Nuclear receptor-mediated repression of human cholesterol 7alpha-hydroxylase gene transcription by bile acids.
Humans; Animals; Rats; Cell Line; Transfection; Liver/metabolism; Reverse Transcriptase Polymerase Chain Reaction; DNA/metabolism; CHO Cells; Cricetinae; Cholesterol 7-alpha-Hydroxylase/*genetics; Bile Acids and Salts/*pharmacology; *Membrane Glycoproteins; *Hydroxysteroid Dehydrogenases; Caco-2 Cells; Carrier Proteins/genetics/physiology; DNA-Binding Proteins/drug effects/genetics/physiology; Gene Expression/*drug effects; Kidney; Luciferases/genetics; Recombinant Fusion Proteins/metabolism; Retinoid X Receptors; Taurocholic Acid/pharmacology; Transcription Factors/drug effects/genetics/physiology; Cultured; Receptors; RNA; Genetic/drug effects; Messenger/analysis; Transcription; Genetic; Tumor Cells; Promoter Regions; Embryo; Cytoplasmic and Nuclear/genetics/*physiology; Mammalian; Retinoic Acid/genetics/physiology
Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated alpha-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species.
Chen W; Owsley E; Yang Y; Stroup D; Chiang J Y
Journal of lipid research
2001
2001-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Black currant anthocyanins abrogate oxidative stress through Nrf2- mediated antioxidant mechanisms in a rat model of hepatocellular carcinoma.
Male; Animals; Rats; Diet; Signal Transduction; NF-E2-Related Factor 2/genetics/*metabolism; *Phytotherapy; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Alkylating Agents/toxicity; Anthocyanins/*therapeutic use; Antioxidants/therapeutic use; Diethylnitrosamine/toxicity; Glutathione Transferase; Immunoenzyme Techniques; Lipid Peroxidation/drug effects; Nitric Oxide Synthase Type II/genetics/metabolism; Oxidative Stress/*drug effects; Ribes/*chemistry; Tyrosine/analogs & derivatives/metabolism; Carcinoma; Sprague-Dawley; Blotting; Western; RNA; Liver Neoplasms; Messenger/genetics; Experimental/chemically induced/metabolism/*prevention & control; Hepatocellular/chemically induced/metabolism/*prevention & control
Hepatocellular carcinoma (HCC), considered to be one of the most lethal cancers with almost \textgreater 1 million deaths reported annually worldwide, remains a devastating disease with no known effective cure. Hence, chemopreventive strategies come into play, offering an effective and safe mode of treatment, ideal to ward off potential cancer risks and mortality. A major predisposing condition, pertinent to the development and progression of HCC is oxidative stress. We previously reported a striking chemopreventive effect of anthocyanin-rich black currant skin extract (BCSE) against diethylnitrosamine (DENA)-initiated hepatocarcinogenesis in rats. The current study aims to elucidate the underlying antioxidant mechanisms of black currant anthocyanins implicated in the previously observed chemopreventive effects against experimental hepatocarcinogenesis. Dietary BCSE (100 and 500 mg/kg) administered four weeks before and 18 weeks after DENA challenge decreased abnormal lipid peroxidation, protein oxidation, and expression of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT) in a dose-responsive fashion. Mechanistic studies revealed that BCSE upregulated the gene expression of a number of hepatic antioxidant and carcinogen detoxifying enzymes, such as NAD(P)H:quinone oxidoreductase, glutathione S-transferase, and uridine diphosphate-glucuronosyltransferase isoenzymes, in DENA-initiated animals. Protein and mRNA expressions of nuclear factor E2-related factor 2 (Nrf2) were substantially elevated with BCSE treatment, providing a direct evidence of a coordinated activation of the Nrf2-regulated antioxidant pathway, which led to the upregulation of a variety of housekeeping genes. The results of our study provide substantial evidence that black currant bioactive anthocyanins exert chemopreventive actions against DENA-inflicted hepatocarcinogenesis by attenuating oxidative stress through activation of Nrf2 signaling pathway.
Thoppil Roslin J; Bhatia Deepak; Barnes Kendra F; Haznagy-Radnai Erzsebet; Hohmann Judit; Darvesh Altaf S; Bishayee Anupam
Current cancer drug targets
2012
2012-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.2174/156800912803987968" target="_blank" rel="noreferrer noopener">10.2174/156800912803987968</a>
A class of genes in the HER2 regulon that is poised for transcription in breast cancer cell lines and expressed in human breast tumors.
