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W1594 Importins α1 and α3 Regulate VEGF Gene Expression and Angiogenesis in Gastric Mucosal Microvascular Endothelial Cells.
AGA Abstracts
2) and akt serine kinases. Results: GHS-R1a and ghrelin mRNA expression were detected in IEC6 cells. Ghrelin, at 10-5 to 10-8 M concentrations, significantly increased proliferation compared with control(0.57±0.09 vs 0.86±0.06 P<0.001). A reproducible stimulatory effect of desoctanoyl ghrelin was also observed (0.57±0.09 vs 0.79±0.10 P<0.001,compared with control). Ghrelin caused a significant increase in phosphorylated ERK 1/2 and akt serine kinases in immunoblotting,while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The proliferative effect of ghrelin and desoctanoyl ghrelin on IEC6 cells was abolished by the MAPK kinase inhibitor U0126, but not PI3 inhibitor Wortman, suggesting that the ghrelin-induced IEC-6 cell proliferation is mediated via a ERK-MAPKdependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by its inhibitors. Conclusion: GHS-R1a mRNA was detected in IEC6 cells. Ghrelin stimulates the proliferation of IEC-6 cell line. ERK and AKT activation is involved in mediating the effects of ghrelin on IEC-6 cell proliferation. Desoctanoyl ghrelin shows a similar effect. In conclusion, ghrelin may play a role in the proliferation of intestinal epithelial cells.
clocks to rhythms in a wide set of genes involved in diurnal regulation of intestinal morphology and function. W1592 Vitamin D3 and Its Nuclear Receptor VDR Regulate the Expression and Activity of the Human Intestinal Proton-Coupled Folate Transporter PCFT Jyrki J. Eloranta, Christian Hiller, Stephanie A. Häusler, Bruno Stieger, Gerd A. KullakUblick Background & Aims: Folates are essential for nucleic acid synthesis and are thus required particularly in rapidly dividing tissues such as intestinal epithelium and hematopoietic cells, as well as during pregnancy. Availability of dietary folates is determined by the rate of their transport across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical membrane of enterocytes. Whereas the transport properties of PCFT have been well characterized, little is known about the regulation of PCFT (gene symbol SLC46A1) gene expression in the intestine. Our aim was to study the mechanisms that regulate human PCFT promoter activity and expression in intestine-derived cells. Methods: All studies were performed in human intestinal Caco-2 cells. TaqMan real-time PCR was used to quantitate mRNA expression levels. Promoter activities were assayed in transient transfections, followed by dual luciferase assays. Protein-DNA binding was investigated in electrophoretic mobility shift assays. Cellular folate uptake was analyzed using [3H]radiolabelled folate. Results: PCFT mRNA levels were increased in cells treated with 1,25dihydroxyvitamin D3 (vitamin D3) in a dose-dependent fashion. The PCFT promoter region was transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-α (RXRα) in the presence of the ligand vitamin D3. In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region between nt -1694 bp and -1680 (-1694/-1680VDRE). DNA-binding assays, in combination with anti-VDR and antiRXRα antibodies and competitor oligonucleotides, showed direct and specific binding of the VDR:RXRα heterodimer to -1694/-1680VDRE, and mutation of this motif led to a reduction in VDR:RXRα binding. Functional analysis of the native PCFT promoter containing mutated -1694/-1680VDRE and heterologous promoter assays confirmed that this motif mediates a transcriptional response to vitamin D3. In functional support of the elucidated regulatory mechanism, treatment with vitamin D3 significantly increased the uptake of folic acid into Caco-2 cells at pH 5.5. Conclusions: Vitamin D3 and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D3 may thus have the added therapeutic benefit of enhancing the intestinal absorption of folate.
W1590 Protein Kinase D Activation and Function in Wounded Monolayers of Rat Intestinal IEC-18 and IEC-6 Cells James Sinnett-Smith, Steven H. Young, Xiaohua Jiang, Enrique Rozengurt Background: Within minutes of gastrointestinal epithelial injury, cells adjacent to a wound migrate over the denuded area to re-establish epithelial continuity, a process known as restitution. Many factors regulating epithelial cell migration have been defined through the use of In Vitro models employing monolayers of intestinal epithelial cells. However, the molecular mechanisms that mediate migration of intestinal epithelial cells in response to wounding and external stimuli remain incompletely understood. Studies with fibroblasts indicated that PKD, the founding member of a novel family of serine/threonine protein kinases, regulates directional migration but the role of PKD in epithelial cell migration remains unknown. Aim: To elucidate the role of PKD signaling in response to woundinduced migration in intestinal epithelial cells. Results: Using IEC18 and IEC6 monolayers, wounding produced a rapid (within 2-3 min) increase in PKD phosphorylated on Ser-916 in cells located at the edge of the wound rather than in the whole cell monolayer. We also determined PKD catalytic activation in response to wounding using In Vitro kinase assays. In order to maximize the number of cells at or near a wound, cell monolayers were injured by multiple parallel scrapes. A marked increase in PKD activity in wounded IEC18 monolayers was evident within 5 min after injury. Since PKD is rapidly activated in cells located at the edge of the wound, we hypothesized that PKD-mediated signaling plays a central role in promoting migration of intestinal epithelial cells. To test this hypothesis, we used siRNAs targeting PKD and non-targeting siRNA (as control) and determined woundinduced IEC-18 cell migration as the number of cells across the initial wound. siRNAs targeting PKD produced striking knockdown of PKD, as shown by Western blot analysis of cell lysates with an antibody directed against the C-terminal region of PKD. This antibody detected a doublet consisting of PKD (upper band, 110 kDa) and PKD2 (lower band, 105 kDa). The intensity of the PKD2 band was not changed by siRNAs targeting PKD, showing the specificity of the siRNAs used. We found that knockdown of PKD strikingly inhibited the migration of IEC-18 cells into the denuded area of a wound. In contrast, non-targeting siRNA did not prevent IEC-18 cell migration. Conclusion: Since PKD activation is one of the early signaling events initiated by wounding monolayers of intestinal epithelial cells and given that PKD is required for the migration of intestinal epithelial into the denuded area of the wound, we propose that PKD is one of the signaling pathways involved in restitution.
