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Expression Profiling of the Proximal Small Intestine in Mature Mice With Intestine-Specific PDX1 Inactivation
genome-wide transcriptional profiles in the proximal small intestine. Differential expression of selected genes as identified by the microarray analysis was validated by real-time RTqPCR. Biological functions emphasized and potential networks formed by differentially expressed genes were analyzed by computer software. Results: Among more than 39,000 mouse transcripts screened by microarray analysis, expression of 86 transcripts is decreased or increased greater than 1.5-fold in mature proximal small intestine of Pdx1flox/flox;VilCre mice (p<0.05). Analysis of the differentially expressed genes with known functional annotations reveals that intestine-specific Pdx1 inactivation significantly alters expression of genes involved in biological functions including cellular movement, gene expression, cell death, cellular growth and proliferation, and lipid metabolism. Examination of the differentially expressed genes based on known relational interactions forms interconnected gene networks and reveals factors and signaling molecules that may be central to the alteration of gene expression in mature proximal small intestine by Pdx1 inactivation in mice. The factors and signaling molecules include p38 mitogen-activated protein kinases, cytokines tumor necrosis factor and growth hormone 1, transcription regulators jun proto-oncogene and hepatocyte nuclear factor 4 alpha, and alkaline phenyl phosphatases. Conclusions: Pdx1 inactivation in the epithelium of mature proximal small intestine in mice has a focused effect on altering the steady-state mRNA content of intestinal genes. The differential expression of genes associated with cell death, cellular movement, growth and proliferation implicates a role of Pdx1 in epithelial homeostasis in mature proximal small intestine. Moreover, expression profiling also reveals that Pdx1 may be involved in modulating lipid metabolism in mouse proximal small intestine.
Su1682 Transgenic Protein Kinase D1 Stimulates the Rate of Epithelial Cell Proliferation in Intestinal Crypts James Sinnett-Smith, Nora Rozengurt, Robert K. Kui, Carlos Huang, Enrique Rozengurt Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is tightly regulated by regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms. Despite its importance for understanding normal homeostasis and pathogenesis of disease states, the intracellular signal transduction mechanisms involved remain incompletely understood. Here, we tested the hypothesis that PKD1 plays a key role in intestinal crypt cell proliferation In Vivo. Results: To clarify the role of PKD1 in intestinal epithelial cell proliferation In Vivo, we generated transgenic mice that express elevated PKD1 protein in the distal small intestinal and proximal colonic epithelium. The catalytic activity and multi-site phosphorylation (Ser744, Ser748 and Ser916) of PKD1 was strikingly higher in extracts from ileal mucosa of transgenic mice as compared with non-transgenic littermates. These results indicate that transgenic PKD1 is functional in the intestinal epithelium. PKD1 signaling stimulated intestinal cell proliferation, as shown by detection of 5-bromo-2-deoxyuridine (BrdU) incorporated into the cell nuclei of crypt cells of the ileum and proximal colon, where PKD1 protein is maximally expressed. Our results demonstrated a highly statistically significant increase (p<0.005) in DNA synthesizing cells in the crypts of the PKD1 transgenic mice as compared with non-transgenic littermates. In the intestine, normal cell numbers are maintained by balancing rates of cell proliferation, differentiation, migration and apoptosis. Consequently, we determined whether transgenic PKD1 leads to a change in tissue architecture, manifested by an increase in the size and total number of epithelial cells in the crypts. We measured crypt height (in micrometer and cell number) and crypt circumference (in micrometer and cell number) in histological sections of control and PKD1 transgenic mice. The data was used to calculate the size of individual cells and the total number of cells per crypt. Our results show a significant increase in the depth (either in μm or in number of cells) and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the nontransgenic littermates (276 total cells per ileal crypt in transgenic mice versus 192 in nontransgenic mice; p< 0.005). These results indicate that the expression of the PKD1 transgene led to a marked increase (44%) in the total number of intestinal epithelial cells per crypt. Conclusion: Transgenic PKD1 expression increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. These results support the hypothesis that PKD1 signaling plays a role in a pathway leading to proliferation in intestinal epithelial cells.
