tumor necrosis factor-alpha production by human monocyte/macrophages.

Science.gov (United States)

Taherzadeh, S; Sharma, S; Chhajlani, V; Gantz, I; Rajora, N; Demitri, M T; Kelly, L; Zhao, H; Ichiyama, T; Catania, A; Lipton, J M

1999-05-01

The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of alpha-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-alpha was inhibited in relation to alpha-MSH concentration. Similar inhibitory effects on TNF-alpha were observed with ACTH peptides that contain the alpha-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-alpha concentration; the antibody also markedly reduced the inhibitory influence of alpha-MSH on TNF-alpha in macrophages treated with LPS. These results in cells known to produce alpha-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury

Directory of Open Access Journals (Sweden)

Mushfiquddin Khan

2017-01-01

Full Text Available Mild traumatic brain injury (TBI, also called concussion, initiates sequelae leading to motor deficits, cognitive impairments and subtly compromised neurobehaviors. While the acute phase of TBI is associated with neuroinflammation and nitroxidative burst, the chronic phase shows a lack of stimulation of the neurorepair process and regeneration. The deficiency of nitric oxide (NO, the consequent disturbed NO metabolome, and imbalanced mechanisms of S-nitrosylation are implicated in blocking the mechanisms of neurorepair processes and functional recovery in the both phases. Hypoxia inducible factor-1 alpha (HIF-1α, a master regulator of hypoxia/ischemia, stimulates the process of neurorepair and thus aids in functional recovery after brain trauma. The activity of HIF-1α is regulated by NO via the mechanism of S-nitrosylation of HIF-1α. S-nitrosylation is dynamically regulated by NO metabolites such as S-nitrosoglutathione (GSNO and peroxynitrite. GSNO stabilizes, and peroxynitrite destabilizes HIF-1α. Exogenously administered GSNO was found not only to stabilize HIF-1α and to induce HIF-1α-dependent genes but also to stimulate the regeneration process and to aid in functional recovery in TBI animals.

Expression of Estrogen Receptor Alpha in Malignant Melanoma

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Parvin Rajabi

2017-01-01

Full Text Available Background: Features of malignant melanoma (MM vary in the different geographic regions of the world. This may be attributable to environmental, ethnic, and genetic factors. The aim of this study was to determine the expression of estrogen receptor alpha (ER-α in MM in Isfahan, Iran. Materials and Methods: This study was planned as a descriptive, analytical, cross-sectional investigation. During this study, paraffin-embedded tissue blocks of patients with a histopathologic diagnosis of MM was studied for ER-α using immunohistochemistry (IHC. Results: In this study, 38 patients (female/male; 20/18 with a definite diagnosis of malignant cutaneous melanoma and mean age of 52.4 ± 11.2 years were investigated. Using envision IHC staining, there were not any cases with ER-α expression. Conclusion: In confirmation to the most previous studies, expression of ER-α was negative in MM. It is recommended to investigate the expression of estrogen receptor beta and other markers in MM.

Anti-obesogenic effects of WY14643 (PPAR-alpha agonist): Hepatic mitochondrial enhancement and suppressed lipogenic pathway in diet-induced obese mice.

Science.gov (United States)

Veiga, Flavia Maria Silva; Graus-Nunes, Francielle; Rachid, Tamiris Lima; Barreto, Aline Barcellos; Mandarim-de-Lacerda, Carlos Alberto; Souza-Mello, Vanessa

2017-09-01

Non-alcoholic fatty liver disease (NAFLD) presents with growing prevalence worldwide, though its pharmacological treatment remains to be established. This study aimed to evaluate the effects of a PPAR-alpha agonist on liver tissue structure, ultrastructure, and metabolism, focusing on gene and protein expression of de novo lipogenesis and gluconeogenesis pathways, in diet-induced obese mice. Male C57BL/6 mice (three months old) received a control diet (C, 10% of lipids, n = 10) or a high-fat diet (HFD, 50% of lipids, n = 10) for ten weeks. These groups were subdivided to receive the treatment (n = 5 per group): C, C-alpha (PPAR-alpha agonist, 2.5 mg/kg/day mixed in the control diet), HFD and HFD-alpha group (PPAR-alpha agonist, 2.5 mg/kg/day mixed in the HFD). The effects were compared with biometrical, biochemical, molecular biology and transmission electron microscopy (TEM) analyses. HFD showed greater body mass (BM) and insulinemia than C, both of which were tackled by the treatment in the HFD-alpha group. Increased hepatic protein expression of glucose-6-phosphatase, CHREBP and gene expression of PEPCK in HFD points to increased gluconeogenesis. Treatment rescued these parameters in the HFD-alpha group, eliciting a reduced hepatic glucose output, confirmed by the smaller GLUT2 expression in HFD-alpha than in HFD. Conversely, favored de novo lipogenesis was found in the HFD group by the increased expression of PPAR-gamma, and its target gene SREBP-1, FAS and GK when compared to C. The treatment yielded a marked reduction in the expression of all lipogenic factors. TEM analyses showed a greater numerical density of mitochondria per area of tissue in treated than in untreated groups, suggesting an increase in beta-oxidation and the consequent NAFLD control. PPAR-alpha activation reduced BM and treated insulin resistance (IR) and NAFLD by increasing the number of mitochondria and reducing hepatic gluconeogenesis and de novo lipogenesis protein and gene

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor alpha combination treatment of EL4-lymphoma-bearing C57BL/6 mice.

Science.gov (United States)

Krawczyk, C M; Verstovsek, S; Ujházy, P; Maccubbin, D; Ehrke, M J

1995-06-01

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor alpha (TNF alpha, 1000 units injection) on days 13, 16, 18, 21, and 23, resulted in about 60% long-term survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNF alpha was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNF alpha. These results indicate that treatment with a single moderate dose of CY in combination with TNF alpha is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus.

The temporal expression pattern of alpha-synuclein modulates olfactory neurogenesis in transgenic mice.

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Sebastian R Schreglmann

Full Text Available Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS under the murine Thy1 (mThy1 promoter, a model known to have a particularly high tg expression associated with impaired olfaction.Survival of newly generated neurons (NeuN-positive in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.

The Effect of Bladder Outlet Obstruction on alpha(1)- and beta-Adrenoceptor Expression and Function

NARCIS (Netherlands)

Barendrecht, Maurits M.; Frazier, Elfaridah P.; Vrydag, Wim; Alewijnse, Astrid E.; Peters, Stephan L. M.; Michel, Martin C.

2009-01-01

Aims: To explore possible changes in expression and/or function of alpha(1)- and beta-adrenoceptor subtypes as a cause for bladder dysfunction in a rat model of bladder outlet obstruction (BOO). Methods: BOO was induced in rats by partial urethral ligature. Contraction and relaxation experiments

High Nuclear Hypoxia-Inducible Factor 1 Alpha Expression Is a Predictor of Distant Recurrence in Patients With Resected Pancreatic Adenocarcinoma

International Nuclear Information System (INIS)

Colbert, Lauren E.; Fisher, Sarah B.; Balci, Serdar; Saka, Burcu; Chen, Zhengjia; Kim, Sungjin; El-Rayes, Bassel F.; Adsay, N. Volkan; Maithel, Shishir K.; Landry, Jerome C.

2015-01-01

Purpose: To evaluate nuclear hypoxia-inducible factor 1α (HIF-1α) expression as a prognostic factor for distant recurrence (DR) and local recurrence (LR) after pancreatic adenocarcinoma resection. Methods and Materials: Tissue specimens were collected from 98 patients with pancreatic adenocarcinoma who underwent resection without neoadjuvant therapy between January 2000 and December 2011. Local recurrence was defined as radiographic or pathologic evidence of progressive disease in the pancreas, pancreatic bed, or associated nodal regions. Distant recurrence was defined as radiographically or pathologically confirmed recurrent disease in other sites. Immunohistochemical staining was performed and scored by an independent pathologist blinded to patient outcomes. High HIF-1α overall expression score was defined as high percentage and intensity staining and thus score >1.33. Univariate analysis was performed for HIF-1α score with LR alone and with DR. Multivariate logistic regression was used to determine predictors of LR and DR. Results: Median follow-up time for all patients was 16.3 months. Eight patients (8%) demonstrated isolated LR, 26 patients (26.5%) had isolated DR, and 13 patients had both LR and DR. Fifty-three patients (54%) had high HIF-1α expression, and 45 patients (46%) had low HIF-1α expression. High HIF-1α expression was significantly associated with DR (P=.03), and low HIF-1α expression was significantly associated with isolated LR (P=.03). On multivariate logistic regression analysis, high HIF-1α was the only significant predictor of DR (odds ratio 2.46 [95% confidence interval 1.06-5.72]; P=.03). In patients with a known recurrence, an HIF-1α score ≥2.5 demonstrated a specificity of 100% for DR. Conclusions: High HIF-1α expression is a significant predictor of distant failure versus isolated local failure in patients undergoing resection of pancreatic adenocarcinoma. Expression of HIF-1α may have utility in determining candidates for

Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

International Nuclear Information System (INIS)

Wada, Hiroshi; Tanemura, Masahiro; Umeshita, Koji; Doki, Yuichiro; Mori, Masaki; Nagano, Hiroaki; Yamamoto, Hirofumi; Noda, Takehiro; Murakami, Masahiro; Kobayashi, Shogo; Marubashi, Shigeru; Eguchi, Hidetoshi; Takeda, Yutaka

2009-01-01

The combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells. The direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined. IFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells. These results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC

Arsenite enhances tumor necrosis factor-α-induced expression of vascular cell adhesion molecule-1

International Nuclear Information System (INIS)

Tsou, T.-C.; Yeh, Szu Ching; Tsai, E.-M.; Tsai, F.-Y.; Chao, H.-R.; Chang, Louis W.

2005-01-01

Epidemiological studies demonstrated a high association of vascular diseases with arsenite exposure. We hypothesize that arsenite potentiates the effect of proinflammatory cytokines on vascular endothelial cells, and hence contributes to atherosclerosis. In this study, we investigated the effect of arsenite and its induction of glutathione (GSH) on vascular cell adhesion molecule-1 (VCAM-1) protein expression in human umbilical vein endothelial cells (HUVECs) in response to tumor necrosis factor-α (TNF-α), a typical proinflammatory cytokine. Our study demonstrated that arsenite pretreatment potentiated the TNF-α-induced VCAM-1 expression with up-regulations of both activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). To elucidate the role of GSH in regulation of AP-1, NF-κB, and VCAM-1 expression, we employed L-buthionine (S,R)-sulfoximine (BSO), a specific γ-glutamylcysteine synthetase (γ-GCS) inhibitor, to block intracellular GSH synthesis. Our investigation revealed that, by depleting GSH, arsenite attenuated the TNF-α-induced VCAM-1 expression as well as a potentiation of AP-1 and an attenuation of NF-κB activations by TNF-α. Moreover, we found that depletion of GSH would also attenuate the TNF-α-induced VCAM-1 expression with a down-regulation of the TNF-α-induced NF-κB activation and without significant effect on AP-1. On the other hand, the TNF-α-induced VCAM-1 expression could be completely abolished by inhibition of AP-1 or NF-κB activity, suggesting that activation of both AP-1 and NF-κB was necessary for VCAM-1 expression. In summary, we demonstrate that arsenite enhances the TNF-α-induced VCAM-1 expression in HUVECs via regulation of AP-1 and NF-κB activities in a GSH-sensitive manner. Our present study suggested a potential mechanism for arsenite in the induction of vascular inflammation and vascular diseases via modulating the actions of proinflammatory cytokines

Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

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Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L

2016-10-04

Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

Increased expression of hepatocyte nuclear factor 4 alpha transcribed by promoter 2 indicates a poor prognosis in hepatocellular carcinoma

OpenAIRE

Cai, Shao-hang; Lu, Shi-xun; Liu, Li-li; Zhang, Chris Zhiyi; Yun, Jing-ping

2017-01-01

Background: Hepatocyte nuclear factor 4 alpha (HNF4α) plays an important role in tumourigenesis. There is growing evidence indicating that HNF4α transcribed by promoter 1 (P1-HNF4α) is expressed at relatively low levels in HCC and its presence predicts a favourable outcome for hepatocellular carcinoma (HCC) patients. However, the role of HNF4α transcribed by promoter 2 (P2-HNF4α) in HCC remains unclear. Methods: A total of 615 HCC specimens were obtained to construct tissue microarrays and pe...

Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

LENUS (Irish Health Repository)

Greene, C M

2010-05-01

alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

International Nuclear Information System (INIS)

Miettinen, Johanna A.; Pietilae, Mika; Salonen, Riikka J.; Ohlmeier, Steffen; Ylitalo, Kari; Huikuri, Heikki V.; Lehenkari, Petri

2011-01-01

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.