W1593 Genome-Wide Analysis of Androgen Receptor Binding in Hepatocellular Carcinoma Cells Hai Feng, Alfred S. Cheng, Minnie Y. Go, Jun Yu, Joseph J. Sung Background and Aim: Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide with gender prevalence observed in men. The gender difference has been attributed to the elevated androgen level and enhanced androgen receptor (AR) activity in male. Recent studies suggest that AR may promote hepatocarcinogenesis via increased cellular oxidative stress as well as suppression of p53-mediated DNA damage sensing/repairing system and cell apoptosis. However, the direct molecules executing the AR procarcinogenic activities remain largely undefined. In this study, we aimed to dissect the molecular mechanism by profiling the genome-wide binding pattern of AR in HCC cells. Methods: The expression and localization of AR in HCC cell lines were examined by Western blot and immunofluorescence, respectively. AR-over-expressing HCC cells were hormone-deprived before stimulation with AR agonist dihydrotestosterone (DHT, 100nM, 1hr). Chromatin immunoprecipitation microarray (or ChIP-chip) was performed in conjunction with tiled promoter arrays (Agilent Technologies) representing >488,000 loci in over ~17,000 best defined human transcripts. Candidate targets were confirmed by ChIP-PCR. Results: AR was over-expressed in Huh7 and PLC/PRF/5 HCC cells. Nuclear localization of AR was already evident before DHT stimulation in both cell lines, indicating active nuclear receptor signaling. ChIP-chip analysis revealed that AR occupied ~3% (500-600) of the interrogated promoters in each liver cancer cell line, similar with the frequency (~5%) observed in the prostate counterpart reported elsewhere. Twenty-five novel genome targets concurrently bound by AR in both cell lines were confirmed by ChIP-PCR. Many of the new AR target genes are either putative oncogenes (FLT1, KLHL2, CCRK) or tumor suppressors (NUDT2, STARD13, WWOX), and expressed aberrantly during liver carcinogenesis (CCRK, GADD45β, STARD13, WWOX). Notably, 32% of the validated AR targets possess Gene Ontology annotations for transcription, suggesting complex transcriptional changes downstream of AR that might be activated in HCC. Consistent with the tumorigenic role of AR as modulators of oxidative stress, DNA damage and cell apoptosis, genes associated with those processes were identified among our direct AR targets. Conclusion: Our results provide new insights into transcriptional regulation in AR gene network. Further studies are ongoing to functionally characterize the novel direct AR transcriptional targets in hepatocarcinogenesis. It will be interesting to assess whether these genes that regulate cancer-related functions emerge as therapeutic targets or diagnostic biomarkers for HCC.
W1591 Diurnally-Expressed MicroRNA Mir-16 Mediates Enterocyte Proliferation and Is Transciptionally Regulated By Circadian Clock Genes Anita Balakrishnan, Adam T. Stearns, David B. Rhoads, Stanley W. Ashley, Ali Tavakkolizadeh Introduction: microRNAs are short non-coding RNAs that regulate multiple genes simultaneously, altering cellular phenotype and function. The intestine undergoes significant diurnal rhythmicity in morphology and function, regulated in part by local clock genes acting through unknown pathways. We hypothesize that rhythmicity in gene expression and cellular proliferation is coordinated by rhythmicity in microRNAs, which are in turn regulated by clock genes in the intestine. Methods: Morphology, proliferation (BrdU labeling), and RNA and protein expression were examined in rat jejunal sections harvested at 3-h intervals. Temporal expression of microRNAs was profiled on microarrays and validated by qPCR. Regulation of mir-16 transcription by clock genes was examined by promoter-reporter assays in HEK293 cells (luciferase). Site-directed mutagenesis was used to test functionality of 5 putative Reverb binding sites. Regulation of mir-16 target genes cyclin D1 and cyclin E was assessed in IEC-6 cells by immunoblotting following mir-16 knockdown. Statistical analysis was carried out using ANOVA or t-tests. Results: Microarrays revealed diurnal rhythmicity in the anti-proliferative microRNA mir-16 in the jejunum (peak 10am, p<0.05). Of clock genes examined, Reverb A oscillated in the jejunum with a phase similar to mir-16 (peak 10am, p<0.01). The mir-16 promoter was stimulated 2.1-fold (p<0.01) by co-transfection with a Reverb A expression vector. Mutation of specific Reverb A response elements abolished this increase, suggesting that this clock gene regulates mir-16 expression in the intestine. Knockdown of mir-16 in IEC-6 cells increased protein levels of cyclins D1 and E by 1.9 and 2.2-fold respectively, identifying anti-proliferative activity of mir-16 in enterocytes (p<0.05). Cyclins D1 and E exhibited rhythmicity in native jejunum, peaking at 7pm (p<0.05) in antiphase to mir-16. Peak morphologic changes (crypt depth, villus height, enterocytes per crypt) occurred between 4-10pm, 6 hours after peak S-phase (10am, p<0.05). Conclusions: This is the first study to show that microRNAs regulate rhythms of proliferation on a diurnal basis. We show for the first time that mir-16 regulates intestinal proliferation by controlling G1/S transition via downregulation of key cell cycle regulators in enterocytes. Furthermore we show activity of clock genes in regulating microRNA expression. We therefore propose that microRNAs may act as essential effectors linking rhythmicity in local molecular
