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W1590 Protein Kinase D Activation and Function in Wounded Monolayers of Rat Intestinal IEC-18 and IEC-6 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
A-698
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
A-698