The contraction induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species

DEFF Research Database (Denmark)

Silveira, Leonardo R.; Pilegaard, Henriette; Kusuhara, Keiko

2006-01-01

We evaluated the role of reactive oxygen species (ROS) for the contraction induced increase in expression of PGC-1alpha, HKII and UCP3 mRNA. Rat skeletal muscle cells were subjected to acute or repeated electrostimulation in the presence and absence of antioxidants. Contraction of muscle cells lead...... to an increased H2O2 formation, as measured by oxidation of H2HFF. Acute contraction of the muscle cells lead to a transient increase in PGC-1alpha and UCP3 mRNA by 172 and 65%, respectively (pantioxidants. Repeated contraction sessions induced...... a sustained elevation in PGC-1alpha and UCP3 mRNA and a transient increase in HKII (pantioxidant cocktail or with GPX+GSH. Incubation of cells for 10 days with ROS produced by xanthine oxidase/xanthine increased the level of PGC-1...

Tumor necrosis factor-alpha and its receptors in epithelial ovarian cancer.

Directory of Open Access Journals (Sweden)

Jacek Nikliński

2010-05-01

Full Text Available The aim of the present study was to characterize the expression pattern of tumor necrosis factor (TNF-alpha and its receptors (TNF-Rs in the epithelial ovarian cancer (EOC and compare these results with the outcome of 126 patients. Presence of TNF-alpha, TNFR-1 and TNFR-2 were studied by Western blotting and immunohistochemistry. The proportion of samples positive for TNF-alpha and TNF-R2 was higher in epithelial ovarian cancer patients than in benign ovarian diseases (p<0.001 and p=0.016, respectively. Immunostaining intensity of TNF-R2 were correlated with tumor stage (p<0.001 and with reduced mean survival time (MST (p=0.002. The results of the present study suggested that tissue expression of TNF-R2 in epithelial ovarian cancer was correlated with the highest risk of cancer progression. Thus, the clinical value of activated TNF system in epithelial ovarian cancer needs to be further investigated.

Interleukin-4 and 13 induce the expression and release of monocyte chemoattractant protein 1, interleukin-6 and stem cell factor from human detrusor smooth muscle cells: synergy with interleukin-1beta and tumor necrosis factor-alpha

DEFF Research Database (Denmark)

Bouchelouche, Kirsten; Andresen, Lars; Alvarez, Susana

2006-01-01

Interstitial cystitis is characterized by an increased number of activated MCs in the detrusor muscle. However, to our knowledge the factors that influence the anatomical relationship between MCs and HDSMCs are unknown. MCP-1, IL-6 and SCF have a critical role in the regulation of MC development,......, signaling and function. We investigated whether HDSMCs are capable of expressing and releasing MCP-1, IL-6 and SCF in response to IL-4, IL-13, IL-1beta and tumor necrosis factor-alpha.......Interstitial cystitis is characterized by an increased number of activated MCs in the detrusor muscle. However, to our knowledge the factors that influence the anatomical relationship between MCs and HDSMCs are unknown. MCP-1, IL-6 and SCF have a critical role in the regulation of MC development...

Compensatory increase in alpha 1-globin gene expression in individuals heterozygous for the alpha-thalassemia-2 deletion.

OpenAIRE

Liebhaber, S A; Cash, F E; Main, D M

1985-01-01

alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expressi...

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

Science.gov (United States)

Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

2014-10-17

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

Science.gov (United States)

Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

2002-08-01

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

The serum concentration of tumor necrosis factor alpha is not an index of growth-hormone- or obesity-induced insulin resistance.

Science.gov (United States)

Pincelli, A I; Brunani, A; Scacchi, M; Dubini, A; Borsotti, R; Tibaldi, A; Pasqualinotto, L; Maestri, E; Cavagnini, F

2001-01-01

The tumor necrosis factor alpha (TNF-alpha) might play a central role in insulin resistance, a frequent correlate of obesity likely contributing to some obesity-associated complications. Adult growth hormone (GH) deficiency syndrome (GHDA) shares with obesity excessive fat mass, hyperlipidemia, increased cardiovascular risk, and insulin resistance. On the other hand, GH has been shown to induce transient deterioration of glucose metabolism and insulin resistance when administered in normal humans and in GHDA patients. No information is presently available on the relationship between serum TNF-alpha levels and insulin sensitivity in GHDA. We compared the serum TNF-alpha levels found in 10 GHDA patients before and after a 6-month recombinant human GH therapy (Genotropin), in an insulin resistance prone population of 16 obese (OB) patients and in 38 normal-weight healthy blood donors (controls). The insulin sensitivity was assessed by a euglycemic-hyperinsulinemic glucose clamp in all the GHDA patients and in 10 OB and in 6 control subjects. The serum TNF-alpha levels were not significantly different in OB patients (42.2 +/- 12.81 pg/ml), in GHDA patients at baseline (71.3 +/- 23.97 pg/ml), and in controls (55.3 +/- 14.28 pg/ml). A slight decrease of TNF-alpha values was noted in GHDA patients after 6 months of recombinant human GH treatment (44.5 +/- 20.19 pg/ml; NS vs. baseline). The insulin sensitivity (M) was significantly reduced in OB patients (2.4 +/- 0.30 mg/kg/min) as compared with control subjects (7.5 +/- 0.39 mg/kg/min) and in GHDA patients both at baseline (6.6 +/- 0.6 mg/kg/min) and after recombinant human GH therapy (5.6 +/- 0.7 mg/kg/min). The insulin sensitivity in the GHDA patients, similar to that of controls at baseline, worsened after recombinant human GH treatment (p < 0.05 vs. baseline; p = 0.05 vs. controls). Linear regression analysis showed no correlation between TNF-alpha and M values (see text) in all patient groups. These data indicate

Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

International Nuclear Information System (INIS)

Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

2013-01-01

Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis

Inducible alpha-synuclein expression affects human Neural Stem Cell behavior.

Science.gov (United States)

Zasso, Jacopo; Mastad, Ahmed; Cutarelli, Alessandro; Conti, Luciano

2018-04-19

Converging evidence suggest that levels of alpha-Synuclein (aSyn) expression play a critical role in Parkinson's disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular systems is a crucial step. Here we present a novel human Tet-on hNSC cell line, in which aSyn timing and level of expression can be tightly experimentally tuned. Induction of aSyn in self-renewing hNSCs leads to progressive formation of aSyn aggregates and impairs their proliferation and cell survival. Furthermore, aSyn induction during the neuronal differentiation process results in reduced neuronal differentiation and increased number astrocytes and undifferentiated cells in culture. Finally, acute aSyn induction in hNSC-derived dopaminergic neuronal cultures results in cell toxicity. This novel conditional in vitro cell model system may be a valuable tool for dissecting of aSyn pathogenic effects in hNSCs and neurons and in developing new potential therapeutic strategies.

Partial Oxygen Pressure Affects the Expression of Prognostic Biomarkers HIF-1 Alpha, Ki67, and CK20 in the Microenvironment of Colorectal Cancer Tissue.

Science.gov (United States)

Zhang, Lirong; Hu, Yu; Xi, Ning; Song, Jie; Huang, Wenjing; Song, Shanshan; Liu, Yiting; Liu, Xianying; Xie, Yingjun

2016-01-01

Hypoxia is prognostically important in colorectal cancer (CRC) therapy. Partial oxygen pressure (pO 2 ) is an important parameter of hypoxia. The correlation between pO 2 levels and expression levels of prognostic biomarkers was measured in CRC tissues. Human CRC tissues were collected and pO 2 levels were measured by OxyLite. Three methods for tissue fixation were compared, including formalin, Finefix, and Finefix-plus-microwave. Immunohistochemistry (IHC) staining was conducted by using the avidin-biotin complex technique for detecting the antibodies to hypoxia inducible factor-1 (HIF-1) alpha, cytokeratin 20 (CK20), and cell proliferation factor Ki67. The levels of pO 2 were negatively associated with the size of CRC tissues. Finefix-plus-microwave fixation has the potential to replace formalin. Additionally, microwave treatment improved Finefix performance in tissue fixation and protein preservation. The percentage of positive cells and gray values of HIF-1 alpha, CK20, and Ki67 were associated with CRC development ( P < 0.05). The levels of pO 2 were positively related with the gray values of Ki67 and negatively related with the values of HIF-1 alpha and CK20 ( P < 0.05). Thus, the levels of microenvironmental pO 2 affect the expression of predictive biomarkers HIF-1 alpha, CK20, and Ki67 in the development of CRC tissues.

Partial Oxygen Pressure Affects the Expression of Prognostic Biomarkers HIF-1 Alpha, Ki67, and CK20 in the Microenvironment of Colorectal Cancer Tissue

Directory of Open Access Journals (Sweden)

Lirong Zhang

2016-01-01

Full Text Available Hypoxia is prognostically important in colorectal cancer (CRC therapy. Partial oxygen pressure (pO2 is an important parameter of hypoxia. The correlation between pO2 levels and expression levels of prognostic biomarkers was measured in CRC tissues. Human CRC tissues were collected and pO2 levels were measured by OxyLite. Three methods for tissue fixation were compared, including formalin, Finefix, and Finefix-plus-microwave. Immunohistochemistry (IHC staining was conducted by using the avidin-biotin complex technique for detecting the antibodies to hypoxia inducible factor-1 (HIF-1 alpha, cytokeratin 20 (CK20, and cell proliferation factor Ki67. The levels of pO2 were negatively associated with the size of CRC tissues. Finefix-plus-microwave fixation has the potential to replace formalin. Additionally, microwave treatment improved Finefix performance in tissue fixation and protein preservation. The percentage of positive cells and gray values of HIF-1 alpha, CK20, and Ki67 were associated with CRC development (P<0.05. The levels of pO2 were positively related with the gray values of Ki67 and negatively related with the values of HIF-1 alpha and CK20 (P<0.05. Thus, the levels of microenvironmental pO2 affect the expression of predictive biomarkers HIF-1 alpha, CK20, and Ki67 in the development of CRC tissues.

Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

Directory of Open Access Journals (Sweden)

Hiroaki Inoue

2015-01-01

Full Text Available Hypoxia-inducible factor (HIF-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP 70, HIF-2α, nuclear factor kappa B (NF-κB, matrix metalloproteinase (MMP-13, MMP-3, and vascular endothelial growth factor (VEGF gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

Science.gov (United States)

Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

2015-01-05

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

miR-19a promotes colitis-associated colorectal cancer by regulating tumor necrosis factor alpha-induced protein 3-NF-κB feedback loops.

Science.gov (United States)

Wang, T; Xu, X; Xu, Q; Ren, J; Shen, S; Fan, C; Hou, Y

2017-06-08

Chronic inflammation is believed to have a crucial role in colon cancer development. MicroRNA (miRNA) deregulation is common in human colorectal cancers, but little is known regarding whether miRNA drives tumor progression by regulating inflammation. Here, we showed that miR-19a can promote colitis and colitis-associated colon cancer (CAC) development using a CAC mouse model and an acute colitis mouse model. Tumor necrosis factor-α (TNF-α) stimulation can increase miR-19a expression, and upregulated miR-19a can in turn activate nuclear factor (NF)-κB signaling and TNF-α production by targeting TNF alpha-induced protein 3 (TNFAIP3). miR-19a inhibition can also alleviate CAC in vivo. Moreover, the regulatory effects of miR-19a on TNFAIP3 and NF-κB signaling were confirmed using tumor samples from patients with colon cancer. These new findings demonstrate that miR-19a has a direct role in upregulating NF-κB signaling and that miR-19a has roles in inflammation and CAC.

Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal.

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Pretto, Chrystel M; Gaide Chevronnay, Héloïse P; Cornet, Patricia B; Galant, Christine; Delvaux, Denis; Courtoy, Pierre J; Marbaix, Etienne; Henriet, Patrick

2008-10-01

Endometrial breakdown during menstruation and dysfunctional bleeding is triggered by the abrupt expression of matrix metalloproteinases (MMPs), including interstitial collagenase (MMP-1). The paracrine induction of MMP-1 in stromal cells via epithelium-derived IL-1alpha is repressed by ovarian steroids. However, the control by estradiol (E) and progesterone (P) of endometrial IL-1alpha expression and bioactivity remains unknown. Variations of endometrial IL-1alpha mRNA and protein along the menstrual cycle and during dysfunctional bleeding were determined using RT-PCR, in situ hybridization, and immunolabeling. The mechanism of EP control was analyzed using culture of explants, laser capture microdissection, and purified cells. Data were compared with expression changes of IL-1beta and IL-1 receptor antagonist. IL-1alpha is synthesized by epithelial cells throughout the cycle but E and/or P prevents its release. In contrast, endometrial stromal cells produce IL-1alpha only at menses and during irregular bleeding in areas of tissue breakdown. Stromal expression of IL-1alpha, like that of MMP-1, is repressed by P (alone or with E) but triggered by epithelium-derived IL-1alpha released upon EP withdrawal. Our experiments in cultured endometrium suggest that IL-1alpha released by epithelial cells triggers the production of IL-1alpha by stromal cells in a paracrine amplification loop to induce MMP-1 expression during menstruation and dysfunctional bleeding. All three steps of this amplification cascade are repressed by EP.

Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency.

Science.gov (United States)

Misra, Sanjay; Shergill, Uday; Yang, Binxia; Janardhanan, Rajiv; Misra, Khamal D

2010-08-01

A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. The authors tested the hypothesis that there is increased gene expression of hypoxia-inducible factor-1 alpha (HIF-1alpha); vascular endothelial growth factor-A (VEGF-A) and its receptors (VEGFR-1, -2); matrix metalloproteinase-2 (MMP-2), -9 (MMP-9); tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2); and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis. Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal function characterized in eight animals. Twenty eight days later, an AVF (n = 11) was created from the right carotid artery to ipsilateral jugular vein, and the mice were killed at day 7 (n = 4) and day 14 (n = 4). The outflow and control veins were removed for gene expression. Three mice were killed at day 28 for histologic analysis. The mean serum blood urea nitrogen level remained significantly elevated for 8 weeks when compared with baseline (P < .05). By day seven, there was a significant increase in the expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein, with HIF-1alpha and TIMP-1 levels significantly elevated at day 14 (P < .05). By day 28, the venous stenosis was characterized by a thickened vein wall and neointima. A mouse model of renal insufficiency with AVF was developed that had increased expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with venous stenosis by day 28. Copyright (c) 2010 SIR. Published by Elsevier Inc. All rights reserved.

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

Energy Technology Data Exchange (ETDEWEB)

Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China); Li, Ruisheng [Institute of Infectious Diseases, 302 Military Hospital, Beijing 100039 (China); Jia, Ying; Zhao, Yun; Xiao, Dongjie [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China); Dang, Ningning [Department of Dermatology, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China); Wang, Yunshan [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013 (China)

2014-07-15

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

International Nuclear Information System (INIS)

Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan; Li, Ruisheng; Jia, Ying; Zhao, Yun; Xiao, Dongjie; Dang, Ningning; Wang, Yunshan

2014-01-01

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

Growth hormone-releasing factor induces c-fos expression in cultured primary pituitary cells

DEFF Research Database (Denmark)

Billestrup, Nils; Mitchell, R L; Vale, W

1987-01-01

GH-releasing factor (GRF) and somatostatin regulates the secretion and biosynthesis of GH as well as the proliferation of GH-producing cells. In order to further characterize the mitogenic effect of GRF, we studied the expression of the proto-oncogene c-fos in primary pituitary cells. Maximal...... induction of c-fos mRNA was observed 20-60 min after stimulation with 5 nM GRF, returning to basal levels after 2 h. Somatostatin-14 (5 nM) partially inhibited the GRF induced c-fos expression. Forskolin and phorbol 12, 13 dibutyrate induced c-fos gene in cultured primary pituitary cells with similar...

Hepatocyte growth factor limits autoimmune neuroinflammation via glucocorticoid-induced leucine zipper expression in dendritic cells.

Science.gov (United States)

Benkhoucha, Mahdia; Molnarfi, Nicolas; Dunand-Sauthier, Isabelle; Merkler, Doron; Schneiter, Gregory; Bruscoli, Stefano; Riccardi, Carlo; Tabata, Yasuhiko; Funakoshi, Hiroshi; Nakamura, Toshikazu; Reith, Walter; Santiago-Raber, Marie-Laure; Lalive, Patrice H

2014-09-15

Autoimmune neuroinflammation, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity, is believed to result from immune tolerance dysfunction leading to demyelination and substantial neurodegeneration. We previously showed that CNS-restricted expression of hepatocyte growth factor (HGF), a potent neuroprotective factor, reduced CNS inflammation and clinical deficits associated with EAE. In this study, we demonstrate that systemic HGF treatment ameliorates EAE through the development of tolerogenic dendritic cells (DCs) with high expression levels of glucocorticoid-induced leucine zipper (GILZ), a transcriptional repressor of gene expression and a key endogenous regulator of the inflammatory response. RNA interference-directed neutralization of GILZ expression by DCs suppressed the induction of tolerance caused by HGF. Finally, adoptive transfer of HGF-treated DCs from wild-type but not GILZ gene-deficient mice potently mediated functional recovery in recipient mice with established EAE through effective modulation of autoaggressive T cell responses. Altogether, these results show that by inducing GILZ in DCs, HGF reproduces the mechanism of immune regulation induced by potent immunomodulatory factors such as IL-10, TGF-β1, and glucocorticoids and therefore that HGF therapy may have potential in the treatment of autoimmune dysfunctions. Copyright © 2014 by The American Association of Immunologists, Inc.

Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

Energy Technology Data Exchange (ETDEWEB)

Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2015-09-25

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

Science.gov (United States)

Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

2010-08-01

In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Relative workload determines exercise-induced increases in PGC-1alpha mRNA

DEFF Research Database (Denmark)

Nordsborg, Nikolai Baastrup; Lundby, Carsten; Leick, Lotte

2010-01-01

INTRODUCTION:: The hypothesis that brief intermittent exercise induced increases in human skeletal muscle metabolic mRNA is dependent on relative workload was investigated. METHODS:: Trained (n=10) and untrained (n=8) subjects performed exhaustive intermittent cycling exercise (4x4 min @ 85% of VO2...... peak, interspersed by 3 min). Trained subjects also performed the intermittent exercise at the same absolute workload as untrained, corresponding to 70% of VO2 peak (n=6). RESULTS:: Exercise at 85% of VO2 peak elevated (P... and untrained, respectively. PGC-1alpha mRNA expression was increased (Pelevated (3.1+/-0.7 mM) and PGC-1alpha mRNA content was less (P

Delayed changes in gene expression in human fibroblasts after alpha irradiation

International Nuclear Information System (INIS)

Salo, A.; Peraelae, M.; Mustonen, R.; Kadhim, M.; Marsden, S.; Sabatier, L.; Martins, L.

2003-01-01

endpoints with radiation-induced cancer. Gene expression changes in human fibroblast cells at delayed time points after alpha particle irradiation were studied. The aim was to identify genes playing pivotal role in inducing genomic instability. (orig.)

Fanconi anemia protein, FANCG, is a phosphoprotein and is upregulated with FANCA after TNF-alpha treatment.

Science.gov (United States)

Futaki, M; Watanabe, S; Kajigaya, S; Liu, J M

2001-02-23

Fanconi anemia (FA) is a genetic syndrome characterized by bone marrow failure, birth defects, and a predisposition to malignancy. At this time, six FA genes have been identified, and several gene products have been found to interact in a protein complex. FA cells appear to overexpress the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). We therefore examined the effects of TNF-alpha on the regulation of FA complementation group proteins, FANCG and FANCA. We found that treatment with TNF-alpha induced FANCG protein expression. FANCA was induced concurrently with FANCG, and the FANCA/FANCG complex was increased in the nucleus following TNF-alpha treatment. Inactivation of inhibitory kappa B kinase-2 modulated the expression of FANCG. We also found that both nuclear and cytoplasmic FANCG fractions were phosphorylated. These results show that FANCG is a phosphoprotein and suggest that the cellular accumulation of FA proteins is subject to regulation by TNF-alpha signaling.

Effect of tumor necrosis factor-alpha infusion on the incretin effect in healthy volunteers

DEFF Research Database (Denmark)

Nielsen, Signe Tellerup; Lehrskov-Schmidt, Louise; Krogh-Madsen, Rikke

2013-01-01

Type 2 diabetes mellitus (T2DM) is associated with peripheral insulin resistance, impaired incretin effect, and increased plasma levels of tumor necrosis factor-alpha (TNF-α). Whereas TNF-α infusion at a dose that induces systemic inflammation in healthy volunteers has been demonstrated to induce...

Evaluation of Cucurbita maxima extract against scopolamine-induced amnesia in rats: implication of tumour necrosis factor alpha.

Science.gov (United States)

Jawaid, Talha; Shakya, Ashok K; Siddiqui, Hefazat Hussain; Kamal, Mehnaz

2014-01-01

Cucurbita maxima (CM) seed oil is commonly used in Indian folk medicine to treat various ailments. We have investigated the effect of CM seed oil on memory impairment induced by scopolamine in rats. Male adult Wistar rats were administered scopolamine 1 mg/kg body weight, i.p. or 1.25 mg/kg body weight, s.c. to induce memory impairment. The nootropic agent piracetam 100 mg/kg body weight, i.p. and CM seed oil 100 and 200 mg/kg body weight, p.o. were administered daily for five consecutive days. The memory function was evaluated in the Morris water maze (MWM) test, the social recognition test (SRT), the elevated plus maze (EPM) test, and the pole climbing test (PCT). Acetylcholinesterase (AChE) activity and oxidative stress parameters were estimated in the cortex, hippocampus, and cerebellum of the brains after completion of the behavioural studies. The effects of scopolamine on the levels of the tumour necrosis factor alpha (TNF-α) transcript were also investigated. Scopolamine caused memory impairment in all the behavioural paradigms along with a significant increase in the AChE activity and oxidative stress in the brain. Scopolamine also caused a significant increase in the expression of TNF-α in the hippocampus. CM seed oil exhibited antiamnesic activity as indicated by a significant reduction in the latency time in the MWM test and decreased social interaction during trial 2 in the SRT. Further, treatment with CM seed oil significantly decreased the AChE activity and malondialdehyde levels and increased the glutathione level in brain regions. CM seed oil also significantly decreased the expression of TNF-α in the hippocampus. The effect of CM seed oil on behavioural and biochemical parameters was comparable to that observed in rats treated with piracetam. These results indicate that CM seed oil may exert antiamnesic activity which may be attributed to the inhibition of AChE and inflammation as well as its antioxidant activity in the brain.

Early growth response protein 1 (EGR1) regulates pro-inflammatory gene expression in response to palmitate and TNF alpha in human placenta cells and is induced in obese placenta

Science.gov (United States)

Maternal obesity has been hypothesized to induce a pro-inflammatory response in the placenta. However, the specific factors contributing to this pro-infalmmatory response are yet to be determined. Our objective was to examine the effects of palmitic acid (PA), tumor necrosis factor alpha (TNF alph...

NF-kappaB is involved in SHetA2 circumvention of TNF-alpha resistance, but not induction of intrinsic apoptosis.

Science.gov (United States)

Chengedza, Shylet; Benbrook, Doris Mangiaracina

2010-03-01

Treatment of cancer with tumor necrosis factor-alpha (TNF-alpha) is hindered by resistance and toxicity. The flexible heteroarotinoid, SHetA2, sensitizes resistant ovarian cancer cells to TNF-alpha-induced extrinsic apoptosis, and also induces intrinsic apoptosis as a single agent. This study tested the hypothesis that nuclear factor-kappaB (NF-kappaB) is involved in SHetA2-regulated intrinsic and extrinsic apoptosis. SHetA2 inhibited basal and TNF-alpha-induced or hydrogen peroxide-induced NF-kappaB activity through counter-regulation of upstream kinase (IkappaB kinase) activity, inhibitor protein (IkappaB-alpha) phosphorylation, and p-65 NF-kappaB subunit nuclear translocation, but independently of reactive oxygen species generation. Ectopic over-expression of p-65, or treatment with TNF-alpha receptor 1 (TNFR1) small interfering RNA or a caspase-8 inhibitor, each attenuated synergistic apoptosis by SHetA2 and TNF-alpha, but did not affect intrinsic apoptosis caused by SHetA2. In conclusion, NF-kappaB repression is involved in SHetA2 circumvention of resistance to TNF-alpha-induced extrinsic apoptosis, but not in SHetA2 induction of intrinsic apoptosis.

Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

International Nuclear Information System (INIS)

Jeong, Wooyoung; Bazer, Fuller W.; Song, Gwonhwa; Kim, Jinyoung

2016-01-01

The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

Energy Technology Data Exchange (ETDEWEB)

Jeong, Wooyoung [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of); Bazer, Fuller W. [Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A& M University, College Station, TX (United States); Song, Gwonhwa, E-mail: ghsong@korea.ac.kr [Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Jinyoung, E-mail: jinyoungkim@dankook.ac.kr [Department of Animal Resources Science, Dankook University, Cheonan (Korea, Republic of)

2016-01-08

The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

Science.gov (United States)

Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

2009-09-01

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Ultraviolet B irradiation of skin induces mast cell degranulation and release of tumour necrosis factor-{alpha}

Energy Technology Data Exchange (ETDEWEB)

Walsh, L.J. [University of Queensland, Brisbane, QLD (Australia). Dept. of Dentistry, Immunopathology Unit

1995-06-01

In the `sunburn` response in skin, dermal blood vessels are activated and traffic of dendritic Langerhans` cells altered. While these changes have been attributed to the cytokine TNF-{alpha}, the source of this acutely released TNF has not been identified. This report demonstrates that the `sunburn` response, both in vivo and in vitro, is accompanied by rapid degranulation of cutaneous mast cells, with consequential release of intracellular stores of TNF. Epidermal keratinocytes were only minor contributors to local TNF production. Expression of the TNF-inducible CD62E (E-selectin/ELAM-1) and CD54 adhesion molecules on cutaneous endothelium occurred 2 hours following mast cell degranulation, and this event was sensitive to blockade of mast cells with disodium cromoglycate. These results indicate that TNF release in skin in the acute sunburn response can largely be attributed to mast cells. 47 refs., 5 tabs., 2 figs.

Integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance

Energy Technology Data Exchange (ETDEWEB)

Sansing, Hope A. [Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center-New Orleans, School of Dentistry, New Orleans, LA (United States); Sarkeshik, Ali; Yates, John R. [Department of Chemical Physiology, Scripps Research Institute, La Jolla, CA (United States); Patel, Vyomesh; Gutkind, J. Silvio [Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Yamada, Kenneth M. [Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD (United States); Berrier, Allison L., E-mail: allison.berrier@gmail.com [Department of Oral and Craniofacial Biology, Louisiana State University Health Sciences Center-New Orleans, School of Dentistry, New Orleans, LA (United States)

2011-03-11

Research highlights: {yields} Proteomics of clustered integrin {alpha}{beta}1, {alpha}{sub v}{beta}, {alpha}{sub 6}{beta} receptors in oral carcinoma. {yields} p130Cas, Dek, Src and talin regulate oral carcinoma invasion. {yields} 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 {alpha}{beta}1, {alpha}{sub v}{beta} or {alpha}{sub 6}{beta} 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.

Epidermal growth factor receptor expression in radiation-induced dog lung tumors by immunocytochemical localization

Energy Technology Data Exchange (ETDEWEB)

Leung, F.L.; Park, J.F.; Dagle, G.E.

1993-06-01

In studies to determine the role of growth factors in radiation-induced lung cancer, epidermal growth factor (EGFR) expression was examined by immunocytochemistry in 51 lung tumors from beagle dogs exposed to inhaled plutonium; 21 of 51 (41%) tumors were positive for EGFR. The traction of tumors positive for EGFR and the histological type of EGFR-positive tumors in the plutonium-exposed dogs were not different from spontaneous dog lung tumors, In which 36% were positive for EGFR. EGFR involvement in Pu-induced lung tumors appeared to be similar to that in spontaneous lung tumors. However, EGFR-positive staining was observed in only 1 of 16 tumors at the three lowest Pu exposure levels, compared to 20 of 35 tumors staining positive at the two highest Pu exposure levels. The results in dogs were in good agreement with the expression of EGFR reported in human non-small cell carcinoma of the lung, suggesting that Pu-induced lung tumors in the dog may be a suitable animal model to investigate the role of EGFR expression in lung carcinogenesis. In humans, EGFR expression in lung tumors has been primarily related to histological tumor types. In individual dogs with multiple primary lung tumors, the tumors were either all EGFR positive or EGFR negative, suggesting that EGFR expression may be related to the response of the individual dog as well as to the histological type of tumor.

TCDD Induces the Hypoxia-Inducible Factor (HIF-1α Regulatory Pathway in Human Trophoblastic JAR Cells

Directory of Open Access Journals (Sweden)

Tien-Ling Liao

2014-09-01

Full Text Available The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K inhibitor or N-acetylcysteine (a ROS scavenger. The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ, PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

Science.gov (United States)

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Glutathione regulation of redox-sensitive signals in tumor necrosis factor-α-induced vascular endothelial dysfunction

International Nuclear Information System (INIS)

Tsou, T.-C.; Yeh, S.C.; Tsai, F.-Y.; Chen, J.-W.; Chiang, H.-C.

2007-01-01

We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-α)-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte-endothelial monolayer binding. Since TNF-α induces various biological effects on vascular cells, TNF-α dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-α concentrations, we adopted the lower TNF-α (0.2 ng/ml) to rule out the possible involvement of other TNF-α-induced biological effects. Inhibition of glutathione synthesis by L-buthionine-(S,R)-sulfoximine (BSO) resulted in down-regulations of the TNF-α-induced adhesion molecule expression and monocyte-endothelial monolayer binding. BSO attenuated the TNF-α-induced nuclear factor-kappaB (NF-κB) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-α. Inhibition of ERK, JNK, or NF-κB attenuates TNF-α-induced ICAM-1 promoter activation and monocyte-endothelial monolayer binding. Our study indicates that TNF-α induces adhesion molecule expression and monocyte-endothelial monolayer binding mainly via activation of NF-κB in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-α. Although AP-1 activation by the lower TNF-α was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-α-induced adhesion molecule expression

Endothelial Cell Migration and Vascular Endothelial Growth Factor Expression Are the Result of Loss of Breast Tissue Polarity

Energy Technology Data Exchange (ETDEWEB)

Chen, Amy; Cuevas, Ileana; Kenny, Paraic A; Miyake, Hiroshi; Mace, Kimberley; Ghajar, Cyrus; Boudreau, Aaron; Bissell, Mina; Boudreau, Nancy

2009-05-26

Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1{alpha}-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

Expression of human lymphotoxin alpha in Aspergillus niger

NARCIS (Netherlands)

Krasevec, N.; Hondel, C.A.M.J.J. van de; Komel, R.

2000-01-01

A gene-fusion expression strategy was applied for heterologous expression of human lymphotoxin alpha (LTα) in the Aspergillus niger AB1.13 protease-deficient strain. The LTα gene was fused with the A. niger glucoamylase GII-form as a carrier-gene, behind its transcription control and secretion

17-AAG induces cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy.

Science.gov (United States)

Riedel, Michael; Goldbaum, Olaf; Schwarz, Lisa; Schmitt, Sebastian; Richter-Landsberg, Christiane

2010-01-18

The accumulation and aggregation of alpha-synuclein in nerve cells and glia are characteristic features of a number of neurodegenerative diseases termed synucleinopathies. alpha-Synuclein is a highly soluble protein which in a nucleation dependent process is capable of self-aggregation. The causes underlying aggregate formation are not yet understood, impairment of the proteolytic degradation systems might be involved. In the present study the possible aggregate clearing effects of the geldanamycin analogue 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) was investigated. Towards this, an oligodendroglial cell line (OLN-93 cells), stably expressing human alpha-synuclein (A53T mutation) was used. In these cells small punctate aggregates, not staining with thioflavine S, representing prefibrillary aggregates, occur characteristically. Our data demonstrate that 17-AAG attenuated the formation of alpha-synuclein aggregates by stimulating macroautophagy. By blocking the lysosomal compartment with NH(4)Cl the aggregate clearing effects of 17-AAG were abolished and alpha-synuclein deposits were enlarged. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that 17-AAG led to the recruitment of LC3-II and to the formation of LC3 positive puncta. This effect was also observed in cultured oligodendrocytes derived from the brains of newborn rats. Inhibition of macroautophagy by 3-methyladenine prevented 17-AAG induced occurrence of LC3 positive puncta as well as the removal of alpha-synuclein aggregates in OLN-A53T cells. Our data demonstrate for the first time that 17-AAG not only causes the upregulation of heat shock proteins, but also is an effective inducer of the autophagic pathway by which alpha-synuclein can be removed. Hence geldanamycin derivatives may provide a means to modulate autophagy in neural cells, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.

Alpha-Driven MHD and MHD-Induced Alpha Loss in TFTR DT Experiments

Science.gov (United States)

Chang, Zuoyang

1996-11-01

Theoretical calculation and numerical simulation indicate that there can be interesting interactions between alpha particles and MHD activity which can adversely affect the performance of a tokamak reactor (e.g., ITER). These interactions include alpha-driven MHD, like the toroidicity-induced-Alfven-eigenmode (TAE) and MHD induced alpha particle losses or redistribution. Both phenomena have been observed in recent TFTR DT experiments. Weak alpha-driven TAE activity was observed in a NBI-heated DT experiment characterized by high q0 ( >= 2) and low core magnetic shear. The TAE mode appears at ~30-100 ms after the neutral beam turning off approximately as predicted by theory. The mode has an amplitude measured by magnetic coils at the edge tildeB_p ~1 mG, frequency ~150-190 kHz and toroidal mode number ~2-3. It lasts only ~ 30-70 ms and has been seen only in DT discharges with fusion power level about 1.5-2.0 MW. Numerical calculation using NOVA-K code shows that this type of plasma has a big TAE gap. The calculated TAE frequency and mode number are close to the observation. (2) KBM-induced alpha particle loss^1. In some high-β, high fusion power DT experiments, enhanced alpha particle losses were observed to be correlated to the high frequency MHD modes with f ~100-200 kHz (the TAE frequency would be two-times higher) and n ~5-10. These modes are localized around the peak plasma pressure gradient and have ballooning characteristics. Alpha loss increases by 30-100% during the modes. Particle orbit simulations show the added loss results from wave-particle resonance. Linear instability analysis indicates that the plasma is unstable to the kinetic MHD ballooning modes (KBM) driven primarily by strong local pressure gradients. ----------------- ^1Z. Chang, et al, Phys. Rev. Lett. 76 (1996) 1071. In collaberation with R. Nazikian, G.-Y. Fu, S. Batha, R. Budny, L. Chen, D. Darrow, E. Fredrickson, R. Majeski, D. Mansfield, K. McGuire, G. Rewoldt, G. Taylor, R. White, K

Transforming growth factor alpha, Shope fibroma growth factor, and vaccinia growth factor can replace myxoma growth factor in the induction of myxomatosis in rabbits.

Science.gov (United States)

Opgenorth, A; Nation, N; Graham, K; McFadden, G

1993-02-01

The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle

DEFF Research Database (Denmark)

Lundby, Carsten; Pilegaard, Henriette; Andersen, Jesper L.

2004-01-01

growth factor (VEGF), a known target gene for hypoxia inducible factor 1 (HIF-1). We hypothesised that prolonged exposure to high altitude increases muscle capillary density and that this can be explained by an enhanced HIF-1alpha expression inducing an increase in VEGF expression. We measured mRNA...... or VEGF mRNA was not changed with prolonged hypoxic exposure in SLR, and both genes were similarly expressed in SLR and HAN. In SLR, whole body mass, mean muscle fibre area and capillary to muscle fibre ratio remained unchanged during acclimatization. The capillary to fibre ratio was lower in HAN than...... in SLR (2.4+/-0.1 vs 3.6+/-0.2; PRNA expression and capillary density are not significantly increased by 8 weeks of exposure to high altitude and are not increased in Aymara high-altitude natives compared with sea level residents....

Forkhead box protein A2, a pioneer factor for hepatogenesis, is involved in the expression of hepatic phenotype of alpha-fetoprotein-producing adenocarcinoma.

Science.gov (United States)

Yamamura, Nobuhisa; Fugo, Kazunori; Kishimoto, Takashi

2017-09-01

Alpha-fetoprotein (AFP)-producing adenocarcinoma is a high-malignant variant of adenocarcinoma with a hepatic or fetal-intestinal phenotype. The number of cases of AFP-producing adenocarcinomas is increasing, but the molecular mechanism underlying the aberrant production of AFP is unclear. Here we sought to assess the role of Forkhead box A (FoxA)2, which is a pioneer transcription factor in the differentiation of hepatoblasts. FoxA2 expression was investigated in five cases of AFP-producing gastric adenocarcinomas by immunohistochemistry, and all cases showed FoxA2 expression. Chromatin immunoprecipitation revealed the DNA binding of FoxA2 on the regulatory element of AFP gene in AFP-producing adenocarcinoma cells. The inhibition of FoxA2 expression with siRNA reduced the mRNA expression of liver-specific proteins, including AFP, albumin, and transferrin. The inhibition of FoxA2 also reduced the expressions of liver-enriched nuclear factors, i.e., hepatocyte nuclear factor (HNF) 4α and HNF6, although the expressions of HNF1α and HNF1β were not affected. The same effect as FoxA2 knockdown in AFP producing adenocarcinoma cells was also observed in hepatocellular carcinoma cells. Our results suggest that FoxA2 plays a key role in the expression of hepatic phenotype of AFP-producing adenocarcinomas. Copyright © 2017 Elsevier GmbH. All rights reserved.

