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673 Identification of a Novel Crosstalk Mechanism Between Protein Kinase D1 (PKD1) and p21-Activated Kinase (PAK) in Intestinal Epithelial Cells
AGA Abstracts
with increased GLP-1 positive L-cells (DIO:0.57±0.09 vs. RYGB:1.22±0.05 cells/crypt, p<0.0001) and 5-HT containing enterochromaffin (EC) cells (DIO:0.5±0.05 vs. RYGB:0.7±0.06, p=0.02) following RYGB. Furthermore, we explored expression of nutrient sensing GPCRs as we have previously shown fatty and amino acids receptors are found in the colon[3]. A significant increase in mRNA expression of fatty acid sensing receptors GPR40, GPR41, GPR43, GPR84, GPR120 and GPR119 was observed in RYGB mice compared to all other groups (Fig 1). Similar increase in amino acid sensing receptor GPR92 was observed (Fig 1). Finally, IHC staining for pan-neuronal marker PGP9.5 (lean:121.2±32.4 vs. DIO:197.3±39.8 vs. RYGB:149.8±33.7 pixels/image), and GLP-1 receptor (lean:64.7±18.5 vs. DIO:64.1±16.7 vs. RYGB:63.4±18.1 pixels/image) showed no difference between lean, DIO or RYGB. However, calretinin which labels vagal afferent fibres [4], significantly decreased in RYBG compared to lean (lean: 49.8±13.2 vs. RYGB:10.6±5.7 pixels/image, p= 0.0098). Conclusion: Expression of gut hormones and nutrient sensing receptors shows a key adaptive change accompanying increased numbers of L and EC cells after RYGB. This may serve to bolster effects of increased nutrient exposure on hormone responses. Despite up- regulation of enteroendocrine nutrient sensing mechanisms after RYGB, overall mucosal innervation is unaltered, with a persistent deficit in putative vagal afferent innervation. References: [1]Miras & le Roux. Nat Rev Gastroenterol Hepatol, 2013. [2]Shin et al, Endocrinology, 2010. [3]Symonds et al, Gut, 2015. [4]Dütsch et al, J Comp Neurol, 1998.
673 Identification of a Novel Crosstalk Mechanism Between Protein Kinase D1 (PKD1) and p21-Activated Kinase (PAK) in Intestinal Epithelial Cells James Sinnett-Smith, Jia Wang, Enrique Rozengurt Background: Protein kinase D1 (PKD1) is a critical node in the control of signal transduction in intestinal cells. Although the N-terminal region of PKD1 mediates auto-inhibition, membrane translocation, nuclear import and Golgi localization, surprisingly little is known about its regulation by post-translational modifications. Results: To determine whether activation of endogenous Gq-coupled receptors induces PKD1 phosphorylation in the N-terminal region of PKD1, cultures of intestinal epithelial IEC-18 cells were stimulated with the mitogenic agonist angiotensin II (ANGII). Cell lysates were analyzed by Western blotting for PKD1 phosphorylated at Ser203. ANGII induced a rapid (detectable within 30 s), sustained (persisted for at least 5 h) and striking increase in PKD1 phosphorylation at Ser203 in IEC18 cells. We also demonstrated PKD1 phosphorylation at Ser203 in response to GPCR activation in a variety of other cells, including Swiss 3T3 cells stimulated with the GI peptide bombesin. Treatment with the PKD family catalytic inhibitors kb NB 142-70 or CRT0066101 or with multiple PKC inhibitors prior to GPCR activation did not prevent Ser203 phosphorylation indicating that Ser203 phosphorylation is not due to autophosphorylation or phosphorylation by members of the PKC family. In striking contrast, treatment with structurally different PAK inhibitors, including PF-3758309 or FRAX597 prevented GPCR-induced PKD1 phosphorylation at Ser203 in all cell types tested, including IEC-18 or Swiss 3T3 cells. PF-3758309 is an ATP-competitive inhibitor of both PAK group I and II whereas FRAX597 is an ATPcompetitive inhibitor of group I PAKs. To corroborate the results obtained with the ATPcompetitive inhibitors PF-3758309 and FRAX597, we also tested the compound IPA-3, a highly specific allosteric inhibitor that binds covalently to the N-terminal regulatory domain of the PAK group I and inhibits activation by Rac1/Cdc42. Treatment with IPA-3, at concentrations that are biologically active in intact cells (5-20 mM), virtually abolished PKD1 phosphorylation at Ser203 in intestinal epithelial IEC-18 cells stimulated with ANGII or in Swiss 3T3 fibroblasts challenged with bombesin. Conclusions: The results presented here demonstrate that activation of GPCRs in intestinal epithelial cells and other cell systems induces rapid and striking phosphorylation of PKD1 at a novel site, the highly conserved Ser203 in the Nterminal region of PKD1. Results obtained with three structurally unrelated inhibitors that act via different mechanisms (PF-3758309, FRAX597 and IPA-3) indicate that group I PAK mediate PKD1 phosphorylation at Ser203 in intact cells, thus revealing a new crosstalk in signal transduction.