Humans; Cell Line; *Gene Expression Regulation; Reverse Transcriptase Polymerase Chain Reaction; *Gene Expression Profiling; Breast Neoplasms/genetics/pathology; Gene Regulatory Networks; Homeodomain Proteins/genetics/metabolism; MCF-7 Cells; Nanog Homeobox Protein; Neoplastic Stem Cells/metabolism; Octamer Transcription Factor-3/genetics/metabolism; Regulon/*genetics; RNA Polymerase II/metabolism; SOXB1 Transcription Factors/genetics/metabolism; Tumor Microenvironment/genetics; Receptor; Blotting; Western; Tumor; Neoplastic; ErbB-2/*genetics/metabolism
HER2-positive breast cancer accounts for 25% of all cases and has a poor prognosis. Although progress has been made in understanding signal transduction, little is known of how HER2 achieves gene regulation. We performed whole genome expression analysis on a HER2(+) and HER2(-) breast cancer cell lines and compared these results to expression in 812 primary tumors stratified by their HER2 expression level. Chip-on-chip with anti-RNA polymerase II was compared among breast cancer cell lines to identify genes that are potentially activated by HER2. The expression levels of these HER2-dependent POL II binding genes were determined for the 812 HER2+/- breast cancer tissues. Genes differentially expressed between HER2+/- cell lines were generally regulated in the same direction as in breast cancer tissues. We identified genes that had POLII binding in HER2(+) cell lines, but without significant gene expression. Of 737 such genes "poised" for expression in cell lines, 113 genes were significantly differentially expressed in breast tumors in a HER2-dependent manner. Pathway analysis of these 113 genes revealed that a large group of genes were associated with stem cell and progenitor cell control as indicated by networks centered on NANOG, SOX2, OCT3/4. HER2 directs POL II binding to a large number of genes in breast cancer cells. A "poised" class of genes in HER2(+) cell lines with POLII binding and low RNA expression but is differentially expressed in primary tumors, strongly suggests a role of the microenvironment and further suggests a role for stem cells proliferation in HER2-regulated breast cancer tissue.
Rahmatpanah Farah B; Jia Zhenyu; Chen Xin; Char Jessica E; Men Bozhao; Franke Anna-Clara; Jones Frank E; McClelland Michael; Mercola Dan
Oncotarget
2015
2015-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.18632/oncotarget.2676" target="_blank" rel="noreferrer noopener">10.18632/oncotarget.2676</a>
Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening.
Female; Animals; Immunohistochemistry; Pregnancy; Rats; Microcirculation; Phosphorylation; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Reverse Transcriptase Polymerase Chain Reaction; Cervical Ripening/*metabolism; Cervix Uteri/blood supply/innervation/*metabolism; Denervation; Nitric Oxide Synthase Type III; Nitric Oxide Synthase/biosynthesis; Protein Isoforms/biosynthesis/metabolism; Protein-Serine-Threonine Kinases/biosynthesis; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins/biosynthesis; Vascular Endothelial Growth Factor A/biosynthesis/*metabolism; Vascular Endothelial Growth Factor Receptor-2/biosynthesis; Wistar; Animal/*metabolism
Bilateral neurectomy of the pelvic nerve (BLPN) that carries uterine cervix-related sensory nerves induces dystocia, and administration of its vasoactive neuropeptides induces changes in the cervical microvasculature, resembling those that occur in the ripening cervix. This study was designed to test the hypothesis that (a) the cervix of pregnant rats expresses vascular endothelial growth factor (VEGF) and components of the angiogenic signaling pathway [VEGF receptors (Flt-1, KDR), activity of protein kinase B, Akt (phosphorylated Akt), and endothelial nitric oxide synthase (eNOS)] and von Willebrand Factor (vWF) and that these molecules undergo changes with pregnancy, and (b) bilateral pelvic neurectomy (BLPN) alters levels of VEGF concentration in the cervix. Using RT-PCR and sequencing, two VEGF isoforms, 120 and 164, were identified in the rat cervix. VEGF, VEGF receptor-1 (Flt-1), eNOS, and vWF immunoreactivities (ir) were localized in the microvasculature of cervical stroma. Their protein levels increased during pregnancy but decreased to control levels by 2 days postpartum. VEGF receptor-2 (KDR)-ir was confined to the epithelium of the endocervix. BLPN downregulated levels of VEGF by a third. Therefore, the components of the angiogenic signaling pathway are expressed in the cervix and change over pregnancy. Furthermore, angiogenic and sensory neuronal factors may be important in regulating the dynamic microvasculature in the ripening cervix and may subsequently play a role in cervical ripening and the birth process.