AGA Abstracts
W1594 Importins α1 and α3 Regulate VEGF Gene Expression and Angiogenesis in Gastric Mucosal Microvascular Endothelial Cells. Amrita Ahluwalia, Xiaoming Deng, Vipal Gandhi, Sushrut S. Thiruvengadam, Andrzej S. Tarnawski Background: Healing of gastric mucosal injury requires angiogenesis - new capillary blood vessel formation - that is critical for delivery of oxygen and nutrients to the healing site. VEGF is the most potent and endothelial specific regulator of angiogenesis. VEGF gene activation is dependent on translocation of transcription factors (e.g. HIF1 α) into the
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nucleus. Importins are proteins that stabilize and facilitate nuclear transport of transcription factors, but their roles in angiogenesis in general, and, specifically in angiogenesis in gastric mucosa remain unexplored. Material and Methods: We isolated gastric mucosal microvascular endothelial cells (GMMECs) from Fisher F-344 rats, 3 months of age using anti-PECAM-1 selection (Chemicon) and magnetic bead separation. PECAM-1 positive cells were separated by using a miniMACS column and grown on collagen coated dishes in endothelial cell growth media. The isolated GMMECs were characterized by immunohistochemical staining for the endothelial markers - von Willebrand's factor (Factor VIII -related antigen), PECAM1 (CD31) and VEGFR2. Cells were treated with either: 100nM control siRNA, importinα1 or - α3 specific siRNAs (Qiagen) for 48 or 72 hrs. Studies: 1) Importin α and VEGF mRNA expression using reverse transcription real-time PCR method with prevalidated QuantiTect assays (Qiagen, Valencia, CA), and, expression and subcellular localization of importin α and VEGF proteins using immunoflorescence studies; 2) In-vitro angiogenesis on matrigel and quantification of capillary-like endothelial tube formation using Metamorph image analysis system. Results: In control GMMECs, importin α and VEGF were strongly expressed and localized to the cell nuclei, with the former also expressed in the nuclear membrane. GMMECs plated on matrigel exhibited endothelial cell migration and capillary tube network and lumen formation at 6 hrs, reflecting in-vitro angiogenesis. Treatment of young GMMECs with importin α1 and α3 specific siRNAs significantly downregulated expression of importin α 1 and α3 (by 2.7-fold; p <0.05 and 2.0-fold; p <0.05 respectively), significantly reduced VEGF gene expression (by 3.3-fold; p <0.05 and 2.4-fold; p <0.05 respectively), and reduced in-vitro angiogenesis in GMMECs (by 1.7-fold; p<0.05 and 1.5-fold; p<0.05 respectively) vs. controls. Conclusions: 1) Knockdown of importin α1 and importin α3 significantly downregulates expression of VEGF and inhibits angiogenesis in gastric mucosal microvascular endothelial cells. 2) These data demonstrate a mechanistic role of importin α as one of the major players in gastric angiogenesis and also as a potential therapeutic target.
W1597 Site-Specific Expression and Modulation of P2X7 Receptor in the Gut and Gut-Associated Lymphoid Tissues Nathalia E. Campos, Carolina Souza, Morgana T. Castelo-Branco, Robson Coutinho-Silva, Heitor Souza Background and aims: The P2 purinergic receptor P2X7 is expressed on cells of the innate immune system and mediates responses of these cells to extracellular nucleotides, and is characterized as a regulator of pro-inflammatory cytokines. Extra-cellular nucleotides are released in conditions of cell stress or inflammation, and may function to alert the immune response. We assessed the sensitivity to ATP of gut-associated mononuclear cells, and the modulation of P2X7 in the intestinal mucosa. Methods: BALB/c, C57BL/6, and P2X7 deficient mice were used in this study. For isolation of mononuclear cells, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and peritoneum, were homogenized in PBS and plated in culture medium. For the permeabilization assay, mononuclear cells were incubated at 37oC for 15 minutes in the presence of different concentrations of ATP, and 2.5 microM ethidium bromide. Immediately after the incubation, ethidium bromide uptake was analyzed by flow cytometry. Mucosal samples of the colon were treated with 2 ng/mL interferon-gamma in organ cultures for 24 h and analyzed for the expression of P2X7 by immunohistochemistry. Results: In the MLNs and PPs, lymphocytes were more sensitive to ATP than macrophages, and the maximum response occurred with 500 microM ATP for lymphocytes and 5 mM for macrophages. Dose-response curves showed that the lymphocytes and, in a lesser extent, macrophages of MLNs and PPs are more sensitive to ATP than those from the peritoneum. Pre-incubation with the inhibitory cations Cd2+, Cu2+, or Zn2+, and with BBG, a specific P2X7 antagonist confirmed that P2X7 is one of the P2 receptors present in MLNs and PPs mononuclear cells. Incubation with interferon-gamma induced an up-regulation of P2X7 in the intestinal mucosa, in particular within the superficial epithelium. Conclusion: Our findings indicate a site-specific modulation of P2X7 receptors on immune cells and define purinergic signaling as a new regulatory element in the control of cell fate in the gutassociated lymphoid tissues. The epithelial P2X7 up-regulation in response to IFN-gamma is consistent with the involvement of purinoceptors in ATP-induced apoptotic cell death. Taken together, these observations may implicate P2X7 in the pathogenesis of diseases based on dysregulation of the immune response, such as inflammatory bowel disease.