674 CREPT Plays an Oncogenic Role in Colorectal Cancer Through Promoting Wnt/b-Catenin Pathway via Enhancing Acetylation of b-catenin Yanquan Zhang, Shiyan Wang, Zhijie Chang, Joseph Sung, Jun Yu Background & aims: We recently reported that CREPT accelerates tumorigenesis by regulating the transcription of cell-cycle-related factors (Cancer Cell 2012). We aimed to elucidate the functional role and molecular mechanism of CREPT in colorectal cancer (CRC). Method: Copy number variation of CREPT was evaluated by Taqman Copy number assay. The biological function of CREPT was determined by cell proliferation, migration and invasion assays. The co-operator of CREPT was identified by co-immunoprecipitation (co-IP) and mass spectrometry (MS). The molecular mechanisms of CREPT were elucidated by luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and Western blot. Result: We first evaluated the expression of CREPT in CRC cell lines and primary CRC tissues. CREPT is upregulated in 5/9 CRC cells and in 13 primary CRC tumors as compared with adjacent normal tissues (p<0.01). Immunohistochemistry staining in tissue microarray ( n=160) demonstrated that CREPT staining is mainly located in nucleus; 47% (75/160) cases showed strong nuclear staining, 29.68% (47/160) cases showed moderate staining, 18.06% (29/160) showed weak staining, and only 5.16% (8/160) cases with negative staining. As CREPT gene localizes at the Ch20q11.23, a highly amplified region in CRC, we examined its copy number variation in 95 CRC tumor tissues using a specific Taqman probe against CREPT. Copy number gain or amplification of CREPT gene was found in 44% (42/95) CRC cases, which is positively correlated with its mRNA expression (p<0.01). These results suggested that copy number amplification of CREPT contributes to its upregulation in CRC. The functional role of CREPT in CRC was thus investigated. Ectopic expression of CREPT resulted in a significant increase in cell viability, colony formation, migration and invasion abilities in DLD-1 and HCT116 cells; whilst knockdown of CREPT had the opposite effects in HT29 and SW480 cells. We further revealed that CREPT enhanced transcriptional activity of bcatenin/TCF4 complex in CRC cells, consequently leading to increased expression of Cyclin D1 and c-Myc, downstream targets of b-catenin/TCF4. p300 was identified as a co-operator of CREPT by Co-IP followed by MS analyses. The direct interaction between CREPT and p300 was further confirmed by overexpression and endogenous IP experiments. CREPT improved the interaction between p300 and b-catenin, which promoted acetylation-mediated stabilization of b-catenin. Finally, we revealed that CREPT enhanced the complex formation of p300, b-catenin and TCF4, as evidenced by IP and EMSA analyses, thereby promoting the transcriptional activity of Wnt/ b-catenin signaling. Conclusion:CREPT plays a critical oncogenic role in CRC by activating the Wnt/ b-catenin pathway, an effect mediated by the recruitment of p300 to b-catenin, which leads to b-catenin acetylation and stabilization.