Mowa C N; Jesmin S; Sakuma I; Usip S; Togashi H; Yoshioka M; Hattori Y; Papka R
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
2004
2004-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1369/jhc.4A6455.2004" target="_blank" rel="noreferrer noopener">10.1369/jhc.4A6455.2004</a>
Induction of vascular progenitor cells from endothelial cells stimulates coronary collateral growth.
*Collateral Circulation; *Coronary Circulation; Animal; Animals; Biomarkers/metabolism; Cell Differentiation; Cell Lineage; Cells; Coronary Occlusion/genetics/metabolism/pathology/physiopathology/*surgery; Coronary Vessels/metabolism/pathology/*physiopathology; Cultured; Developmental; Disease Models; Endothelial Cells/metabolism/pathology/*transplantation; Epigenesis; Gene Expression Profiling; Gene Expression Regulation; Genetic; Induced Pluripotent Stem Cells/metabolism/*transplantation; Mice; Muscle; Myocytes; Neovascularization; Physiologic; Rats; Regenerative Medicine/methods; Regional Blood Flow; Reverse Transcriptase Polymerase Chain Reaction; SCID; Smooth; Smooth Muscle/metabolism/pathology/*transplantation; Sprague-Dawley; Teratoma/metabolism/pathology; Time Factors; Transcription Factors/genetics/metabolism; Transduction; Vascular/metabolism/pathology/*physiopathology
RATIONALE: A well-developed coronary collateral circulation improves the morbidity and mortality of patients following an acute coronary occlusion. Although regenerative medicine has great potential in stimulating vascular growth in the heart, to date there have been mixed results, and the ideal cell type for this therapy has not been resolved. OBJECTIVE: To generate induced vascular progenitor cells (iVPCs) from endothelial cells, which can differentiate into vascular smooth muscle cells (VSMCs) or endothelial cells (ECs), and test their capability to stimulate coronary collateral growth. METHODS AND RESULTS: We reprogrammed rat ECs with the transcription factors Oct4, Klf4, Sox2, and c-Myc. A population of reprogrammed cells was derived that expressed pluripotent markers Oct4, SSEA-1, Rex1, and AP and hemangioblast markers CD133, Flk1, and c-kit. These cells were designated iVPCs because they remained committed to vascular lineage and could differentiate into vascular ECs and VSMCs in vitro. The iVPCs demonstrated better in vitro angiogenic potential (tube network on 2-dimensional culture, tube formation in growth factor reduced Matrigel) than native ECs. The risk of teratoma formation in iVPCs is also reduced in comparison with fully reprogrammed induced pluripotent stem cells (iPSCs). When iVPCs were implanted into myocardium, they engrafted into blood vessels and increased coronary collateral flow (microspheres) and improved cardiac function (echocardiography) better than iPSCs, mesenchymal stem cells, native ECs, and sham treatments. CONCLUSIONS: We conclude that iVPCs, generated by partially reprogramming ECs, are an ideal cell type for cell-based therapy designed to stimulate coronary collateral growth.
Yin Liya; Ohanyan Vahagn; Pung Yuh Fen; Delucia Angelo; Bailey Erin; Enrick Molly; Stevanov Kelly; Kolz Christopher L; Guarini Giacinta; Chilian William M
Circulation research
2012
2012-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1161/CIRCRESAHA.111.250126" target="_blank" rel="noreferrer noopener">10.1161/CIRCRESAHA.111.250126</a>
Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4alpha and coactivators, and suppression of small heterodimer partner gene expression.