Su1685 KrüPpel-Like Factor 4 Regulates Intestinal Epithelial Cell Morphology and Polarity Tianxin Yu, Ren Xu, Walden Ai, Chunming Liu BACKGROUND: KLF4 is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mouse demonstrated decrease in number of goblet cells in the colon and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. We aim to further define the role of KLF4 in differentiated intestinal cells and colon cancer cells, and the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine. RESULTS: KLF4 was partially depleted in the differentiated intestinal epithelial cells by a Tamoxifen-inducible Cre recombinase. We found that there was a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers, probably by inhibiting cell proliferation. The number and position of Paneth cells were also changed. This is consistent with the study that Wnt signaling induces maturation of Paneth cells and that KLF4 crosstalks with Wnt signaling in the intestine. We found that the morphology and polarity of the intestinal epithelial cells were altered by KLF4 deletion. Using a three-dimensional (3D) culture system for Caco-2 colon cancer cells, we found that KLF4 is essential for cell polarity and crypt-cyst formation. CONCLUSION: We demonstrated that KLF4 not only regulates cells differentiation, but also regulates the number and polarity of intestinal epithelial cells. KLF4 plays an important role in lumencyst formation in 3D cultures.
Su1683 Wnt Ligand Expression and Regulation of Stem Cells During Postnatal Growth of the Small Intestine in Rats Rino Donato, Jane K. Fauser, Imme A. Penttila, Ian C. Roberts-Thomson, Adrian G. Cummins The Wnt/β-catenin pathway is thought to play an important role in promoting crypt stem cells that mediate fission for intestinal growth. In rats, intestinal crypt fission peaks at day 14 of life (Dig Dis Sci 51:718-23, 2006). The expression of 84 Wnt pathway genes (including 18 of the 19 Wnt ligands) was investigated in the jejunum of 14 day old infant and 72 day old adult Hooded Wistar rats by real-time PCR array analysis. Wnt ligands with higher expression at day 14 were further investigated by real-time PCR analysis using different inhouse primers in 2, 7, 14, 21, 35 and 72 day old rats. Laser capture micro-dissection of jejunal sections from 14 day old rats was used to investigate Wnt expression in the crypt epithelium, pericryptal mesenchyme, villous epithelium and villous mesenchyme. In another experiment the expression of the stem cell marker Lgr5 was assessed in 14 day old rats with and without dickkopf-1 mediated blockade of Wnt signalling (100 ng per rat ip from days 9 to 13). Wnt3, Wnt4, Wnt5b, Wnt6, Wnt8b, Wnt9b and Wnt10a had 2-fold or greater mRNA expression in intestinal homogenates of full thickness jejunal segments of 14 day old compared to 72 day old rats. Expression of all these Wnts was low at day 2, increased at day 7, peaked at day 14 and decreased thereafter. Wnt3 was expressed in the crypt epithelium and Wnt5b in the pericryptal mesenchyme. Other Wnts were predominantly expressed in the villous epithelium or villous mesenchyme. In dickkopf-1 treated rats Lgr5 expression was decreased more than 2-fold. The results indicate enhanced expression of several Wnt ligands with crypt fission but only Wnt3 and Wnt5b were expressed in the region of crypt stem cells. Inhibition of the Wnt/β-catenin pathway by dickkopf-1 appears to suppress stem cell proliferation as indicated by reduced expression of Lgr5.
Su1686 Beta-Catenin Activation in the Developing Intestinal Tract Edwin K. McDonald, Tatiana Goretsky, Rebecca B. Katzman, Terrence A. Barrett Background and AIMS: Mechanisms responsible for normal crypt growth and development in the small bowel and colon are not well understood. Previous studies suggest that protein kinase Akt (Akt) signaling activates phosphorylated β-catenin(P-β-catenin552) in intestinal stem and progenitor cells in the progression of inflammation to dysplasia to colitis associated cancer. We sought to investigate β-catenin activation in the small bowel and colon. Methods: C57BL/6 mice were sacrificed at 7,14, and 21 days post birth. Alterations in intestinal morphology were assessed by H&E staining. The effects of early development on P-β catenin and Akt activation in the small bowel and colon were assessed by immunohistochemistry. Epithelial cell apoptosis was detected by TUNEL. Epithelial cell proliferation was assessed by Brdu staining. Results: Histologic analysis revealed paneth cell hypertrophy in 7 and 14 day old mice that resolved by 21 days. Crypt size and submucosal thickness progressed significantly between the time points. Brdu staining demonstrated reduced proliferation in the small bowel and colon as through the time points. There were no significant differences in apotosis as detected by TUNEL between the groups. Akt levels detected by IHC were also unchanged throughout the time points. P-β-catenin activation was significantly more predominant in the small bowel at 7 and 14 days. There were no significant differences in P-β-catenin in the small bowel and colon at 21 days. Conclusions: P-β-catenin activation in the developing intestinal tract involves signaling besides Akt.