mRNA Expression Relative to 18s (2-DCt)
672 Zinc Transporter SLC39A7/ZIP7 Is Essential for Intestinal Homeostatic SelfRenewal Wakana Ohashi, Koji Hase, Toshiyuki Fukada Background: The essential trace element zinc is required for the normal functions of the many organs including the intestine. Dysregulation of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology. However, the molecular mechanisms by which zinc regulates intestinal physiology are largely unknown. A zinc-uptake transporter, Zrt-Irt-like protein 7 (ZIP7) is highly expressed in the intestinal crypt, but little is known about its role. Hence, we aimed to investigate the role of ZIP7 in intestinal epithelium in mice. Methods: We assessed the expressions of ZIP7 in intestinal epithelium by Q-PCR, western blot, and in situ hybridization analysis. We generated the mice with an inducible, intestinal epithelium-specific knockout of Zip7. To investigate the role of Zip7 in crypt cells, Intestinal stem cells and transit-amplifying (TA) cells from Zip7deficient mice were isolated and subjected to gene expression analysis. Results: In situ hybridization analysis revealed that Zip7 was distributed in the middle and lower crypt regions in a pattern much like that of TA cells. Immunohistochemistry confirmed that Zip7expressing cells were positive for Ki67, a marker for TA cells. Furthermore, Zip7 was also expressed in Lgr5+ stem cells, and post-mitotic Paneth cells. Zip7 deletion in intestinal epithelium caused a significant impairment of epithelial integrity. Electron microscopy revealed that Zip7 deletion triggered the death of massive numbers of TA cells. This abnormality was accompanied with loss of intestinal stem cells, suggesting an essential role of Zip7 in TA cell proliferation and stem cell maintenance. In gene expression profiling, unfolded protein response (UPR)-related genes were highly upregulated in the Zip7-deficient TA cells. Conclusions: UPR plays a key role in regulation of proliferative property of intestinal epithelium, whereas excessive UPR evokes unresolved ER stress. Our data demonstrated that Zip7-deficiency abnormally increases ER stress, leading to the massive death of TA cells followed by the loss of intestinal stem cells. These observations uncover a novel link between intracellular zinc regulation by ZIP7 and resolving ER stress to maintain homeostatic selfrenewal of intestinal epithelium.
675 Krüppel-Like Factor 4 Suppresses Intestinal Tumorigenesis by Regulating mTOR Signaling Pathway and DNA Repair Mechanisms Amr Ghaleb, Enas Elkarim, Agnieszka Bialkowska, Vincent W. Yang BACKGROUND: The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that KLF4 is a tumor suppressor in the intestinal tract and is important in DNA damage-repair mechanisms. In many cancer types, including colorectal cancer, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in DNA damage repair mechanisms are associated
AGA Abstracts
S-138
with increased GLP-1 positive L-cells (DIO:0.57±0.09 vs. RYGB:1.22±0.05 cells/crypt, p<0.0001) and 5-HT containing enterochromaffin (EC) cells (DIO:0.5±0.05 vs. RYGB:0.7±0.06, p=0.02) following RYGB. Furthermore, we explored expression of nutrient sensing GPCRs as we have previously shown fatty and amino acids receptors are found in the colon[3]. A significant increase in mRNA expression of fatty acid sensing receptors GPR40, GPR41, GPR43, GPR84, GPR120 and GPR119 was observed in RYGB mice compared to all other groups (Fig 1). Similar increase in amino acid sensing receptor GPR92 was observed (Fig 1). Finally, IHC staining for pan-neuronal marker PGP9.5 (lean:121.2±32.4 vs. DIO:197.3±39.8 vs. RYGB:149.8±33.7 pixels/image), and GLP-1 receptor (lean:64.7±18.5 vs. DIO:64.1±16.7 vs. RYGB:63.4±18.1 pixels/image) showed no difference between lean, DIO or RYGB. However, calretinin which labels vagal afferent fibres [4], significantly decreased in RYBG compared to lean (lean: 49.8±13.2 vs. RYGB:10.6±5.7 pixels/image, p= 0.0098). Conclusion: Expression of gut hormones and nutrient sensing receptors shows a key adaptive change accompanying increased numbers of L and EC cells after RYGB. This may serve to bolster effects of increased nutrient exposure on hormone responses. Despite up- regulation of enteroendocrine nutrient sensing mechanisms after RYGB, overall mucosal innervation is unaltered, with a persistent deficit in putative vagal afferent innervation. References: [1]Miras & le Roux. Nat Rev Gastroenterol Hepatol, 2013. [2]Shin et al, Endocrinology, 2010. [3]Symonds et al, Gut, 2015. [4]Dütsch et al, J Comp Neurol, 1998.