Antibiotics; Antitubercular/*pharmacology; Blotting; Cell Line; Chromatin/metabolism; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System/*biosynthesis; Cytoplasmic and Nuclear/*biosynthesis/*drug effects/genetics; Electrophoretic Mobility Shift Assay; Enzyme Induction/drug effects; Glutathione Transferase/metabolism; Hepatocyte Nuclear Factor 4/genetics/*metabolism; Hepatocytes/drug effects/metabolism; Humans; Immunoprecipitation; Messenger/biosynthesis; Plasmids/genetics; Pregnane X Receptor; Receptor Cross-Talk/drug effects; Receptors; Reverse Transcriptase Polymerase Chain Reaction; Rifampin/*pharmacology; RNA; Steroid/*drug effects/genetics; Transfection; Tumor; Western
Bile acids and drugs activate pregnane X receptor (PXR) to induce CYP3A4, which is the predominant cytochrome P450 enzyme expressed in the liver and intestine and plays a critical role in detoxifying bile acids and drugs, and protecting against cholestasis. The aim of this study is to investigate the molecular mechanism of PXR cross talk with other nuclear receptors and coactivators in regulating human CYP3A4 gene transcription. Rifampicin dose dependently induced the CYP3A4 but inhibited small heterodimer partner (SHP) mRNA expression levels in primary human hepatocytes. Rifampicin strongly stimulated PXR and hepatocyte nuclear factor 4alpha (HNF4alpha) interaction, and CYP3A4 reporter activity, which was further stimulated by peroxisome proliferators-activated receptorgamma co-activator 1alpha (PGC-1alpha) and steroid receptor coactivator-1 (SRC-1) but inhibited by SHP. Mutation of the putative HNF4alpha binding site in the distal xenobiotic responsive element module did not affect CYP3A4 basal promoter activity and synergistic stimulation by PXR and HNF4alpha. Chromatin immunoprecipitation assays revealed that rifampicin-activated PXR recruited HNF4alpha and SRC-1 to the CYP3A4 chromatin. On the other hand, SHP reduced PXR recruitment of HNF4alpha and SRC-1 to the CYP3A4 chromatin. The human SHP promoter was stimulated by HNF4alpha and PGC-1alpha. Upon activation by rifampicin, PXR inhibited SHP promoter activity. Results suggest that PXR strongly induces CYP3A4 gene transcription by interacting with HNF4alpha, SRC-1, and PGC-1alpha. PXR concomitantly inhibits SHP gene transcription and maximizes the PXR induction of the CYP3A4 gene in human livers. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs.
Li Tiangang; Chiang John Y L
Drug metabolism and disposition: the biological fate of chemicals
2006
2006-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1124/dmd.105.007575" target="_blank" rel="noreferrer noopener">10.1124/dmd.105.007575</a>
Effect of Pregnancy and Delivery on Cytokine Expression in a Mouse Model of Pelvic Organ Prolapse.
Amino Acid Oxidoreductases/*genetics/metabolism; Animal; Animals; Chemokine CCL7/genetics/metabolism; Chemokine CXCL12/genetics/metabolism; Delivery; Female; Humans; Knockout; Mice; Models; Obstetric/*adverse effects/methods; Pelvic Organ Prolapse/*genetics/metabolism/pathology; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; Urethra/metabolism; Urinary Bladder/metabolism; Vagina/metabolism
OBJECTIVES: The aim of this study was to determine the effect of pregnancy and delivery mode on cytokine expression in the pelvic organs and serum of lysyl oxidase like-1 knockout (LOXL1 KO) mice, which develop pelvic organ prolapse after delivery. METHODS: Bladder, urethra, vagina, rectum, and blood were harvested from female LOXL1 KO mice during pregnancy, after vaginal or cesarean delivery, and from sham cesarean and unmanipulated controls. Pelvic organs and blood were also harvested from pregnant and vaginally delivered wild-type (WT) mice and from unmanipulated female virgin WT controls. Specimens were assessed using quantitative real-time reverse transcription polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS: Both CXCL12 and CCL7 mRNA were significantly up-regulated in the vagina, urethra, bladder, and rectum of pregnant LOXL1 KO mice compared with pregnant WT mice, suggesting systemic dysregulation of both of these cytokines in LOXL1 KO mice as a response to pregnancy.The differences in cytokine expression between LOXL1 KO and WT mice in pregnancy persisted after vaginal delivery. CCL7 gene expression increases faster and to a greater extent in LOXL1 KO mice, translating to longer lasting increases in CCL7 in serum of LOXL1 KO mice after vaginal delivery, compared with pregnant mice. CONCLUSIONS: Lysyl oxidase like-1 KO mice have an increased cytokine response to pregnancy perhaps because they are less able to reform and re-cross-link stretched elastin to accommodate pups, and this resultant tissue stretches during pregnancy. The up-regulation of CCL7 after delivery could provide an indicator of level of childbirth injury, to which the urethra and vagina seem to be particularly vulnerable.