Modulating factors in the expression of radiation-induced oncogenic transformation

International Nuclear Information System (INIS)

Hall, E.J.; Hei, T.K.

1990-01-01

Many assays for oncogenic transformation have been developed ranging from those in established rodent cell lines where morphological alteration is scored, to those in human cells growing in nude mice where tumor invasiveness is scored. In general, systems that are most quantitaive are also the least relevant in terms of human carcinogenesis and human risk estimation. The development of cell culture systems has made it possible to assess at the cellular level the oncogenic potential of a variety of chemical, physical and viral agents. Cell culture systems afford the opportunity to identify factors and conditions that may prevent or enhance cellular transformation by radiation and chemicals. Permissive and protective factors in radiation-induced transformation include thyroid hormone and the tumor promoter TPA that increase the transformation incidence for a given dose of radiation, and retinoids, selenium, vitamin E, and 5-aminobenzamide that inhibit the expression of transformation. Densely ionizing α-particles, similar to those emitted by radon daughters, are highly effective in inducing transformations and appear to interact in a supra-additive fashion with asbestos fibers. The activation of a known dominant oncogene has not yet been demonstrated in radiation-induced oncogenic transformation. The most likely mechanism for radiation activation of an oncogene would be via the production of a chromosomal translocation. Radiation also efficiently induces deletions and may thus lead to the loss of a suppressor gene

Substance P ameliorates tumor necrosis factor-alpha-induced endothelial cell dysfunction by regulating eNOS expression in vitro.

Science.gov (United States)

Piao, Jiyuan; Hong, Hyun Sook; Son, Youngsook

2018-04-01

The aim of this study was to explore the beneficial effects of SP on NO production and inflammation-induced vascular endothelium cell death. To mimic the inflammatory environment, TNF-α was treated with HUVECs, and SP was added prior to TNF-α to determine its protective effect. WST-1 assay was performed to detect cell viability. NO level in conditioned medium was measured by Griess Reagent System. The protein level of cleaved caspase-3, eNOS, and phosphorylated Akt was detected by Western blot analysis. TNF-α declined endothelial cell viability by downregulating Akt and NO production. TNF-α-induced cell death was reliably restored by NO, confirming the requirement of NO for cell survival. By contrast, pretreatment of SP attenuated TNF-α-induced cellular apoptosis, accompanied by an increase in the phosphorylation of Akt, eNOS expression, and NO production. Blockage of NK-1R, phosphorylated Akt or eNOS by CP-96345, A6730, or L-NAME entirely eliminated the effect of SP. SP can protect the vascular endothelium against inflammation-induced damage through modulation of the Akt/eNOS/NO signaling pathway. © 2018 John Wiley & Sons Ltd.

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

Science.gov (United States)

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.

Inhibition of TNF-alpha production contributes to the attenuation of LPS-induced hypophagia by pentoxifylline.

Science.gov (United States)

Porter, M H; Hrupka, B J; Altreuther, G; Arnold, M; Langhans, W

2000-12-01

Cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are assumed to mediate anorexia during bacterial infections. To improve our understanding of the role that these two cytokines serve in mediating infection during anorexia, we investigated the ability of pentoxifylline (PTX), a potent inhibitor of TNF-alpha production, to block the anorectic effects of the bacterial products lipopolysaccharide (LPS) and muramyl dipeptide (MDP) in rats. Intraperitoneally injected PTX (100 mg/kg body wt) completely eliminated the anorectic effect of intraperitoneally injected LPS (100 microg/kg body wt) and attenuated the anorectic effect of a higher dose of intraperitoneally injected LPS (250 microg/kg body wt). Concurrently, PTX pretreatment suppressed low-dose LPS-induced TNF-alpha production by more than 95% and IL-1beta production 39%, as measured by ELISA. Similarly, high-dose LPS-induced TNF-alpha production was reduced by approximately 90%. PTX administration also attenuated the tolerance that is normally observed with a second injection of LPS. In addition, PTX pretreatment attenuated the hypophagic effect of intraperitoneally injected MDP (2 mg/kg body wt) but had no effect on the anorectic response to intraperitoneally injected recombinant human TNF-alpha (150 ug/kg body wt). The results suggest that suppression of TNF-alpha production is sufficient to attenuate LPS- and MDP-induced anorexia. This is consistent with the hypothesis that TNF-alpha plays a major role in the anorexia associated with bacterial infection.

HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells

Czech Academy of Sciences Publication Activity Database

Kudová, Jana; Procházková, J.; Vašíček, Ondřej; Perečko, Tomáš; Sedláčková, M.; Pešl, M.; Pachernik, J.; Kubala, Lukáš

2016-01-01

Roč. 11, č. 6 (2016) E-ISSN 1932-6203 R&D Projects: GA ČR GA13-29358S Grant - others:Grantová agentura ČR - GA ČR(CZ) GJ15-13443Y Institutional support: RVO:68081707 Keywords : INDUCIBLE FACTOR 1- ALPHA * GENE-EXPRESSION * CARDIAC DIFFERENTIATION Subject RIV: BO - Biophysics Impact factor: 2.806, year: 2016

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.

Correlation between spontaneous apoptosis and the expression of angiogenic factors in advanced gastric adenocarcinoma.

Science.gov (United States)

Ikeguchi, M; Cai, J; Fukuda, K; Oka, S; Katano, K; Tsujitani, S; Maeta, M; Kaibara, N

2001-06-01

The aim of this study was to investigate whether angiogenic factors influence the occurrence of spontaneous apoptosis in advanced gastric cancer. The apoptotic indices (AIs) of 97 tumors from 97 patients with advanced gastric cancer (pT3, pN0, pM0, Stage II) were analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. Intratumoral microvessel densities (IMVDs) of tumors stained with anti-CD34 monoclonal antibody were quantified under x 200 magnification using computer-assisted image analysis. The expressions of angiogenic factors, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (dThdPase), transforming growth factor-alpha (TGF-alpha), and p53 were analyzed immunohistochemically and compared with IMVDs and AIs. The mean IMVD of the 97 tumors was 365/mm2 (range 147-990/mm2). The mean AI of tumors was 2.1% (range 0-11.3%). A significant inverse correlation between the AIs and the IMVDs was shown (p = -0.278, P = 0.0064). The mean IMVDs of tumors with high expressions of dThdPase, TGF-alpha, or p53 were significantly higher than those of tumors with low expressions of these factors. The mean AI of tumors with high expressions of dThdPase was significantly lower than that of tumors with low expressions of dThdPase (P = 0.023). However, no significant correlations were detected between AIs and the expression levels of VEGF, TGF-alpha, or p53. In gastric cancer, dThdPase may play an important role in tumor progression by increasing microvessels and by suppressing apoptosis of cancer cells.

Cytosolic phospholipase A2-alpha expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours.

LENUS (Irish Health Repository)

Caiazza, F

2012-02-01

BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)alpha expression correlated with EGFR\\/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)alpha activity in clinical breast cancer was established by relating the expression of cPLA(2)alpha in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)alpha mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)alpha expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)alpha expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)alpha in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.

Supernatants from Staphylococcus epidermidis grown in the presence of different antibiotics induce differential release of tumor necrosis factor alpha from human monocytes.

OpenAIRE

Mattsson, E; Van Dijk, H; Verhoef, J; Norrby, R; Rollof, J

1996-01-01

Bacterial products from gram-positive bacteria, such as peptidoglycan, teichoic acid, and toxins, activate mononuclear cells to produce tumor necrosis factor alpha (TNF). The present study evaluated the release of soluble cell wall components from Staphylococcus epidermidis capable of inducing TNF after exposure of the bacteria to various antibiotics. A clinical S. epidermidis isolate (694) was incubated with either penicillin, oxacillin, vancomycin, or clindamycin at five times the MIC. Supe...

Mechanoactive scaffold induces tendon remodeling and expression of fibrocartilage markers.

Science.gov (United States)

Spalazzi, Jeffrey P; Vyner, Moira C; Jacobs, Matthew T; Moffat, Kristen L; Lu, Helen H

2008-08-01

Biological fixation of soft tissue-based grafts for anterior cruciate ligament (ACL) reconstruction poses a major clinical challenge. The ACL integrates with subchondral bone through a fibrocartilage enthesis, which serves to minimize stress concentrations and enables load transfer between two distinct tissue types. Functional integration thus requires the reestablishment of this fibrocartilage interface on reconstructed ACL grafts. We designed and characterized a novel mechanoactive scaffold based on a composite of poly-alpha-hydroxyester nanofibers and sintered microspheres; we then used the scaffold to test the hypothesis that scaffold-induced compression of tendon grafts would result in matrix remodeling and the expression of fibrocartilage interface-related markers. Histology coupled with confocal microscopy and biochemical assays were used to evaluate the effects of scaffold-induced compression on tendon matrix collagen distribution, cellularity, proteoglycan content, and gene expression over a 2-week period. Scaffold contraction resulted in over 15% compression of the patellar tendon graft and upregulated the expression of fibrocartilage-related markers such as Type II collagen, aggrecan, and transforming growth factor-beta3 (TGF-beta3). Additionally, proteoglycan content was higher in the compressed tendon group after 1 day. The data suggest the potential of a mechanoactive scaffold to promote the formation of an anatomic fibrocartilage enthesis on tendon-based ACL reconstruction grafts.

Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

Energy Technology Data Exchange (ETDEWEB)

Tandle, Anita T. [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Calvani, Maura; Uranchimeg, Badarch [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Zahavi, David [Tumor Angiogenesis Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892 (United States); Melillo, Giovanni [DTP-Tumor Hypoxia Laboratory, SAIC Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702 (United States); Libutti, Steven K., E-mail: slibutti@montefiore.org [Department of Surgery, Montefiore-Einstein Center for Cancer Care, Albert Einstein College of Medicine, Greene Medical Arts Pavilion, 4th Floor 3400, Bainbridge Avenue, Bronx, New York 10467 (United States)

2009-07-01

The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

Calcineurin signaling and PGC-1alpha expression are suppressed during muscle atrophy due to diabetes.

Science.gov (United States)

Roberts-Wilson, Tiffany K; Reddy, Ramesh N; Bailey, James L; Zheng, Bin; Ordas, Ronald; Gooch, Jennifer L; Price, S Russ

2010-08-01

PGC-1alpha is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1alpha expression and oxidative capacity are decreased in skeletal muscle of patients and animals undergoing atrophy, suggesting that PGC-1alpha participates in the regulation of muscle mass. PGC-1alpha gene expression is controlled by calcium- and cAMP-sensitive pathways. However, the mechanism regulating PGC-1alpha in skeletal muscle during atrophy remains unclear. Therefore, we examined the mechanism responsible for decreased PGC-1alpha expression using a rodent streptozotocin (STZ) model of chronic diabetes and atrophy. After 21days, the levels of PGC-1alpha protein and mRNA were decreased. We examined the activation state of CREB, a potent activator of PGC-1alpha transcription, and found that phospho-CREB was paradoxically high in muscle of STZ-rats, suggesting that the cAMP pathway was not involved in PGC-1alpha regulation. In contrast, expression of calcineurin (Cn), a calcium-dependent phosphatase, was suppressed in the same muscles. PGC-1alpha expression is regulated by two Cn substrates, MEF2 and NFATc. Therefore, we examined MEF2 and NFATc activity in muscles from STZ-rats. Target genes MRF4 and MCIP1.4 mRNAs were both significantly reduced, consistent with reduced Cn signaling. Moreover, levels of MRF4, MCIP1.4, and PGC-1alpha were also decreased in muscles of CnAalpha-/- and CnAbeta-/- mice without diabetes indicating that decreased Cn signaling, rather than changes in other calcium- or cAMP-sensitive pathways, were responsible for decreased PGC-1alpha expression. These findings demonstrate that Cn activity is a major determinant of PGC-1alpha expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass.