W1595 Regulation of TFF and MUC Expression By SOX2 in Gastric Epithelial Cells Tadahito Shimada, Makoto Suzuki, Mariko Uchizono, Kyoko Tabei, Hideyuki Hiraishi Background and Aim: It is well established that SOX2, a SRY-related HMG box family transcription factor, is critically involved in organ development and cell differentiation in various tissues. Although previous studies demonstrated the expression of SOX2 in gastric epithelial cells, little is known about the regulation of stomach-specific gene expression by SOX2. In this study, we examined the effects of SOX2 on the expression of trefoil factor family peptides (TFF1, TFF2, and TFF3) and mucin core proteins (MUC2, MUC5AC, and MUC6) in gastric epithelial cells. Methods: MKN45 and AGS, cell lines derived from human gastric cancer, were used. SOX2 protein expression was examined by Western blot analysis. SOX2 expression vector was made by inserting the cording sequence of human SOX2 gene into pcDNA3.1/V5/His vector. Reporter genes were made by inserting PCR-amplified human TFF1 (-963 to +36), TFF2 (-912 to +24), TFF3 (-956 to +12), MUC2 (-1139 to +22), MUC5AC (-1363 to +24), and MUC6 (-1051 to +55) promoter sequences into pGL3basic vector. Expression of endogenous TFF1 and MUC6 mRNA was analyzed by real-time quantitative RT-PCR. Results: Both cell lines expressed endogenous SOX2 proteins. Among these TFFs and MUCs, reporter gene analyses showed that forced overexpression of SOX2 significantly down-regulates the transcription of TFF1 (60% suppression in MKN45 cells and 90% suppression in AGS cells) and up-regulates the transcription of MUC6 (1.8-fold increase in MKN45 cells and 2.3-fold increase in AGS cells). TFF2 and MUC5AC transcription was slightly repressed by SOX2 (about 20-25% suppression in both cell lines). TFF3 and MUC2 transcription was slightly up-regulated by SOX2 in MKN45 cells but slightly downregulated in AGS cells. Real-time quantitative RT-PCR also confirmed the down-regulation of endogenous TFF1 mRNA expression and up-regulation of MUC6 mRNA expression by SOX2 overexpression. Conclusions: These results suggest that SOX2 differentially affects the expression of stomach-specific TFFs (TFF1 and TFF2) and MUCs (MUC5AC and MUC6), and that SOX2 is not likely to be a key factor for the repression of intestine-specific TFF (TFF3) and MUC (MUC2) expression in gastric epithelial cells.
W1598 CD98 Regulates Cell-Cell Interactions Through Its Extracellular Phosphorylation Hang Thi Thu Nguyen, Guillaume Dalmasso, Yutao Yan, Tracy S. Obertone, Shanthi V. Sitaraman, Didier Merlin Extracellular (Ecto-) phosphorylation is emerging as an important mechanism in the regulation of many physiological processes, such as cell-cell interactions, cellular differentiation and proliferation, ion fluxes and cellular activation. Many cell-surface proteins, extracellular matrix proteins and soluble substrates have been shown to be phosphorylated by ectoprotein kinases (ePKs) using extracellular ATP as a phosphate donor. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated In Vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.
W1596 GATA-6 Mediates IL-1β-Induced Up-Regulation of RGS4 in Rabbit Colonic Smooth Muscle Cells Fang Li, Wu Deng, William Yen, Karnam S. Murthy, Wenhui Hu GATA transcription factors play important roles in cell proliferation and differentiation. GATA1/2/3 are predominantly expressed in hematopoietic cells and GATA4/5/6 are present in cardiovascular system and the gut. We have recently shown that the pro-inflammatory cytokine, interleukin-1β (IL-1β) induces up-regulation of RGS4 mRNA and protein expression in rabbit colonic smooth muscle cells by activating NF-κB signaling and MAPK pathways. Sequence analysis of rabbit RGS4 promoter (1.4 kb) predicts three sites for GATA transcription factors within the proximal region. Aim. To characterize the GATA sites within RGS4 promoter and define the role of GATA in regulating IL-1β-induced up-regulation of RGS4 expression. Methods. Various deletion mutants and site-directed mutants of RGS4 promoter (1.4 kb) were generated using pMluc3 secreted renilla luciferase reporter vector. Cultured rabbit colonic circular smooth muscle cells were transfected with Lipofectamine 2000 or infected with adenovirus carrying GATA-1 or GATA-6 vector. Adeno-EGFP control confirmed the transduction efficiency of over 95% in cultured colonic smooth muscle cells. The promoter activity was determined by measuring secreted renilla luciferase activity normalized by the activity of CMV-promoted firefly-luciferase. RGS4 mRNA expression was measured by realtime RT-PCR. Results. Removal of the first and second GATA sites including other transcription factor sites led to the increase of RGS4 promoter activity from 8-fold to 60-fold. In contrast, site-directed mutagenesis of the third GATA site (16 bp from the core TATA box) reduced the promoter activity to 15-fold. Adenovirus-mediated expression of GATA-6 but not GATA-1 significantly increased the promoter activity of RGS4(1.4 kb), which was further increased by IL-1β treatment. In addition, GATA-6 not GATA-1 significantly increased the constitutive and IL-1β-induced mRNA expression of the endogenous RGS4 in colonic smooth muscle cells. RT-PCR analysis demonstrated that only GATA-6 and GATA-4 were expressed in rabbit colonic smooth muscle cells. Conclusion. GATA site within the proximal region
W1599 Crohn's Disease Susceptibility Loci NOD2/CARD15/IBD1 and DEFB2 Are Direct and Indirect Targets of 1,25-Dihydroxyvitamin D3 Signaling John H. White, Tian-Tian Wang, Basel Dabbas, David Laperriere, Ari J. Bitton, Luz E. Tavera-Mendoza, Serge Dionne, Marc J. Servant, Marcel Behr, Ernest G. Seidman, Sylvie Mader, Hafid Soualhine BACKGROUND: Crohn's disease (CD), a chronic incurable inflammatory bowel condition, is believed to arise from defective innate immune regulation of intestinal bacterial load. Genome-wide association studies have identified polymorphisms associated with increased risk of CD pathogenesis in a number of loci, many of which encode proteins implicated in innate immune responses. Recent studies revealed that vitamin D signaling is a key regulator of innate immunity in humans. Notably, epidemiological studies and data from animal models of IBD have suggested that vitamin D deficiency increases the risk of development of CD. Vitamin D is produced by photochemical conversion of 7-dehydrocholesterol in skin by adequate levels of UVB radiation, which is increasingly limited at higher latitudes. Thus,
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AGA Abstracts
AGA Abstracts
is critical for RGS4 promoter activity. GATA-6 regulates the constitutive expression of RGS4 and mediates IL-1β-induced up-regulation of RGS4 expression in colonic smooth muscle cells. These data suggest that GATA factors may affect G protein signaling through regulating RGS expression.