Couri Bruna M; Lenis Andrew T; Borazjani Ali; Balog Brian M; Kuang Mei; Butler Robert S; Penn Marc S; Damaser Margot S
Female pelvic medicine & reconstructive surgery
2017
2017-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1097/SPV.0000000000000394" target="_blank" rel="noreferrer noopener">10.1097/SPV.0000000000000394</a>
A Prospero-related homeodomain protein is a novel co-regulator of hepatocyte nuclear factor 4alpha that regulates the cholesterol 7alpha-hydroxylase gene.
*Gene Expression Regulation; Aged; Amino Acid Motifs; Bile Acids and Salts/metabolism; Cell Line; Cell Nucleus/metabolism; Cells; Cholesterol 7-alpha-Hydroxylase/*chemistry/*genetics; Cultured/metabolism; Enzymologic; Female; Genes; Genetic; Gluconeogenesis; Glutathione Transferase/metabolism; Hepatocyte Nuclear Factor 4/metabolism/*physiology; Hepatocytes/metabolism; Homeodomain Proteins/metabolism/*physiology; Humans; Immunoprecipitation; Liver/metabolism; Luciferases/metabolism; Male; Messenger/metabolism; Middle Aged; Phosphoenolpyruvate Carboxykinase (ATP)/metabolism; Plasmids/metabolism; Protein Structure; Reporter; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA; Small Interfering/metabolism; Tertiary; Time Factors; Transcription; Transcriptional Activation; Transfection; Tumor Suppressor Proteins; Two-Hybrid System Techniques
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.
Song Kwang-Hoon; Li Tiangang; Chiang John Y L
The Journal of biological chemistry
2006
2006-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M513420200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M513420200</a>
Bcl-2 positively regulates Sox9-dependent chondrocyte gene expression by suppressing the MEK-ERK1/2 signaling pathway.
*Gene Expression Regulation; Adenoviridae/genetics; Animals; Apoptosis; beta-Galactosidase/metabolism; Blotting; Butadienes/pharmacology; Caspase Inhibitors; Cell Differentiation; Cell Line; Chondrocytes/*metabolism; Collagen Type II/metabolism; Down-Regulation; Enzyme Inhibitors/pharmacology; Fibroblasts/metabolism; Fluorescence; Genetic; High Mobility Group Proteins/*metabolism; Lac Operon; Luciferases/metabolism; MAP Kinase Kinase Kinases/*metabolism; Messenger/metabolism; Microscopy; Mitogen-Activated Protein Kinase 1/*metabolism; Mitogen-Activated Protein Kinase 3/*metabolism; NF-kappa B/metabolism; Nitriles/pharmacology; Phenotype; Phosphorylation; Promoter Regions; Protein Kinase C-alpha; Protein Kinase C/antagonists & inhibitors; Proteoglycans/metabolism; Proto-Oncogene Proteins c-bcl-2/*metabolism; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Small Interfering/metabolism; SOX9 Transcription Factor; Sprague-Dawley; Time Factors; Transcription; Transcription Factors/*metabolism; Transfection; Western
Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase C alpha and NFkappaB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype.
Yagi Rieko; McBurney Denise; Horton Walter E Jr
The Journal of biological chemistry
2005
2005-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M502751200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M502751200</a>
Substance P in the uterine cervix, dorsal root ganglia and spinal cord during pregnancy and the effect of estrogen on SP synthesis.