Comparative study of TGF-alpha and P53 markers′ expression in odontogenic keratocyst and orthokeratinaized odontogenic cyst

Directory of Open Access Journals (Sweden)

Parviz Deyhimi

2012-01-01

Full Text Available Background: Odontogenic keratocyst (OKC is an aggressive cyst and its recurrence is higher than other odontogenic cysts, orthokeratinized odontogenic cyst (OOC is a cyst with moderate biological behavior in comparison with OKC, but with the probability of carcinomatous changes. The present study aims to evaluate the quantity and intensity of the expression of P53 protein and transforming growth factor alpha (TGF-alpha in OKC and OOC in order to compare the biologic behavior of these two cysts. Materials and Methods: This is a cross-sectional study. The samples include 30 cysts (15 OKC and 15 OOC, all stained immunohistochemically for P53 protein and TGF-alpha by the Novolinke polymer method. Then, all the cases were examined with an optical microscope with Χ400 magnification and the stained cells were counted in the basal and parabasal layers. Finally the results were analyzed by the Mann–and Wilcoxon tests (P value < 0.05. Results: The difference between the expression of P53 protein in the basal layer in OKC and OOC was not statistically significant (P value = 0.076. The difference between the expression of P53 protein in the parabasal layer in OKC and OOC was statistically significant (P value = 0.003; moreover, the difference between the expression of TGF-alpha in the basal layer in OKC and OOC was not statistically significant (P value = 0.284. The difference between the expression of TGF-alpha in the parabasal layer in OKC and OOC was statistically significant (P value = 0.015. Conclusion: Since there was a higher expression of P53 protein and TGF-alpha in OKC compared to those in OOC, the probability of carcinomatous changes was at least theoretically higher in OKC than in OOC.

Shifts in renin-angiotensin system components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa region of streptozotocin-induced diabetic mice.

Science.gov (United States)

Qian, Xiaobing; Lin, Leilei; Zong, Yao; Yuan, Yongguang; Dong, Yanmin; Fu, Yue; Shao, Wanwen; Li, Yujie; Gao, Qianying

2018-03-01

This study aimed to analyse shifts in renin-angiotensin system (RAS) components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa (LC) region in streptozotocin (STZ)-induced diabetic mice. Six months after diabetes induction, the retinal vessels of male C57BL/6 J mice were observed by colour photography, fundus fluorescein angiography (FFA), and immunofluorescent staining following incubation with CD31. Immunofluorescence for glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA),and NG2 was also performed. Angiotensin-converting enzyme 1 (ACE1), angiotensin II type I receptor (AT1R), renin, hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and haeme oxygenase 1 (HO-1) expression levels were confirmed by immunohistochemical and western blotting analyses. Compared with control mice, diabetic mice had significantly higher blood glucose concentrations (p diabetic mice; however, immunostaining of whole-mount retinas revealed an increased number of retinal vessels. Furthermore, histopathological staining showed significant reduction in the whole retinal thickness. GFAP expression was slightly higher, whereas fewer NG2 + pericytes were observed in diabetic mice than in control mice. ACE1, AT1R, renin, HIF-1α, VEGF, VEGFR2, and HO-1 expression were up-regulated in the LC of the STZ-induced diabetic mice. Collectively, ACE 1, AT1R, HIF-1α, VEGF, VEGFR2, and HO-1 activation in the LC region in diabetic mice may be involved in diabetes via the RAS and induction of angiogenesis and oxidative stress.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2-Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells.

Science.gov (United States)

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state.

Insights into Alpha-Hemolysin (Hla) Evolution and Expression among Staphylococcus aureus Clones with Hospital and Community Origin

DEFF Research Database (Denmark)

Tavares, Ana; Nielsen, Jesper B; Boye, Kit

2014-01-01

BACKGROUND: Alpha-hemolysin (Hla) is a major virulence factor in the pathogenesis of Staphylococcus aureus infection, being active against a wide range of host cells. Although hla is ubiquitous in S. aureus, its genetic diversity and variation in expression in different genetic backgrounds...... and SCCmec typing. The internal regions of hla and the hla promoter were sequenced and gene expression was assessed by RT-PCR. RESULTS: Alpha-hemolysin encoding- and promoter sequences were diverse, with 12 and 23 different alleles, respectively. Based on phylogenetic analysis, we suggest that hla may have...... in the RNAIII binding site were not associated to hla expression. Although expression rates of hla were in general strain-specific, we observed CA clones showed significantly higher hla expression (p = 0.003) when compared with HA clones. CONCLUSION: We propose that the hla gene has evolved together...

Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways.

Science.gov (United States)

Jahan, Naima; Hannila, Sari S

2015-01-01

The expression of chondroitin sulfate proteoglycans (CSPGs) by reactive astrocytes is a major factor contributing to glial scarring and regenerative failure after spinal cord injury, but the molecular mechanisms underlying CSPG expression remain largely undefined. One contributing factor is transforming growth factor β (TGFβ), which is upregulated after injury and has been shown to induce expression of CSPGs in vitro. TGFβ typically mediates its effects through the Smad2/3 signaling pathway, and it has been suggested that this pathway is responsible for CSPG expression. However, there is evidence that TGFβ can also activate non-Smad signaling pathways. In this study, we report that TGFβ-induced expression of three different CSPGs--neurocan, brevican, and aggrecan--is mediated through non-Smad signaling pathways. We observed significant increases in TGFβ-induced expression of neurocan, brevican, and aggrecan following siRNA knockdown of Smad2 or Smad4, which indicates that Smad signaling is not required for the expression of these CSPGs. In addition, we show that neurocan, aggrecan, and brevican levels are significantly reduced when TGFβ is administered in the presence of either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin, but not the MEK1/2 inhibitor U0126. This suggests that TGFβ mediates this effect through non-Smad-dependent activation of the PI3K-Akt-mTOR signaling pathway, and targeting this pathway may therefore be an effective means of reducing CSPG expression in the injured CNS. Copyright © 2014 Elsevier Inc. All rights reserved.

Cytokine vaccination: neutralising IL-1alpha autoantibodies induced by immunisation with homologous IL-1alpha

DEFF Research Database (Denmark)

Svenson, M; Hansen, M B; Thomsen, Allan Randrup

2000-01-01

with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding...... in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients...

Epidermal growth factor protects squamous cell carcinoma against cisplatin-induced cytotoxicity through increased interleukin-1β expression.

Directory of Open Access Journals (Sweden)

Shian-Chin Ko

Full Text Available The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression was confirmed by knockdown of RelA and Akt in cells or treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. The expression of dominant negative IκB also repressed the activation of NF-κB and inhibited EGF-induced IL-1β expression. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65 was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment.

Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

Energy Technology Data Exchange (ETDEWEB)

Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of); Park, Jong-Wan, E-mail: parkjw@snu.ac.kr [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of)

2012-02-10

Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation.

LENUS (Irish Health Repository)

Li, Chong Hui

2012-02-03

Tolerance to bacterial cell-wall components may represent an essential regulatory mechanism during bacterial infection. We have demonstrated previously that the inhibition of nuclear factor (NF)-kappaB and mitogen-activated protein kinase activation was present in bacterial lipoprotein (BLP) self-tolerance and its cross-tolerance to lipopolysaccharide (LPS). In this study, the effect of BLP-induced tolerance on the myeloid differentiation factor 88 (MyD88)-dependent upstream signaling pathway for NF-kappaB activation in vitro was examined further. When compared with nontolerant human monocytic THP-1 cells, BLP-tolerant cells had a significant reduction in tumor necrosis factor alpha (TNF-alpha) production in response to a high-dose BLP (86+\\/-12 vs. 6042+\\/-245 ng\\/ml, P < 0.01) or LPS (341+\\/-36 vs. 7882+\\/-318 ng\\/ml, P < 0.01) stimulation. The expression of Toll-like receptor 2 (TLR2) protein was down-regulated in BLP-tolerant cells, whereas no significant differences in TLR4, MyD88, interleukin-1 receptor-associated kinase 4 (IRAK-4), and TNF receptor-associated factor 6 expression were observed between nontolerant and BLP-tolerant cells, as confirmed by Western blot analysis. The IRAK-1 protein was reduced markedly in BLP-tolerant cells, although IRAK-1 mRNA expression remained unchanged as revealed by real-time reverse transcriptase-polymerase chain reaction analysis. Furthermore, decreased MyD88-IRAK immunocomplex formation, as demonstrated by immunoprecipitation, was observed in BLP-tolerant cells following a second BLP or LPS stimulation. BLP pretreatment also resulted in a marked inhibition in total and phosphorylated inhibitor of kappaB-alpha (IkappaB-alpha) expression, which was not up-regulated by subsequent BLP or LPS stimulation. These results demonstrate that in addition to the down-regulation of TLR2 expression, BLP tolerance is associated with a reduction in IRAK-1 expression, MyD88-IRAK association, and IkappaB-alpha phosphorylation. These

Expression of alpha V integrin is modulated by Epstein-Barr virus nuclear antigen 3C and the metastasis suppressor Nm23-H1 through interaction with the GATA-1 and Sp1 transcription factors

International Nuclear Information System (INIS)

Choudhuri, Tathagata; Verma, Subhash C.; Lan, Ke; Robertson, Erle S.

2006-01-01

Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C

Chronic liver injury in mice promotes impairment of skin barrier function via tumor necrosis factor-alpha.

Science.gov (United States)

Yokoyama, Satoshi; Hiramoto, Keiichi; Koyama, Mayu; Ooi, Kazuya

2016-09-01

Alcohol is frequently used to induce chronic liver injury in laboratory animals. Alcohol causes oxidative stress in the liver and increases the expression of inflammatory mediators that cause hepatocellular damage. However, during chronic liver injury, it is unclear if/how these liver-derived factors affect distal tissues, such as the skin. The purpose of this study was to evaluate skin barrier function during chronic liver injury. Hairless mice were administered 5% or 10% ethanol for 8 weeks, and damages to the liver and skin were assessed using histological and protein-analysis methods, as well as by detecting inflammatory mediators in the plasma. After alcohol administration, the plasma concentration of the aspartate and alanine aminotransferases increased, while albumin levels decreased. In mice with alcohol-induced liver injury, transepidermal water loss was significantly increased, and skin hydration decreased concurrent with ceramide and type I collagen degradation. The plasma concentrations of [Formula: see text]/[Formula: see text] and tumor necrosis factor-alpha (TNF-α) were significantly increased in mice with induced liver injury. TNF receptor (TNFR) 2 expression was upregulated in the skin of alcohol-administered mice, while TNFR1 levels remained constant. Interestingly, the impairment of skin barrier function in mice administered with 10% ethanol was ameliorated by administering an anti-TNF-α antibody. We propose a novel mechanism whereby plasma TNF-α, via TNFR2 alone or with TNFR1, plays an important role in skin barrier function during chronic liver disease in these mouse models.

The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells

DEFF Research Database (Denmark)

Muñoz, A; Höppner, W; Sap, J

1990-01-01

To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon...

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

Science.gov (United States)

Mehrotra nee Tandon, P; Lind, D S; Bear, H D; Susskind, B M

1992-08-01

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.

Lack of co-ordinate expression of the alpha1(I) and alpha1(III) procollagen genes in fibroblast clonal cultures.

Science.gov (United States)

Yamaguchi, Y; Crane, S; Zhou, L; Ochoa, S M; Falanga, V

2000-12-01

Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.

Epidermal growth factor and active caspase-3 expression in the levator ani muscle of dogs with and without perineal hernia.

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Pérez-Gutiérrez, J F; Argüelles, J C; Iglesias-Núñez, M; Oliveira, K S; De La Muela, M Sánchez

2011-07-01

To perform a histological and immunohistochemical study of epidermal growth factor, transforming growth factor-alpha and their receptor, as well as the apoptotic signal active caspase-3 in the levator ani muscle of dogs with and without perineal hernia. Biopsy specimens of the levator ani muscle were obtained from 25 dogs with perineal hernia and 4 non-affected dogs and were processed for Masson and immunohistochemical staining. The affected dogs exhibited myopathological features, internalised nuclei, destruction and abnormal size of muscle fibres, which were replaced by collagen. The immunohistochemical study revealed active caspase-3, epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor in the levator ani. Compared to the healthy muscle, transforming growth factor-alpha staining intensity was lower in the affected muscle, whereas epidermal growth factor receptor and active caspase-3 staining were higher. Pelvic diaphragm muscle weakening is the leading cause of perineal hernia in the dog. Survival and death signals expressed in these muscles may contribute to the pathogenesis of this disease. This study reports epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor immunohistochemical expression in the skeletal muscle and suggests that perineal hernia in the dog is accompanied by levator ani muscle atrophy, increased expression of epidermal growth factor receptor, caspase-3 activation, and decreased expression of transforming growth factor-alpha. © 2011 British Small Animal Veterinary Association.

Anti-inflammatory effects of compounds alpha-humulene and (-)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea.