Su1684 Expression Profiling of the Proximal Small Intestine in Mature Mice With Intestine-Specific PDX1 Inactivation Chin Chen, Eric Sibley
Su1687 Genetically-Altered Enteric Nervous System Results in an Amplified Adaptation Response to Massive Small Bowel Resection Meredith C. Hitch, Jennifer A. Leinicke, Jun Guo, Christopher R. Erwin, Robert O. Heuckeroth, Brad W. Warner
Background: Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, less is known about the function of Pdx1 in the small intestine. Objective: We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated following inactivation of Pdx1 specifically in the intestinal epithelium. Methods: Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1flox/flox;VilCre mice, by crossing mutant mice homozygous for loxP site-flanked Pdx1 alleles with transgenic mice expressing Cre recombinase under the control of the mouse villin 1 gene promoter. Total RNA was isolated from the first five centimeters of the small intestine from adult Pdx1flox/ flox ;VilCre and littermate control mice. Microarray analysis was performed to investigate
Introduction: Intestinal adaptation following massive small bowel resection (SBR) is a crucial compensatory response to enhance absorptive and digestive surface area within the remnant bowel. Adaptation is characterized by increased villus height and crypt depth as a consequence of a mitogenic stimulus to the intestinal crypt cells via unknown mechanisms. A few prior studies suggested that the enteric nervous system (ENS) might regulate enterocyte proliferation. The purpose of the present study was to elucidate the contribution of the
S-473
AGA Abstracts
AGA Abstracts
population. The presence of autophagy in this specific cell population could be essential for the maintenance of homeostasis and for the prevention of malignant transformation via regulation of the Wnt/β-catenin pathway.
Su1682 Transgenic Protein Kinase D1 Stimulates the Rate of Epithelial Cell Proliferation in Intestinal Crypts James Sinnett-Smith, Nora Rozengurt, Robert K. Kui, Carlos Huang, Enrique Rozengurt Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is tightly regulated by regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms. Despite its importance for understanding normal homeostasis and pathogenesis of disease states, the intracellular signal transduction mechanisms involved remain incompletely understood. Here, we tested the hypothesis that PKD1 plays a key role in intestinal crypt cell proliferation In Vivo. Results: To clarify the role of PKD1 in intestinal epithelial cell proliferation In Vivo, we generated transgenic mice that express elevated PKD1 protein in the distal small intestinal and proximal colonic epithelium. The catalytic activity and multi-site phosphorylation (Ser744, Ser748 and Ser916) of PKD1 was strikingly higher in extracts from ileal mucosa of transgenic mice as compared with non-transgenic littermates. These results indicate that transgenic PKD1 is functional in the intestinal epithelium. PKD1 signaling stimulated intestinal cell proliferation, as shown by detection of 5-bromo-2-deoxyuridine (BrdU) incorporated into the cell nuclei of crypt cells of the ileum and proximal colon, where PKD1 protein is maximally expressed. Our results demonstrated a highly statistically significant increase (p<0.005) in DNA synthesizing cells in the crypts of the PKD1 transgenic mice as compared with non-transgenic littermates. In the intestine, normal cell numbers are maintained by balancing rates of cell proliferation, differentiation, migration and apoptosis. Consequently, we determined whether transgenic PKD1 leads to a change in tissue architecture, manifested by an increase in the size and total number of epithelial cells in the crypts. We measured crypt height (in micrometer and cell number) and crypt circumference (in micrometer and cell number) in histological sections of control and PKD1 transgenic mice. The data was used to calculate the size of individual cells and the total number of cells per crypt. Our results show a significant increase in the depth (either in μm or in number of cells) and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the nontransgenic littermates (276 total cells per ileal crypt in transgenic mice versus 192 in nontransgenic mice; p< 0.005). These results indicate that the expression of the PKD1 transgene led to a marked increase (44%) in the total number of intestinal epithelial cells per crypt. Conclusion: Transgenic PKD1 expression increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. These results support the hypothesis that PKD1 signaling plays a role in a pathway leading to proliferation in intestinal epithelial cells.