Afferent/metabolism; Animals; Cervix Uteri/cytology/*metabolism; Down-Regulation; Estradiol/*analogs & derivatives/pharmacology; Estrogen/antagonists & inhibitors; Estrogens/*pharmacology; Female; Fulvestrant; Ganglia; Immunohistochemistry; In Situ Hybridization; Messenger/genetics/metabolism; Neurons; Postpartum Period; Pregnancy/*metabolism; Radioimmunoassay; Rats; Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA; Spinal Cord/cytology/*metabolism; Spinal/cytology/*metabolism; Sprague-Dawley; Substance P/*biosynthesis/genetics/metabolism; Up-Regulation
Prior to parturition the non-pliable uterine cervix undergoes a ripening process ("softens" and dilates) to allow a timely passage of the fetus at term. The exact mechanism(s) triggering and involved in cervical ripening are unknown, though evidence for a role for sensory neurons and their contained neuropeptides is emerging. Moreover, an apparent increase in neuropeptide immunoreactive nerves occurs in the cervix during pregnancy, maternal serum estrogen levels rise at term and uterine cervix-related L6-S1 dorsal root ganglia (DRG) sensory neurons express estrogen receptor (ER) and neuropeptides. Thus, we sought to test the hypothesis that the neuropeptide substance P (SP) changes biosynthesis and release over pregnancy, that estrogen, acting via the ER pathway, increases synthesis of SP in DRG, and that SP is utilized in cervical ripening at late pregnancy. Using immunohistochemistry, in situ hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR) and radioimmunoassay (RIA), we investigated coexpression of ER-alpha/beta and SP; differential expression of
Mowa C N; Usip S; Storey-Workley M; Amann R; Papka R
Peptides
2003
2003-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/s0196-9781(03)00120-7" target="_blank" rel="noreferrer noopener">10.1016/s0196-9781(03)00120-7</a>
Resveratrol inhibition of varicella-zoster virus replication in vitro.
Antiviral Agents/*pharmacology; Blotting; Cell Line; Dose-Response Relationship; Drug; Fibroblasts/virology; Herpesvirus 3; Human/*drug effects/physiology; Humans; Immediate-Early Proteins/biosynthesis; Messenger/biosynthesis; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA; Stilbenes/*pharmacology; Trans-Activators/biosynthesis; Viral Envelope Proteins/biosynthesis; Viral Plaque Assay; Viral/biosynthesis; Virus Attachment/drug effects; Virus Inactivation/drug effects; Virus Replication/*drug effects; Western
Resveratrol was found to inhibit varicella-zoster virus (VZV) replication in a dose-dependent and reversible manner. This decrease in virus production in the presence of resveratrol was not caused by direct inactivation of VZV or inhibition of virus attachment to MRC-5 cells. The drug effectively limited VZV replication if added during the first 30 h of infection. Western blot analysis and real-time RT-PCR studies demonstrated that protein and mRNA levels of IE62, an essential immediate early viral protein, were reduced when compared to controls. These results demonstrate that VZV replication is adversely affected by resveratrol which is negatively impacting IE62 synthesis.
Docherty John J; Sweet Thomas J; Bailey Erin; Faith Seth A; Booth Tristan
Antiviral research
2006
2006-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.antiviral.2006.07.004" target="_blank" rel="noreferrer noopener">10.1016/j.antiviral.2006.07.004</a>
Resveratrol suppresses nuclear factor-kappaB in herpes simplex virus infected cells.
Animals; Antiviral Agents/*pharmacology; Cell Nucleus/chemistry; Cercopithecus aethiops; Cytoplasm/chemistry; DNA; Electrophoretic Mobility Shift Assay; Fluorescence; Herpesvirus 1; Herpesvirus 2; Human/drug effects/genetics/*growth & development; Human/drug effects/genetics/*physiology; I-kappa B Proteins/metabolism; Messenger/biosynthesis; Microscopy; NF-kappa B/*metabolism; NF-KappaB Inhibitor alpha; Polymerase Chain Reaction; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA; Stilbenes/*pharmacology; Transcription Factor RelA/metabolism; Vero Cells; Viral/biosynthesis; Virus Replication/*drug effects
Resveratrol inhibits herpes simplex virus (HSV) replication by an unknown mechanism. Previously it was suggested that this inhibition may be mediated through a cellular factor essential for HSV replication [Docherty, J.J., Fu, M.M., Stiffler, B.S., Limperos, R.J., Pokabla, C.M., DeLucia, A.L., 1999. Resveratrol inhibition of herpes simplex virus replication. Antivir. Res. 43,
Faith Seth A; Sweet Thomas J; Bailey Erin; Booth Tristan; Docherty John J
Antiviral research
2006
2006-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.antiviral.2006.06.011" target="_blank" rel="noreferrer noopener">10.1016/j.antiviral.2006.06.011</a>
Analysis of connective tissues by laser capture microdissection and reverse transcriptase-polymerase chain reaction.