2) and akt serine kinases. Results: GHS-R1a and ghrelin mRNA expression were detected in IEC6 cells. Ghrelin, at 10-5 to 10-8 M concentrations, significantly increased proliferation compared with control(0.57±0.09 vs 0.86±0.06 P<0.001). A reproducible stimulatory effect of desoctanoyl ghrelin was also observed (0.57±0.09 vs 0.79±0.10 P<0.001,compared with control). Ghrelin caused a significant increase in phosphorylated ERK 1/2 and akt serine kinases in immunoblotting,while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The proliferative effect of ghrelin and desoctanoyl ghrelin on IEC6 cells was abolished by the MAPK kinase inhibitor U0126, but not PI3 inhibitor Wortman, suggesting that the ghrelin-induced IEC-6 cell proliferation is mediated via a ERK-MAPKdependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by its inhibitors. Conclusion: GHS-R1a mRNA was detected in IEC6 cells. Ghrelin stimulates the proliferation of IEC-6 cell line. ERK and AKT activation is involved in mediating the effects of ghrelin on IEC-6 cell proliferation. Desoctanoyl ghrelin shows a similar effect. In conclusion, ghrelin may play a role in the proliferation of intestinal epithelial cells.
clocks to rhythms in a wide set of genes involved in diurnal regulation of intestinal morphology and function. W1592 Vitamin D3 and Its Nuclear Receptor VDR Regulate the Expression and Activity of the Human Intestinal Proton-Coupled Folate Transporter PCFT Jyrki J. Eloranta, Christian Hiller, Stephanie A. Häusler, Bruno Stieger, Gerd A. KullakUblick Background & Aims: Folates are essential for nucleic acid synthesis and are thus required particularly in rapidly dividing tissues such as intestinal epithelium and hematopoietic cells, as well as during pregnancy. Availability of dietary folates is determined by the rate of their transport across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical membrane of enterocytes. Whereas the transport properties of PCFT have been well characterized, little is known about the regulation of PCFT (gene symbol SLC46A1) gene expression in the intestine. Our aim was to study the mechanisms that regulate human PCFT promoter activity and expression in intestine-derived cells. Methods: All studies were performed in human intestinal Caco-2 cells. TaqMan real-time PCR was used to quantitate mRNA expression levels. Promoter activities were assayed in transient transfections, followed by dual luciferase assays. Protein-DNA binding was investigated in electrophoretic mobility shift assays. Cellular folate uptake was analyzed using [3H]radiolabelled folate. Results: PCFT mRNA levels were increased in cells treated with 1,25dihydroxyvitamin D3 (vitamin D3) in a dose-dependent fashion. The PCFT promoter region was transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-α (RXRα) in the presence of the ligand vitamin D3. In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region between nt -1694 bp and -1680 (-1694/-1680VDRE). DNA-binding assays, in combination with anti-VDR and antiRXRα antibodies and competitor oligonucleotides, showed direct and specific binding of the VDR:RXRα heterodimer to -1694/-1680VDRE, and mutation of this motif led to a reduction in VDR:RXRα binding. Functional analysis of the native PCFT promoter containing mutated -1694/-1680VDRE and heterologous promoter assays confirmed that this motif mediates a transcriptional response to vitamin D3. In functional support of the elucidated regulatory mechanism, treatment with vitamin D3 significantly increased the uptake of folic acid into Caco-2 cells at pH 5.5. Conclusions: Vitamin D3 and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D3 may thus have the added therapeutic benefit of enhancing the intestinal absorption of folate.
W1590 Protein Kinase D Activation and Function in Wounded Monolayers of Rat Intestinal IEC-18 and IEC-6 Cells James Sinnett-Smith, Steven H. Young, Xiaohua Jiang, Enrique Rozengurt Background: Within minutes of gastrointestinal epithelial injury, cells adjacent to a wound migrate over the denuded area to re-establish epithelial continuity, a process known as restitution. Many factors regulating epithelial cell migration have been defined through the use of In Vitro models employing monolayers of intestinal epithelial cells. However, the molecular mechanisms that mediate migration of intestinal epithelial cells in response to wounding and external stimuli remain incompletely understood. Studies with fibroblasts indicated that PKD, the founding member of a novel family of serine/threonine protein kinases, regulates directional migration but the role of PKD in epithelial cell migration remains unknown. Aim: To elucidate the role of PKD signaling in response to woundinduced migration in intestinal epithelial cells. Results: Using IEC18 and IEC6 monolayers, wounding produced a rapid (within 2-3 min) increase in PKD phosphorylated on Ser-916 in cells located at the edge of the wound rather than in the whole cell monolayer. We also determined PKD catalytic activation in response to wounding using In Vitro kinase assays. In order to maximize the number of cells at or near a wound, cell monolayers were injured by multiple parallel scrapes. A marked increase in PKD activity in wounded IEC18 monolayers was evident within 5 min after injury. Since PKD is rapidly activated in cells located at the edge of the wound, we hypothesized that PKD-mediated signaling plays a central role in promoting migration of intestinal epithelial cells. To test this hypothesis, we used siRNAs targeting PKD and non-targeting siRNA (as control) and determined woundinduced IEC-18 cell migration as the number of cells across the initial wound. siRNAs targeting PKD produced striking knockdown of PKD, as shown by Western blot analysis of cell lysates with an antibody directed against the C-terminal region of PKD. This antibody detected a doublet consisting of PKD (upper band, 110 kDa) and PKD2 (lower band, 105 kDa). The intensity of the PKD2 band was not changed by siRNAs targeting PKD, showing the specificity of the siRNAs used. We found that knockdown of PKD strikingly inhibited the migration of IEC-18 cells into the denuded area of a wound. In contrast, non-targeting siRNA did not prevent IEC-18 cell migration. Conclusion: Since PKD activation is one of the early signaling events initiated by wounding monolayers of intestinal epithelial cells and given that PKD is required for the migration of intestinal epithelial into the denuded area of the wound, we propose that PKD is one of the signaling pathways involved in restitution.