673 Identification of a Novel Crosstalk Mechanism Between Protein Kinase D1 (PKD1) and p21-Activated Kinase (PAK) in Intestinal Epithelial Cells James Sinnett-Smith, Jia Wang, Enrique Rozengurt Background: Protein kinase D1 (PKD1) is a critical node in the control of signal transduction in intestinal cells. Although the N-terminal region of PKD1 mediates auto-inhibition, membrane translocation, nuclear import and Golgi localization, surprisingly little is known about its regulation by post-translational modifications. Results: To determine whether activation of endogenous Gq-coupled receptors induces PKD1 phosphorylation in the N-terminal region of PKD1, cultures of intestinal epithelial IEC-18 cells were stimulated with the mitogenic agonist angiotensin II (ANGII). Cell lysates were analyzed by Western blotting for PKD1 phosphorylated at Ser203. ANGII induced a rapid (detectable within 30 s), sustained (persisted for at least 5 h) and striking increase in PKD1 phosphorylation at Ser203 in IEC18 cells. We also demonstrated PKD1 phosphorylation at Ser203 in response to GPCR activation in a variety of other cells, including Swiss 3T3 cells stimulated with the GI peptide bombesin. Treatment with the PKD family catalytic inhibitors kb NB 142-70 or CRT0066101 or with multiple PKC inhibitors prior to GPCR activation did not prevent Ser203 phosphorylation indicating that Ser203 phosphorylation is not due to autophosphorylation or phosphorylation by members of the PKC family. In striking contrast, treatment with structurally different PAK inhibitors, including PF-3758309 or FRAX597 prevented GPCR-induced PKD1 phosphorylation at Ser203 in all cell types tested, including IEC-18 or Swiss 3T3 cells. PF-3758309 is an ATP-competitive inhibitor of both PAK group I and II whereas FRAX597 is an ATPcompetitive inhibitor of group I PAKs. To corroborate the results obtained with the ATPcompetitive inhibitors PF-3758309 and FRAX597, we also tested the compound IPA-3, a highly specific allosteric inhibitor that binds covalently to the N-terminal regulatory domain of the PAK group I and inhibits activation by Rac1/Cdc42. Treatment with IPA-3, at concentrations that are biologically active in intact cells (5-20 mM), virtually abolished PKD1 phosphorylation at Ser203 in intestinal epithelial IEC-18 cells stimulated with ANGII or in Swiss 3T3 fibroblasts challenged with bombesin. Conclusions: The results presented here demonstrate that activation of GPCRs in intestinal epithelial cells and other cell systems induces rapid and striking phosphorylation of PKD1 at a novel site, the highly conserved Ser203 in the Nterminal region of PKD1. Results obtained with three structurally unrelated inhibitors that act via different mechanisms (PF-3758309, FRAX597 and IPA-3) indicate that group I PAK mediate PKD1 phosphorylation at Ser203 in intact cells, thus revealing a new crosstalk in signal transduction.
674 CREPT Plays an Oncogenic Role in Colorectal Cancer Through Promoting Wnt/b-Catenin Pathway via Enhancing Acetylation of b-catenin Yanquan Zhang, Shiyan Wang, Zhijie Chang, Joseph Sung, Jun Yu Background & aims: We recently reported that CREPT accelerates tumorigenesis by regulating the transcription of cell-cycle-related factors (Cancer Cell 2012). We aimed to elucidate the functional role and molecular mechanism of CREPT in colorectal cancer (CRC). Method: Copy number variation of CREPT was evaluated by Taqman Copy number assay. The biological function of CREPT was determined by cell proliferation, migration and invasion assays. The co-operator of CREPT was identified by co-immunoprecipitation (co-IP) and mass spectrometry (MS). The molecular mechanisms of CREPT were elucidated by luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and Western blot. Result: We first evaluated the expression of CREPT in CRC cell lines and primary CRC tissues. CREPT is upregulated in 5/9 CRC cells and in 13 primary CRC tumors as compared with adjacent normal tissues (p<0.01). Immunohistochemistry staining in tissue microarray ( n=160) demonstrated that CREPT staining is mainly located in nucleus; 47% (75/160) cases showed strong nuclear staining, 29.68% (47/160) cases showed moderate staining, 18.06% (29/160) showed weak staining, and only 5.16% (8/160) cases with negative staining. As CREPT gene localizes at the Ch20q11.23, a highly amplified region in CRC, we examined its copy number variation in 95 CRC tumor tissues using a specific Taqman probe against CREPT. Copy number gain