*Microdissection; Animals; Cell Separation/*methods; Chondrocytes/chemistry/cytology; Connective Tissue Cells/chemistry/*cytology; Gene Expression Profiling/*methods; Growth Plate/cytology; Inbred Strains; Lasers; Messenger/analysis/*isolation & purification; Methods; Mice; Newborn; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tibia/cytology
Studies of gene expression from bone, cartilage, and other tissues are complicated by the fact that their RNA, collected and pooled for analysis, often represents a wide variety of composite cells distinct in individual phenotype, age, and state of maturation. Laser capture microdissection (LCM) is a technique that allows specific cells to be isolated according to their phenotype, condition, or other marker from within such heterogeneity. As a result, this approach can yield RNA that is particular to a subset of cells comprising the total cell population of the tissue. This study reports the application of LCM to the gene expression analysis of the cartilaginous epiphyseal growth plate of normal newborn mice. The methodology utilized for this purpose has been coupled with real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) to quantitate the expression of certain genes involved in growth plate development and calcification. In this paper, the approaches used for isolating and purifying RNA from phenotypically specific chondrocyte populations of the murine growth plate are detailed and illustrate and compare both qualitative and quantitative RT-PCR results. The technique will hopefully serve as a guide for the further analysis of this and other connective tissues by LCM and RT-PCR.
Jacquet Robin; Hillyer Jennifer; Landis William J
Analytical biochemistry
2005
2005-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.ab.2004.09.033" target="_blank" rel="noreferrer noopener">10.1016/j.ab.2004.09.033</a>
The presence of extracellular matrix alters the chondrocyte response to endoplasmic reticulum stress.
*Stress; Animals; Apoptosis/drug effects; Articular/cytology; Blotting; Cartilage; Cattle; Cells; Chondrocytes/cytology/drug effects/*metabolism; Cultured; DNA-Binding Proteins/genetics/metabolism; Dose-Response Relationship; Drug; Endoplasmic Reticulum/*metabolism; Extracellular Matrix/*metabolism; Glucose/pharmacology; Heat-Shock Proteins/genetics/metabolism; Physiological; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism; Reverse Transcriptase Polymerase Chain Reaction; Thapsigargin/pharmacology; Time Factors; Transcription Factors/genetics/metabolism; Tunicamycin/pharmacology; Western
The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.
Nugent Ashleigh E; McBurney Denise L; Horton Walter E Jr
Journal of cellular biochemistry
2011
2011-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/jcb.23025" target="_blank" rel="noreferrer noopener">10.1002/jcb.23025</a>
Modified aminosilane substrates to evaluate osteoblast attachment, growth, and gene expression in vitro.
*Cell Adhesion; *Cell Division; *Gene Expression; *Silanes/chemistry; Alkaline Phosphatase/metabolism; Animals; Base Sequence; Chick Embryo; Collagen/metabolism; DNA Primers; Osteoblasts/*cytology/enzymology/metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA/isolation & purification/metabolism
Bone cell-substrate interactions are important to understand in the design, selection, and surface modification of bone implants. To gain insight into such interactions, substrates designed with surface species approximating the physiological environment of bone matrix were studied. Osteoblasts (Ob) grown on three such surfaces were used to evaluate cell-substrate effects on attachment, growth, and gene expression as compared with controls. Initial surface preparation consisted of coating glass slides with aminopropyltriethoxy silane (APTES), after which the coated slides were modified with collagen-rich extracellular matrix components obtained from normally mineralizing avian tendon: the tripeptide arginine-glycine-aspartic acid (arg-gly-asp), or a precipitate formed from a metastable solution containing inorganic ions normally found in blood (simulated body fluid). Each of the modified substrates, as well as the nonmodified (APTES) control, provided distinctly different physical (evidenced by differences in rms roughness) and chemical surfaces for seeding primary osteoblasts obtained from 14-day-old normal embryonic chickens. Cell responses to each of the substrates were evaluated over a 21-day period in terms of Ob growth and growth rate, alkaline phosphatase (ALP) activity, and gene expression of type I collagen (COL I), osteopontin (OPN), osteocalcin (OC), and bone sialoprotein (BSP). From these preliminary experiments, indications are that cell attachment and growth in this study possibly are independent processes, an assumption that compels the need for further studies. Collagen-rich matrix-modified substrates had a distinct advantage over others when cell growth rate, ALP activity, and gene expression were considered; cells on these substrates exhibited increased ALP activity and enhanced expression of BSP, OPN, and OC when compared with those of cells on APTES controls or other modified substrates. These results indicate that matrix-modified substrates such as those used in this study provide favorable templates for tissue generation, suggesting their potential in the design of surfaces for bone implants.