W1593 Genome-Wide Analysis of Androgen Receptor Binding in Hepatocellular Carcinoma Cells Hai Feng, Alfred S. Cheng, Minnie Y. Go, Jun Yu, Joseph J. Sung Background and Aim: Hepatocellular carcinoma (HCC) is one of the leading malignancies worldwide with gender prevalence observed in men. The gender difference has been attributed to the elevated androgen level and enhanced androgen receptor (AR) activity in male. Recent studies suggest that AR may promote hepatocarcinogenesis via increased cellular oxidative stress as well as suppression of p53-mediated DNA damage sensing/repairing system and cell apoptosis. However, the direct molecules executing the AR procarcinogenic activities remain largely undefined. In this study, we aimed to dissect the molecular mechanism by profiling the genome-wide binding pattern of AR in HCC cells. Methods: The expression and localization of AR in HCC cell lines were examined by Western blot and immunofluorescence, respectively. AR-over-expressing HCC cells were hormone-deprived before stimulation with AR agonist dihydrotestosterone (DHT, 100nM, 1hr). Chromatin immunoprecipitation microarray (or ChIP-chip) was performed in conjunction with tiled promoter arrays (Agilent Technologies) representing >488,000 loci in over ~17,000 best defined human transcripts. Candidate targets were confirmed by ChIP-PCR. Results: AR was over-expressed in Huh7 and PLC/PRF/5 HCC cells. Nuclear localization of AR was already evident before DHT stimulation in both cell lines, indicating active nuclear receptor signaling. ChIP-chip analysis revealed that AR occupied ~3% (500-600) of the interrogated promoters in each liver cancer cell line, similar with the frequency (~5%) observed in the prostate counterpart reported elsewhere. Twenty-five novel genome targets concurrently bound by AR in both cell lines were confirmed by ChIP-PCR. Many of the new AR target genes are either putative oncogenes (FLT1, KLHL2, CCRK) or tumor suppressors (NUDT2, STARD13, WWOX), and expressed aberrantly during liver carcinogenesis (CCRK, GADD45β, STARD13, WWOX). Notably, 32% of the validated AR targets possess Gene Ontology annotations for transcription, suggesting complex transcriptional changes downstream of AR that might be activated in HCC. Consistent with the tumorigenic role of AR as modulators of oxidative stress, DNA damage and cell apoptosis, genes associated with those processes were identified among our direct AR targets. Conclusion: Our results provide new insights into transcriptional regulation in AR gene network. Further studies are ongoing to functionally characterize the novel direct AR transcriptional targets in hepatocarcinogenesis. It will be interesting to assess whether these genes that regulate cancer-related functions emerge as therapeutic targets or diagnostic biomarkers for HCC.
W1591 Diurnally-Expressed MicroRNA Mir-16 Mediates Enterocyte Proliferation and Is Transciptionally Regulated By Circadian Clock Genes Anita Balakrishnan, Adam T. Stearns, David B. Rhoads, Stanley W. Ashley, Ali Tavakkolizadeh Introduction: microRNAs are short non-coding RNAs that regulate multiple genes simultaneously, altering cellular phenotype and function. The intestine undergoes significant diurnal rhythmicity in morphology and function, regulated in part by local clock genes acting through unknown pathways. We hypothesize that rhythmicity in gene expression and cellular proliferation is coordinated by rhythmicity in microRNAs, which are in turn regulated by clock genes in the intestine. Methods: Morphology, proliferation (BrdU labeling), and RNA and protein expression were examined in rat jejunal sections harvested at 3-h intervals. Temporal expression of microRNAs was profiled on microarrays and validated by qPCR. Regulation of mir-16 transcription by clock genes was examined by promoter-reporter assays in HEK293 cells (luciferase). Site-directed mutagenesis was used to test functionality of 5 putative Reverb binding sites. Regulation of mir-16 target genes cyclin D1 and cyclin E was assessed in IEC-6 cells by immunoblotting following mir-16 knockdown. Statistical analysis was carried out using ANOVA or t-tests. Results: Microarrays revealed diurnal rhythmicity in the anti-proliferative microRNA mir-16 in the jejunum (peak 10am, p<0.05). Of clock genes examined, Reverb A oscillated in the jejunum with a phase similar to mir-16 (peak 10am, p<0.01). The mir-16 promoter was stimulated 2.1-fold (p<0.01) by co-transfection with a Reverb A expression vector. Mutation of specific Reverb A response elements abolished this increase, suggesting that this clock gene regulates mir-16 expression in the intestine. Knockdown of mir-16 in IEC-6 cells increased protein levels of cyclins D1 and E by 1.9 and 2.2-fold respectively, identifying anti-proliferative activity of mir-16 in enterocytes (p<0.05). Cyclins D1 and E exhibited rhythmicity in native jejunum, peaking at 7pm (p<0.05) in antiphase to mir-16. Peak morphologic changes (crypt depth, villus height, enterocytes per crypt) occurred between 4-10pm, 6 hours after peak S-phase (10am, p<0.05). Conclusions: This is the first study to show that microRNAs regulate rhythms of proliferation on a diurnal basis. We show for the first time that mir-16 regulates intestinal proliferation by controlling G1/S transition via downregulation of key cell cycle regulators in enterocytes. Furthermore we show activity of clock genes in regulating microRNA expression. We therefore propose that microRNAs may act as essential effectors linking rhythmicity in local molecular