Science.gov (United States)

Fernandes, Elizabeth S; Passos, Giselle F; Medeiros, Rodrigo; da Cunha, Fernanda M; Ferreira, Juliano; Campos, Maria M; Pianowski, Luiz F; Calixto, João B

2007-08-27

This study evaluated the anti-inflammatory properties of two sesquiterpenes isolated from Cordia verbenacea's essential oil, alpha-humulene and (-)-trans-caryophyllene. Our results revealed that oral treatment with both compounds displayed marked inhibitory effects in different inflammatory experimental models in mice and rats. alpha-humulene and (-)-trans-caryophyllene were effective in reducing platelet activating factor-, bradykinin- and ovoalbumin-induced mouse paw oedema, while only alpha-humulene was able to diminish the oedema formation caused by histamine injection. Also, both compounds had important inhibitory effects on the mouse and rat carrageenan-induced paw oedema. Systemic treatment with alpha-humulene largely prevented both tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) generation in carrageenan-injected rats, whereas (-)-trans-caryophyllene diminished only TNFalpha release. Furthermore, both compounds reduced the production of prostaglandin E(2) (PGE(2)), as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression, induced by the intraplantar injection of carrageenan in rats. The anti-inflammatory effects of alpha-humulene and (-)-trans-caryophyllene were comparable to those observed in dexamethasone-treated animals, used as positive control drug. All these findings indicate that alpha-humulene and (-)-trans-caryophyllene, derived from the essential oil of C. verbenacea, might represent important tools for the management and/or treatment of inflammatory diseases.

Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

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Yoshioka, Toyo; Inagaki, Kenjiro [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Noguchi, Tetsuya, E-mail: noguchi@med.kobe-u.ac.jp [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Sakai, Mashito; Ogawa, Wataru; Hosooka, Tetsuya [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Iguchi, Haruhisa; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji [Genomic Science Laboratories, DainipponSumitomo Pharma Co. Ltd., 4-2-1 Takatsukasa, Takarazuka 665-8555 (Japan); Kasuga, Masato [Division of Diabetes, Metabolism, and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655 (Japan)

2009-04-17

The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.

Francisella tularensis elicits IL-10 via a PGE₂-inducible factor, to drive macrophage MARCH1 expression and class II down-regulation.

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Danielle Hunt

Full Text Available Francisella tularensis is a bacterial pathogen that uses host-derived PGE₂ to subvert the host's adaptive immune responses in multiple ways. Francisella-induced PGE₂ acts directly on CD4 T cells to blunt production of IFN-γ. Francisella-induced PGE₂ can also elicit production of a >10 kDa soluble host factor termed FTMØSN (F. tularensismacrophage supernatant, which acts on IFN-γ pre-activated MØ to down-regulate MHC class II expression via a ubiquitin-dependent mechanism, blocking antigen presentation to CD4 T cells. Here, we report that FTMØSN-induced down-regulation of MØ class II is the result of the induction of MARCH1, and that MØ expressing MARCH1 "resistant" class II molecules are resistant to FTMØSN-induced class II down-regulation. Since PGE₂ can induce IL-10 production and IL-10 is the only reported cytokine able to induce MARCH1 expression in monocytes and dendritic cells, these findings suggested that IL-10 is the active factor in FTMØSN. However, use of IL-10 knockout MØ established that IL-10 is not the active factor in FTMØSN, but rather that Francisella-elicited PGE₂ drives production of a >10 kDa host factor distinct from IL-10. This factor then drives MØ IL-10 production to induce MARCH1 expression and the resultant class II down-regulation. Since many human pathogens such as Salmonella typhi, Mycobacterium tuberculosis and Legionella pneumophila also induce production of host PGE₂, these results suggest that a yet-to-be-identified PGE₂-inducible host factor capable of inducing IL-10 is central to the immune evasion mechanisms of multiple important human pathogens.

Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

International Nuclear Information System (INIS)

Yasmin, Tania; Takahashi-Yanaga, Fumi; Mori, Jun; Miwa, Yoshikazu; Hirata, Masato; Watanabe, Yutaka; Morimoto, Sachio; Sasaguri, Toshiyuki

2005-01-01

To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of β-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3β (GSK-3β) and inhibition of GSK-3β attenuated the DIF-1-induced β-catenin degradation, indicating the involvement of GSK-3β in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/β-catenin signaling, resulting in the suppression of cyclin D1 promoter activity

Expression of interferon regulatory factor 4 in chronic myeloid leukemia: correlation with response to interferon alfa therapy.

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Schmidt, M; Hochhaus, A; König-Merediz, S A; Brendel, C; Proba, J; Hoppe, G J; Wittig, B; Ehninger, G; Hehlmann, R; Neubauer, A

2000-10-01

Mice experiments have established an important role for interferon regulatory factor (IRF) family members in hematopoiesis. We wanted to study the expression of interferon regulatory factor 4 (IRF4) in various hematologic disorders, especially chronic myeloid leukemia (CML), and its association with response to interferon alfa (IFN-alpha) treatment in CML. Blood samples from various hematopoietic cell lines, different leukemia patients (70 CML, 29 acute myeloid leukemia [AML], 10 chronic myelomonocytic leukemia [CMMoL], 10 acute lymphoblastic leukemia, and 10 chronic lymphoid leukemia patients), and 33 healthy volunteers were monitored for IRF4 expression by reverse transcriptase polymerase chain reaction. Then, with a focus on CML, the IRF4 level was determined in sorted cell subpopulations from CML patients and healthy volunteers and in in vitro-stimulated CML cells. Furthermore, IRF4 expression was compared in the CML samples taken before IFN-alpha therapy and in 47 additional CML samples taken during IFN-alpha therapy. IRF4 expression was then correlated with cytogenetic response to IFN-alpha. IRF4 expression was significantly impaired in CML, AML, and CMMoL samples. The downregulation of IRF4 in CML samples was predominantly found in T cells. In CML patients during IFN-alpha therapy, a significant increase in IRF4 levels was detected, and this was also observed in sorted T cells from CML patients. The increase seen during IFN-alpha therapy was not due to different blood counts. In regard to the cytogenetic response with IFN-alpha, a good response was associated with high IRF4 expression. IRF4 expression is downregulated in T cells of CML patients, and its increase is associated with a good response to IFN-alpha therapy. These data suggest IRF4 expression as a useful marker to monitor, if not predict, response to IFN-alpha in CML.

Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay.

Science.gov (United States)

Wang, Hui; Bi, Xiaohui; Xu, Lei; Li, Yirong

2017-01-01

Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen-anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime

The Ikaros transcription factor regulates responsiveness to IL-12 and expression of IL-2 receptor alpha in mature, activated CD8 T cells.

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Eric T Clambey

Full Text Available The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

Protection against cyanide-induced convulsions with alpha-ketoglutarate.

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Yamamoto, H

1990-04-30

Protection against convulsions induced by cyanide was observed after treatment with alpha-ketoglutarate, either alone or in combination with sodium thiosulfate, a classical antagonist for cyanide intoxication. However, sodium thiosulfate alone did not protect against cyanide (30 mg/kg)-induced convulsions. gamma-Aminobutyric acid (GABA) levels in brain were decreased by 31% in KCN-treated mice exhibiting convulsions. The combined administration of alpha-ketoglutarate and sodium thiosulfate completely abolished the decrease of GABA levels induced by cyanide. Furthermore, sodium thiosulfate alone also completely abolished the decrease of GABA levels. These results suggest that the depletion of brain GABA levels may not directly contribute to the development of convulsions induced by cyanide. On the other hand, cyanide increased calcium levels by 32% in brain crude mitochondrial fractions in mice with convulsions. The increased calcium levels were completely abolished by the combined administration of alpha-ketoglutarate and sodium thiosulfate, but not affected by sodium thiosulfate alone. These findings support the hypothesis proposed by Johnson et al. (Toxicol. Appl. Pharmacol., 84 (1986) 464) and Robinson et al. (Toxicology, 35 (1985) 59) that calcium may play an important role in mediating cyanide neurotoxicity.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

Science.gov (United States)

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages

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Chua Su-Kiat

2009-05-01

Full Text Available Abstract Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.

Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

International Nuclear Information System (INIS)

Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A.

1990-01-01

Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC

Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish.

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Köblitz, Louise; Fiechtner, Birgit; Baus, Katharina; Lussnig, Rebecca; Pelster, Bernd

2015-01-01

The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1α, HIF-2α and HIF-3α. Knock down of either Hif-1α or Hif-2α in mice results in lethality in embryonic or perinatal stages, suggesting that this transcription factor is not only controlling the hypoxic response, but is also involved in developmental phenomena. In the translucent zebrafish embryo the performance of the cardiovascular system is not essential for early development, therefore this study was designed to analyze the expression of the three Hif-isoforms during zebrafish development and to test the hypoxic inducibility of these transcription factors. To complement the existing zfHif-1α antibody we expressed the whole zfHif-2α protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3α protein were used for immunization and generation of a zfHif-3α specific antibody. To demonstrate presence of the Hif-isoforms during development [between 1 day post fertilization (1 dpf) and 9 dpf] affinity-purified antibodies were used. Hif-1α protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2α was also present from 1 dpf onwards, but in post hatching stages (between 5 and 9 dpf) the expression level was significantly higher than prior to hatching. Similarly, Hif-3α was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2α and Hif-3α play a particular role in early development. Hypoxic exposure (oxygen partial pressure = 5 kPa) in turn caused a significant increase in the level of Hif-1α protein even at 1 dpf and in later stages, while neither Hif-2α nor Hif-3α protein level were affected. In these early developmental stages Hif-1α therefore appears to be more important for

Expression of Estrogen Alpha and Beta Receptors in Prostate ...

African Journals Online (AJOL)

Expression of Estrogen Alpha and Beta Receptors in Prostate Cancer and Hyperplasia: Immunohistochemical Analysis. ... Additionally, ER-α was not expressed in either luminal or basal cells in any of the 35 BPH cases. However ... Key Words: ER-α, ER-β, prostate, hyperplasia, premalignant, cancer, immunohistochemistry ...

Heterologous expression of Homo sapiens alpha-folate receptors in E. coli by fusion with a trigger factor for enhanced solubilization.

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Miranda, Beatriz Nogueira Messias; Fotoran, Wesley Luzetti; Canduri, Fernanda; Souza, Dulce Helena Ferreira; Wunderlich, Gerhard; Carrilho, Emanuel

2018-02-01

The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

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Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

2011-04-15

Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNFalpha, anti-IL-1alpha/beta and IL-1Ra.

NARCIS (Netherlands)

Joosten, L.A.B.; Helsen, M.M.A.; Loo, F.A.J. van de; Berg, W.B. van den

2008-01-01

OBJECTIVE: To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis were treated

Co-receptor choice by V alpha14i NKT cells is driven by Th-POK expression rather than avoidance of CD8-mediated negative selection.

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Engel, Isaac; Hammond, Kirsten; Sullivan, Barbara A; He, Xi; Taniuchi, Ichiro; Kappes, Dietmar; Kronenberg, Mitchell

2010-05-10

Mouse natural killer T (NKT) cells with an invariant V alpha14-J alpha18 rearrangement (V alpha14 invariant [V alpha14i] NKT cells) are either CD4(+)CD8(-) or CD4(-)CD8(-). Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor alpha rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8(+) V alpha14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by V alpha14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of V alpha14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of V alpha14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing V alpha14i NKT cells.

Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

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Herr, D; Keck, C; Tempfer, C; Pietrowski, Detlef

2004-12-01

The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells. Copyright 2004 Wiley-Liss, Inc.

Insulin-Like Growth Factor-1 Inscribes a Gene Expression Profile for Angiogenic Factors and Cancer Progression in Breast Epithelial Cells

Directory of Open Access Journals (Sweden)

J.S. Oh

2002-01-01

Full Text Available Activation of the insulin-like growth factor-1 receptor (IGF-11R by IGF-1 is associated with the risk and progression of many types of cancer, although despite this it remains unclear how activated IGF-1 R contributes to cancer progression. In this study, gene expression changes elicited by IGF-1 were profiled in breast epithelial cells. We noted that many genes are functionally linked to cancer progression and angiogenesis. To validate some of the changes observed, the RNA and/or protein was confirmed for c-fos, cytochrome P4501Al, cytochrome P450 1131, interleukin-1 beta, fas ligand, vascular endothelial growth factor, and urokinase plasminogen activator. Nuclear proteins were also temporally monitored to address how gene expression changes were regulated. We found that IGF-1 stimulated the nuclear translocation of phosphorylated AKT, hypoxic-inducible factor-1 alpha, and phosphorylated cAMP-responsive element-binding protein, which correlated with temporal changes in gene expression. Next, the promoter regions of IGF-1-regulated genes were searched in silico. The promoters of genes that clustered together had similar regulatory regions. In summary, IGF-1 inscribes a gene expression profile relevant to cancer progression, and this study provides insight into the mechanism(s whereby some of these changes occur.