or amplification of CREPT gene was found in 44% (42/95) CRC cases, which is positively correlated with its mRNA expression (p<0.01). These results suggested that copy number amplification of CREPT contributes to its upregulation in CRC. The functional role of CREPT in CRC was thus investigated. Ectopic expression of CREPT resulted in a significant increase in cell viability, colony formation, migration and invasion abilities in DLD-1 and HCT116 cells; whilst knockdown of CREPT had the opposite effects in HT29 and SW480 cells. We further revealed that CREPT enhanced transcriptional activity of bcatenin/TCF4 complex in CRC cells, consequently leading to increased expression of Cyclin D1 and c-Myc, downstream targets of b-catenin/TCF4. p300 was identified as a co-operator of CREPT by Co-IP followed by MS analyses. The direct interaction between CREPT and p300 was further confirmed by overexpression and endogenous IP experiments. CREPT improved the interaction between p300 and b-catenin, which promoted acetylation-mediated stabilization of b-catenin. Finally, we revealed that CREPT enhanced the complex formation of p300, b-catenin and TCF4, as evidenced by IP and EMSA analyses, thereby promoting the transcriptional activity of Wnt/ b-catenin signaling. Conclusion:CREPT plays a critical oncogenic role in CRC by activating the Wnt/ b-catenin pathway, an effect mediated by the recruitment of p300 to b-catenin, which leads to b-catenin acetylation and stabilization.
mRNA Expression Relative to 18s (2-DCt)
672 Zinc Transporter SLC39A7/ZIP7 Is Essential for Intestinal Homeostatic SelfRenewal Wakana Ohashi, Koji Hase, Toshiyuki Fukada Background: The essential trace element zinc is required for the normal functions of the many organs including the intestine. Dysregulation of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology. However, the molecular mechanisms by which zinc regulates intestinal physiology are largely unknown. A zinc-uptake transporter, Zrt-Irt-like protein 7 (ZIP7) is highly expressed in the intestinal crypt, but little is known about its role. Hence, we aimed to investigate the role of ZIP7 in intestinal epithelium in mice. Methods: We assessed the expressions of ZIP7 in intestinal epithelium by Q-PCR, western blot, and in situ hybridization analysis. We generated the mice with an inducible, intestinal epithelium-specific knockout of Zip7. To investigate the role of Zip7 in crypt cells, Intestinal stem cells and transit-amplifying (TA) cells from Zip7deficient mice were isolated and subjected to gene expression analysis. Results: In situ hybridization analysis revealed that Zip7 was distributed in the middle and lower crypt regions in a pattern much like that of TA cells. Immunohistochemistry confirmed that Zip7expressing cells were positive for Ki67, a marker for TA cells. Furthermore, Zip7 was also expressed in Lgr5+ stem cells, and post-mitotic Paneth cells. Zip7 deletion in intestinal epithelium caused a significant impairment of epithelial integrity. Electron microscopy revealed that Zip7 deletion triggered the death of massive numbers of TA cells. This abnormality was accompanied with loss of intestinal stem cells, suggesting an essential role of Zip7 in TA cell proliferation and stem cell maintenance. In gene expression profiling, unfolded protein response (UPR)-related genes were highly upregulated in the Zip7-deficient TA cells. Conclusions: UPR plays a key role in regulation of proliferative property of intestinal epithelium, whereas excessive UPR evokes unresolved ER stress. Our data demonstrated that Zip7-deficiency abnormally increases ER stress, leading to the massive death of TA cells followed by the loss of intestinal stem cells. These observations uncover a novel link between intracellular zinc regulation by ZIP7 and resolving ER stress to maintain homeostatic selfrenewal of intestinal epithelium.
675 Krüppel-Like Factor 4 Suppresses Intestinal Tumorigenesis by Regulating mTOR Signaling Pathway and DNA Repair Mechanisms Amr Ghaleb, Enas Elkarim, Agnieszka Bialkowska, Vincent W. Yang BACKGROUND: The zinc finger transcription factor Krüppel-like factor 4 (KLF4) is frequently down-regulated in colorectal cancer. Previous studies showed that KLF4 is a tumor suppressor in the intestinal tract and is important in DNA damage-repair mechanisms. In many cancer types, including colorectal cancer, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in DNA damage repair mechanisms are associated
AGA Abstracts
S-138