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W1594 Importins α1 and α3 Regulate VEGF Gene Expression and Angiogenesis in Gastric Mucosal Microvascular Endothelial Cells. Amrita Ahluwalia, Xiaoming Deng, Vipal Gandhi, Sushrut S. Thiruvengadam, Andrzej S. Tarnawski Background: Healing of gastric mucosal injury requires angiogenesis - new capillary blood vessel formation - that is critical for delivery of oxygen and nutrients to the healing site. VEGF is the most potent and endothelial specific regulator of angiogenesis. VEGF gene activation is dependent on translocation of transcription factors (e.g. HIF1 α) into the
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nucleus. Importins are proteins that stabilize and facilitate nuclear transport of transcription factors, but their roles in angiogenesis in general, and, specifically in angiogenesis in gastric mucosa remain unexplored. Material and Methods: We isolated gastric mucosal microvascular endothelial cells (GMMECs) from Fisher F-344 rats, 3 months of age using anti-PECAM-1 selection (Chemicon) and magnetic bead separation. PECAM-1 positive cells were separated by using a miniMACS column and grown on collagen coated dishes in endothelial cell growth media. The isolated GMMECs were characterized by immunohistochemical staining for the endothelial markers - von Willebrand's factor (Factor VIII -related antigen), PECAM1 (CD31) and VEGFR2. Cells were treated with either: 100nM control siRNA, importinα1 or - α3 specific siRNAs (Qiagen) for 48 or 72 hrs. Studies: 1) Importin α and VEGF mRNA expression using reverse transcription real-time PCR method with prevalidated QuantiTect assays (Qiagen, Valencia, CA), and, expression and subcellular localization of importin α and VEGF proteins using immunoflorescence studies; 2) In-vitro angiogenesis on matrigel and quantification of capillary-like endothelial tube formation using Metamorph image analysis system. Results: In control GMMECs, importin α and VEGF were strongly expressed and localized to the cell nuclei, with the former also expressed in the nuclear membrane. GMMECs plated on matrigel exhibited endothelial cell migration and capillary tube network and lumen formation at 6 hrs, reflecting in-vitro angiogenesis. Treatment of young GMMECs with importin α1 and α3 specific siRNAs significantly downregulated expression of importin α 1 and α3 (by 2.7-fold; p <0.05 and 2.0-fold; p <0.05 respectively), significantly reduced VEGF gene expression (by 3.3-fold; p <0.05 and 2.4-fold; p <0.05 respectively), and reduced in-vitro angiogenesis in GMMECs (by 1.7-fold; p<0.05 and 1.5-fold; p<0.05 respectively) vs. controls. Conclusions: 1) Knockdown of importin α1 and importin α3 significantly downregulates expression of VEGF and inhibits angiogenesis in gastric mucosal microvascular endothelial cells. 2) These data demonstrate a mechanistic role of importin α as one of the major players in gastric angiogenesis and also as a potential therapeutic target.
W1597 Site-Specific Expression and Modulation of P2X7 Receptor in the Gut and Gut-Associated Lymphoid Tissues Nathalia E. Campos, Carolina Souza, Morgana T. Castelo-Branco, Robson Coutinho-Silva, Heitor Souza Background and aims: The P2 purinergic receptor P2X7 is expressed on cells of the innate immune system and mediates responses of these cells to extracellular nucleotides, and is characterized as a regulator of pro-inflammatory cytokines. Extra-cellular nucleotides are released in conditions of cell stress or inflammation, and may function to alert the immune response. We assessed the sensitivity to ATP of gut-associated mononuclear cells, and the modulation of P2X7 in the intestinal mucosa. Methods: BALB/c, C57BL/6, and P2X7 deficient mice were used in this study. For isolation of mononuclear cells, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and peritoneum, were homogenized in PBS and plated in culture medium. For the permeabilization assay, mononuclear cells were incubated at 37oC for 15 minutes in the presence of different concentrations of ATP, and 2.5 microM ethidium bromide. Immediately after the incubation, ethidium bromide uptake was analyzed by flow cytometry. Mucosal samples of the colon were treated with 2 ng/mL interferon-gamma in organ cultures for 24 h and analyzed for the expression of P2X7 by immunohistochemistry. Results: In the MLNs and PPs, lymphocytes were more sensitive to ATP than macrophages, and the maximum response occurred with 500 microM ATP for lymphocytes and 5 mM for macrophages. Dose-response curves showed that the lymphocytes and, in a lesser extent, macrophages of MLNs and PPs are more sensitive to ATP than those from the peritoneum. Pre-incubation with the inhibitory cations Cd2+, Cu2+, or Zn2+, and with BBG, a specific P2X7 antagonist confirmed that P2X7 is one of the P2 receptors present in MLNs and PPs mononuclear cells. Incubation with interferon-gamma induced an up-regulation of P2X7 in the intestinal mucosa, in particular within the superficial epithelium. Conclusion: Our findings indicate a site-specific modulation of P2X7 receptors on immune cells and define purinergic signaling as a new regulatory element in the control of cell fate in the gutassociated lymphoid tissues. The epithelial P2X7 up-regulation in response to IFN-gamma is consistent with the involvement of purinoceptors in ATP-induced apoptotic cell death. Taken together, these observations may implicate P2X7 in the pathogenesis of diseases based on dysregulation of the immune response, such as inflammatory bowel disease.