Effect of ascorbic acid and alpha-tocopherol supplementations on serum leptin, tumor necrosis factor alpha, and serum amyloid A levels in individuals with type 2 diabetes mellitus

Directory of Open Access Journals (Sweden)

Mostafa Jamalan

2015-10-01

Full Text Available Objective: Diabetes mellitus Type 2 is one of the most widespread chronic metabolic diseases. In most cases, this type of diabetes is associated with alterations in levels of some inflammatory cytokines and hormones. Considering anti-inflammatory properties of plant extracts rich in ascorbic acid (vitamin C and alpha-tocopherol (vitamin E, anti-diabetic properties of these two well-known antioxidant vitamins were investigated through measurement of serum levels of high-sensitivity C-reactive protein (hs-CRP, insulin, leptin, tumor necrosis factor alpha (TNF-α, and serum amyloid A (SAA in patients with diabetes mellitus type 2. Methods: Male patients (n=80 were randomly divided into two groups each consisted of 40 subjects. Test groups were supplemented with ascorbic acid (1000 mg/day or alpha-tocopherol (300 mg/day orally during four weeks. Before and after treatment, serum biochemical factors of subjects were measured and compared. Results: Our results showed that both ascorbic acid and alpha-tocopherol could induce significant anti-inflammatory effects by decreasing the level of inflammatory factors such as TNF-α, SAA, and hs-CRP in diabetes mellitus type 2 patients. Effects of alpha-tocopherol and ascorbic acid in decreasing serum leptin level were similar. Ascorbic acid in contrast to alpha-tocopherol diminished fasting insulin and HOMA index but had no effect on LDL serum level. Conclusion: Concerning the obtained results, it is concluded that consumption of supplementary vitamins C and E could decrease induced inflammatory response in patients with diabetes mellitus type 2.  It is also possible that vitamin C and vitamin E supplementation can attenuate incidence of some proposed pathological effects of diabetes mellitus.

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

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Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

2012-05-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats.

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Choi, Miyeon; Lee, Seung Hoon; Park, Min Hyeop; Kim, Yong-Seok; Son, Hyeon

2017-08-05

Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine. Copyright © 2017 Elsevier Inc. All rights reserved.

Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

International Nuclear Information System (INIS)

Chandrasekharan, Sabarinath; Kandasamy, Krishna Kumar; Dayalan, Pavithra; Ramamurthy, Viraragavan

2013-01-01

Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

Estrogen induced concentration dependent differential gene expression in human breast cancer (MCF7) cells: Role of transcription factors

Energy Technology Data Exchange (ETDEWEB)

Chandrasekharan, Sabarinath, E-mail: csab@bio.psgtech.ac.in [Department of Biotechnology, PSG College of Technology, Coimbatore 641004 (India); Kandasamy, Krishna Kumar [Max Planck Institute for Biology of Ageing, Cologne (Germany); Dayalan, Pavithra; Ramamurthy, Viraragavan [Department of Biotechnology, PSG College of Technology, Coimbatore 641004 (India)

2013-08-02

Highlights: •Estradiol (E2) at low dose induced cell proliferation in breast cancer cells. •E2 at high concentration induced cell stress in breast cancer cells. •Estrogen receptor physically interacts only with a few transcription factors. •Differential expression of genes with Oct-1 binding sites increased under stress. •Transcription factor binding sites showed distinct spatial distribution on genes. -- Abstract: Background: Breast cancer cells respond to estrogen in a concentration dependent fashion, resulting in proliferation or apoptosis. The mechanism of this concentration dependent differential outcome is not well understood yet. Methodology: Meta-analysis of the expression data of MCF7 cells treated with low (1 nM) or high (100 nM) dose of estradiol (E2) was performed. We identified genes differentially expressed at the low or the high dose, and examined the nature of regulatory elements in the vicinity of these genes. Specifically, we looked for the difference in the presence, abundance and spatial distribution of binding sites for estrogen receptor (ER) and selected transcription factors (TFs) in the genomic region up to 25 kb upstream and downstream from the transcription start site (TSS) of these genes. Results: It was observed that at high dose E2 induced the expression of stress responsive genes, while at low dose, genes involved in cell cycle were induced. We found that the occurrence of transcription factor binding regions (TFBRs) for certain factors such as Sp1 and SREBP1 were higher on regulatory regions of genes expressed at low dose. At high concentration of E2, genes with a higher frequency of Oct-1 binding regions were predominantly involved. In addition, there were differences in the spatial distribution pattern of the TFBRs in the genomic regions among the two sets of genes. Discussion: E2 induced predominantly proliferative/metabolic response at low concentrations; but at high concentration, stress–rescue responses were induced

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

Energy Technology Data Exchange (ETDEWEB)

Munoz, Juan Pablo; Collao, Andres; Chiong, Mario; Maldonado, Carola; Adasme, Tatiana; Carrasco, Loreto; Ocaranza, Paula; Bravo, Roberto; Gonzalez, Leticia; Diaz-Araya, Guillermo [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Hidalgo, Cecilia [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Lavandero, Sergio, E-mail: slavander@uchile.cl [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile)

2009-10-09

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

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...

Air pollution alters brain and pituitary endothelin-1 and inducible nitric oxide synthase gene expression.

Science.gov (United States)

Thomson, Errol M; Kumarathasan, Prem; Calderón-Garcidueñas, Lilian; Vincent, Renaud

2007-10-01

Recent work suggests that air pollution is a risk factor for cerebrovascular and neurodegenerative disease. Effects of inhaled pollutants on the production of vasoactive factors such as endothelin (ET) and nitric oxide (NO) in the brain may be relevant to disease pathogenesis. Inhaled pollutants increase circulating levels of ET-1 and ET-3, and the pituitary is a potential source of plasma ET, but the effects of pollutants on the expression of ET and NO synthase genes in the brain and pituitary are not known. In the present study, Fischer-344 rats were exposed by nose-only inhalation to particles (0, 5, 50mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Real-time reverse transcription polymerase chain reaction was used to measure mRNA levels in the cerebral hemisphere and pituitary 0 and 24 h post-exposure. Ozone inhalation significantly increased preproET-1 but decreased preproET-3 mRNAs in the cerebral hemisphere, while increasing mRNA levels of preproET-1, preproET-3, and the ET-converting enzyme (ECE)-1 in the pituitary. Inducible NO synthase (iNOS) was initially decreased in the cerebral hemisphere after ozone inhalation, but increased 24 h post-exposure. Particles decreased tumour necrosis factor (TNF)-alpha mRNA in the cerebral hemisphere, and both particles and ozone decreased TNF-alpha mRNA in the pituitary. Our results show that ozone and particulate matter rapidly modulate the expression of genes involved in key vasoregulatory pathways in the brain and pituitary, substantiating the notion that inhaled pollutants induce cerebrovascular effects.

Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation

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Stephan Niebler

2015-01-01

Full Text Available The transcription factor AP-2ε (activating enhancer-binding protein epsilon is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4 strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1, the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2′-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

Regulation of human lung fibroblast C1q-receptors by transforming growth factor-beta and tumor necrosis factor-alpha.

Science.gov (United States)

Lurton, J; Soto, H; Narayanan, A S; Raghu, G

1999-03-01

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are two polypeptide mediators which are believed to play a role in the evolution of idiopathic pulmonary fibrosis (IPF). We have evaluated the effect of these two substances on the expression of receptors for collagen (cC1q-R) and globular (gC1q-R) domains of C1q and on type I collagen in human lung fibroblasts. Two fibroblast subpopulations differing in C1q receptor expression were obtained by culturing human lung explants in medium containing fresh human serum and heated plasma-derived serum and separating them based on C1q binding [Narayanan, Lurton and Raghu: Am J Resp Cell Mol Biol. 1998; 17:84]. The cells, referred to as HH and NL cells, respectively, were exposed to TGF-beta and TNF-alpha in serum-free conditions. The levels of mRNA were assessed by in situ hybridization and Northern analysis, and protein levels compared after SDS-polyacrylamide gel electrophoresis and Western blotting. NL cells exposed to TGF-beta and TNF-alpha contained 1.4 and 1.6 times as much cC1q-R mRNA, respectively, whereas in HH cells cC1q-R mRNA increased 2.0- and 2.4-fold. The gC1q-R mRNA levels increased to a lesser extent in both cells. These increases were not reflected in protein levels of CC1q-R and gC1q-R, which were similar to or less than controls. Both TGF-beta and TNF-alpha also increased procollagen [I] mRNA levels in both cells. Overall, TNF-alpha caused a greater increase and the degree of response by HH fibroblasts to both TGF-beta and TNF-alpha was higher than NL cells. These results indicated that TGF-beta and TNF-alpha upregulate the mRNA levels for cC1q-R and collagen and that they do not affect gC1q-R mRNA levels significantly. They also indicated different subsets of human lung fibroblasts respond differently to inflammatory mediators.

Trovafloxacin-induced replication stress sensitizes HepG2 cells to tumor necrosis factor-alpha-induced cytotoxicity mediated by extracellular signal-regulated kinase and ataxia telangiectasia and Rad3-related

International Nuclear Information System (INIS)

Beggs, Kevin M.; Maiuri, Ashley R.; Fullerton, Aaron M.; Poulsen, Kyle L.; Breier, Anna B.; Ganey, Patricia E.; Roth, Robert A.

2015-01-01

Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress

Interleukin-10 to tumor necrosis factor-alpha ratio is a predictive biomarker in nonalcoholic fatty liver disease: interleukin-10 to tumor necrosis factor-alpha ratio in steatohepatitis.

Science.gov (United States)

Hashem, Reem M; Mahmoud, Mona F; El-Moselhy, Mohamed A; Soliman, Hala M

2008-10-01

Fatty liver disease is commonly associated with diabetes mellitus (DM). Insulin resistance (IR) as an investigative biomarker is only concerned with fatty liver that results from DM type 2 associated with metabolic syndrome. Irrespective of IR, DM is generally characterized by overproduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), whereas action of the latter is modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). The aim of this study was to investigate the efficacy of using TNF-alpha alone or IL-10/TNF-alpha ratio compared to IR, as a promising biomarker for fatty liver assessment in DM. Furthermore, we hypothesized that using garlic as an immunomodulator may decrease TNF-alpha and increase IL-10 production to improve steatohepatitis. DM was induced metabolically by a high-fat diet to bring about IR, or chemically by alloxan, producing insulin deficiency, in male albino rats. Garlic powder was supplemented (15 mg/kg per day) for 3 weeks. Fatty liver was depicted histologically and biochemically (aspartic aminotransferase, alanine aminotransferase, HOMA-IR, TNF-alpha, IL-10, IL-10/TNF-alpha ratio). We found that, in contrast to obese rats, garlic decreased IL-10/TNF-alpha ratio, despite decreasing TNF-alpha in alloxan diabetic rats in agreement with the histology, which revealed more prominent improvement in the obese group. Moreover, the effect of garlic was not linked to improvement of IR in obese rats. We conclude that IL-10/TNF-alpha ratio may be considered as a convenient biomarker for investigation of fatty liver of different grades, apart from being associated with IR, and immunomodulation of this ratio in favor of increasing it may exert significant improvement.

Triiodothyronine (T3) induces HIF1A and TGFA expression in MCF7 cells by activating PI3K.

Science.gov (United States)

Moretto, Fernanda Cristina Fontes; De Sibio, Maria Teresa; Luvizon, Aline Carbonera; Olimpio, Regiane Marques Castro; de Oliveira, Miriane; Alves, Carlos Augusto Barnabe; Conde, Sandro José; Nogueira, Célia Regina

2016-06-01

High expression levels of hypoxia inducing factor 1 alpha are related to mammary carcinogenesis. In previous studies, we demonstrated that expression of transforming growth factor alpha increases upon treatment with triiodothyronine, but this expression does not occur in cellular models that do not express the estrogen receptor, or when cells are co-treated with the anti-estrogen, tamoxifen. The aim of this study was to determine the effect of the hormone triiodothyronine on the expression of the genes HIF1A and TGFA in the breast cancer cell line MCF7. The cell line was subjected to treatment with triiodothyronine at the supraphysiological dose of 10(-8)M for 10min, 30min, 1h, and 4h in the presence or absence of actinomycin D, the gene expression inhibitor, cycloheximide, the protein synthesis inhibitor, and LY294002, the phosphoinositide 3 kinase inhibitor. HIF1A and TGFA mRNA expression was analyzed by reverse transcription polymerase chain reaction. For data analysis, we used analysis of variance complemented by Tukey test and an adopted minimum of 5% significance. We found that HIF1A and TGFA expression increased in the presence of triiodothyronine at all times studied. HIF1A expression decreased in triiodothyronine-treated cells when gene transcription was also inhibited; however, TGFA expression decreased after 10 and 30min of treatment even when transcription was not inhibited. We found that activation of PI3K was necessary for triiodothyronine to modulate HIF1A and TGFA expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of the local kinetics and localization of interleukin-1 alpha, tumour necrosis factor-alpha and transforming growth factor-beta, during the course of experimental pulmonary tuberculosis.