Siperko L M; Jacquet R; Landis W J
Journal of biomedical materials research. Part A
2006
2006-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/jbm.a.30731" target="_blank" rel="noreferrer noopener">10.1002/jbm.a.30731</a>
Bile acids and cytokines inhibit the human cholesterol 7 alpha-hydroxylase gene via the JNK/c-jun pathway in human liver cells.
Bile Acids and Salts/*metabolism; Cells; Chenodeoxycholic Acid/pharmacology; Cholesterol 7-alpha-Hydroxylase/*genetics/metabolism; Cultured; Cytokines/*metabolism; Gene Expression Regulation; Genetic; Hepatocytes/*cytology/drug effects; Humans; Immunoblotting; In Vitro Techniques; Interleukin-1/pharmacology; Messenger/analysis; Probability; Proto-Oncogene Proteins c-jun/*metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sensitivity and Specificity; Signal Transduction/genetics; Transcription
Cholesterol 7 alpha-hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen-activated protein kinase/c-Jun N-terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c-Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c-Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin-1 beta (IL-1 beta) regulation of human CYP7A1 gene expression via real-time polymerase chain reaction, reporter assays, co-immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL-1 beta and CDCA reduced CYP7A1 but induced c-Jun messenger RNA expression in human primary hepatocytes. IL-1beta inhibited human CYP7A1 reporter activity via the HNF4 alpha binding site. A JNK-specific inhibitor blocked the inhibitory effect of IL-1 beta on HNF4 alpha expression and CYP7A1 reporter activity. c-Jun inhibited HNF4 alpha and PPARgamma coactivator-1 alpha (PGC-1 alpha) coactivation of CYP7A1 reporter activity, whereas a dominant negative c-Jun did not. Co-immunoprecipitation and ChIP assays revealed that IL-1 beta and CDCA reduced HNF4 alpha bound to the CYP7A1 chromatin, and that c-Jun interacted with HNF4 alpha and blocked HNF4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin. In conclusion, IL-1 beta and CDCA inhibit HNF4 alpha but induce c-Jun, which in turn blocks HNF 4 alpha recruitment of PGC-1 alpha to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents.
Li Tiangang; Jahan Asmeen; Chiang John Y L
Hepatology (Baltimore, Md.)
2006
2006-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/hep.21183" target="_blank" rel="noreferrer noopener">10.1002/hep.21183</a>
MicroRNA-9 promotion of interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in interleukin-1beta-stimulated human chondrocytes.
Academic Medical Centers; Aged; Articular/drug effects/*metabolism; Cartilage; Cells; Chondrocytes – Physiology; Chondrocytes/drug effects/*metabolism; Cultured; Enzyme-Linked Immunosorbent Assay; Funding Source; Gene Knockdown Techniques; Hip/*genetics/metabolism; Human; Humans; Immunoblotting – Utilization; Immunoprecipitation; Interleukin-1beta/pharmacology; Interleukin-6/*genetics/metabolism; Interleukins – Physiology; Messenger/drug effects/*metabolism; MicroRNAs/drug effects/*genetics/metabolism; Middle Aged; Ohio; Osteoarthritis; Osteoarthritis – Complications; Osteoarthritis – Physiopathology; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases/drug effects/*genetics/metabolism; RNA; T-Tests; Transcription Factors/drug effects/*genetics/metabolism
OBJECTIVE: Enhanced expression of interleukin-6 (IL-6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) is a novel posttranscriptional regulator of IL-6 expression and is targeted by microRNA-9 (miR-9). We investigated the expression of MCPIP-1 in OA cartilage and explored whether targeting of MCPIP-1 by miR-9 contributes to enhanced IL-6 expression in OA. METHODS: Gene and protein expression in
Makki Mohammad S; Haseeb Abdul; Haqqi Tariq M
Arthritis & rheumatology (Hoboken, N.J.)
2015
2015-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/art.39173" target="_blank" rel="noreferrer noopener">10.1002/art.39173</a>