W1595 Regulation of TFF and MUC Expression By SOX2 in Gastric Epithelial Cells Tadahito Shimada, Makoto Suzuki, Mariko Uchizono, Kyoko Tabei, Hideyuki Hiraishi Background and Aim: It is well established that SOX2, a SRY-related HMG box family transcription factor, is critically involved in organ development and cell differentiation in various tissues. Although previous studies demonstrated the expression of SOX2 in gastric epithelial cells, little is known about the regulation of stomach-specific gene expression by SOX2. In this study, we examined the effects of SOX2 on the expression of trefoil factor family peptides (TFF1, TFF2, and TFF3) and mucin core proteins (MUC2, MUC5AC, and MUC6) in gastric epithelial cells. Methods: MKN45 and AGS, cell lines derived from human gastric cancer, were used. SOX2 protein expression was examined by Western blot analysis. SOX2 expression vector was made by inserting the cording sequence of human SOX2 gene into pcDNA3.1/V5/His vector. Reporter genes were made by inserting PCR-amplified human TFF1 (-963 to +36), TFF2 (-912 to +24), TFF3 (-956 to +12), MUC2 (-1139 to +22), MUC5AC (-1363 to +24), and MUC6 (-1051 to +55) promoter sequences into pGL3basic vector. Expression of endogenous TFF1 and MUC6 mRNA was analyzed by real-time quantitative RT-PCR. Results: Both cell lines expressed endogenous SOX2 proteins. Among these TFFs and MUCs, reporter gene analyses showed that forced overexpression of SOX2 significantly down-regulates the transcription of TFF1 (60% suppression in MKN45 cells and 90% suppression in AGS cells) and up-regulates the transcription of MUC6 (1.8-fold increase in MKN45 cells and 2.3-fold increase in AGS cells). TFF2 and MUC5AC transcription was slightly repressed by SOX2 (about 20-25% suppression in both cell lines). TFF3 and MUC2 transcription was slightly up-regulated by SOX2 in MKN45 cells but slightly downregulated in AGS cells. Real-time quantitative RT-PCR also confirmed the down-regulation of endogenous TFF1 mRNA expression and up-regulation of MUC6 mRNA expression by SOX2 overexpression. Conclusions: These results suggest that SOX2 differentially affects the expression of stomach-specific TFFs (TFF1 and TFF2) and MUCs (MUC5AC and MUC6), and that SOX2 is not likely to be a key factor for the repression of intestine-specific TFF (TFF3) and MUC (MUC2) expression in gastric epithelial cells.
W1598 CD98 Regulates Cell-Cell Interactions Through Its Extracellular Phosphorylation Hang Thi Thu Nguyen, Guillaume Dalmasso, Yutao Yan, Tracy S. Obertone, Shanthi V. Sitaraman, Didier Merlin Extracellular (Ecto-) phosphorylation is emerging as an important mechanism in the regulation of many physiological processes, such as cell-cell interactions, cellular differentiation and proliferation, ion fluxes and cellular activation. Many cell-surface proteins, extracellular matrix proteins and soluble substrates have been shown to be phosphorylated by ectoprotein kinases (ePKs) using extracellular ATP as a phosphate donor. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated In Vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.
W1596 GATA-6 Mediates IL-1β-Induced Up-Regulation of RGS4 in Rabbit Colonic Smooth Muscle Cells Fang Li, Wu Deng, William Yen, Karnam S. Murthy, Wenhui Hu GATA transcription factors play important roles in cell proliferation and differentiation. GATA1/2/3 are predominantly expressed in hematopoietic cells and GATA4/5/6 are present in cardiovascular system and the gut. We have recently shown that the pro-inflammatory cytokine, interleukin-1β (IL-1β) induces up-regulation of RGS4 mRNA and protein expression in rabbit colonic smooth muscle cells by activating NF-κB signaling and MAPK pathways. Sequence analysis of rabbit RGS4 promoter (1.4 kb) predicts three sites for GATA transcription factors within the proximal region. Aim. To characterize the GATA sites within RGS4 promoter and define the role of GATA in regulating IL-1β-induced up-regulation of RGS4 expression. Methods. Various deletion mutants and site-directed mutants of RGS4 promoter (1.4 kb) were generated using pMluc3 secreted renilla luciferase reporter vector. Cultured rabbit colonic circular smooth muscle cells were transfected with Lipofectamine 2000 or infected with adenovirus carrying GATA-1 or GATA-6 vector. Adeno-EGFP control confirmed the transduction efficiency of over 95% in cultured colonic smooth muscle cells. The promoter activity was determined by measuring secreted renilla luciferase activity normalized by the activity of CMV-promoted firefly-luciferase. RGS4 mRNA expression was measured by realtime RT-PCR. Results. Removal of the first and second GATA sites including other transcription factor sites led to the increase of RGS4 promoter activity from 8-fold to 60-fold. In contrast, site-directed mutagenesis of the third GATA site (16 bp from the core TATA box) reduced the promoter activity to 15-fold. Adenovirus-mediated expression of GATA-6 but not GATA-1 significantly increased the promoter activity of RGS4(1.4 kb), which was further increased by IL-1β treatment. In addition, GATA-6 not GATA-1 significantly increased the constitutive and IL-1β-induced mRNA expression of the endogenous RGS4 in colonic smooth muscle cells. RT-PCR analysis demonstrated that only GATA-6 and GATA-4 were expressed in rabbit colonic smooth muscle cells. Conclusion. GATA site within the proximal region
W1599 Crohn's Disease Susceptibility Loci NOD2/CARD15/IBD1 and DEFB2 Are Direct and Indirect Targets of 1,25-Dihydroxyvitamin D3 Signaling John H. White, Tian-Tian Wang, Basel Dabbas, David Laperriere, Ari J. Bitton, Luz E. Tavera-Mendoza, Serge Dionne, Marc J. Servant, Marcel Behr, Ernest G. Seidman, Sylvie Mader, Hafid Soualhine BACKGROUND: Crohn's disease (CD), a chronic incurable inflammatory bowel condition, is believed to arise from defective innate immune regulation of intestinal bacterial load. Genome-wide association studies have identified polymorphisms associated with increased risk of CD pathogenesis in a number of loci, many of which encode proteins implicated in innate immune responses. Recent studies revealed that vitamin D signaling is a key regulator of innate immunity in humans. Notably, epidemiological studies and data from animal models of IBD have suggested that vitamin D deficiency increases the risk of development of CD. Vitamin D is produced by photochemical conversion of 7-dehydrocholesterol in skin by adequate levels of UVB radiation, which is increasingly limited at higher latitudes. Thus,
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is critical for RGS4 promoter activity. GATA-6 regulates the constitutive expression of RGS4 and mediates IL-1β-induced up-regulation of RGS4 expression in colonic smooth muscle cells. These data suggest that GATA factors may affect G protein signaling through regulating RGS expression.