Su1685 KrüPpel-Like Factor 4 Regulates Intestinal Epithelial Cell Morphology and Polarity Tianxin Yu, Ren Xu, Walden Ai, Chunming Liu BACKGROUND: KLF4 is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mouse demonstrated decrease in number of goblet cells in the colon and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. We aim to further define the role of KLF4 in differentiated intestinal cells and colon cancer cells, and the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine. RESULTS: KLF4 was partially depleted in the differentiated intestinal epithelial cells by a Tamoxifen-inducible Cre recombinase. We found that there was a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers, probably by inhibiting cell proliferation. The number and position of Paneth cells were also changed. This is consistent with the study that Wnt signaling induces maturation of Paneth cells and that KLF4 crosstalks with Wnt signaling in the intestine. We found that the morphology and polarity of the intestinal epithelial cells were altered by KLF4 deletion. Using a three-dimensional (3D) culture system for Caco-2 colon cancer cells, we found that KLF4 is essential for cell polarity and crypt-cyst formation. CONCLUSION: We demonstrated that KLF4 not only regulates cells differentiation, but also regulates the number and polarity of intestinal epithelial cells. KLF4 plays an important role in lumencyst formation in 3D cultures.
Su1683 Wnt Ligand Expression and Regulation of Stem Cells During Postnatal Growth of the Small Intestine in Rats Rino Donato, Jane K. Fauser, Imme A. Penttila, Ian C. Roberts-Thomson, Adrian G. Cummins The Wnt/β-catenin pathway is thought to play an important role in promoting crypt stem cells that mediate fission for intestinal growth. In rats, intestinal crypt fission peaks at day 14 of life (Dig Dis Sci 51:718-23, 2006). The expression of 84 Wnt pathway genes (including 18 of the 19 Wnt ligands) was investigated in the jejunum of 14 day old infant and 72 day old adult Hooded Wistar rats by real-time PCR array analysis. Wnt ligands with higher expression at day 14 were further investigated by real-time PCR analysis using different inhouse primers in 2, 7, 14, 21, 35 and 72 day old rats. Laser capture micro-dissection of jejunal sections from 14 day old rats was used to investigate Wnt expression in the crypt epithelium, pericryptal mesenchyme, villous epithelium and villous mesenchyme. In another experiment the expression of the stem cell marker Lgr5 was assessed in 14 day old rats with and without dickkopf-1 mediated blockade of Wnt signalling (100 ng per rat ip from days 9 to 13). Wnt3, Wnt4, Wnt5b, Wnt6, Wnt8b, Wnt9b and Wnt10a had 2-fold or greater mRNA expression in intestinal homogenates of full thickness jejunal segments of 14 day old compared to 72 day old rats. Expression of all these Wnts was low at day 2, increased at day 7, peaked at day 14 and decreased thereafter. Wnt3 was expressed in the crypt epithelium and Wnt5b in the pericryptal mesenchyme. Other Wnts were predominantly expressed in the villous epithelium or villous mesenchyme. In dickkopf-1 treated rats Lgr5 expression was decreased more than 2-fold. The results indicate enhanced expression of several Wnt ligands with crypt fission but only Wnt3 and Wnt5b were expressed in the region of crypt stem cells. Inhibition of the Wnt/β-catenin pathway by dickkopf-1 appears to suppress stem cell proliferation as indicated by reduced expression of Lgr5.
Su1686 Beta-Catenin Activation in the Developing Intestinal Tract Edwin K. McDonald, Tatiana Goretsky, Rebecca B. Katzman, Terrence A. Barrett Background and AIMS: Mechanisms responsible for normal crypt growth and development in the small bowel and colon are not well understood. Previous studies suggest that protein kinase Akt (Akt) signaling activates phosphorylated β-catenin(P-β-catenin552) in intestinal stem and progenitor cells in the progression of inflammation to dysplasia to colitis associated cancer. We sought to investigate β-catenin activation in the small bowel and colon. Methods: C57BL/6 mice were sacrificed at 7,14, and 21 days post birth. Alterations in intestinal morphology were assessed by H&E staining. The effects of early development on P-β catenin and Akt activation in the small bowel and colon were assessed by immunohistochemistry. Epithelial cell apoptosis was detected by TUNEL. Epithelial cell proliferation was assessed by Brdu staining. Results: Histologic analysis revealed paneth cell hypertrophy in 7 and 14 day old mice that resolved by 21 days. Crypt size and submucosal thickness progressed significantly between the time points. Brdu staining demonstrated reduced proliferation in the small bowel and colon as through the time points. There were no significant differences in apotosis as detected by TUNEL between the groups. Akt levels detected by IHC were also unchanged throughout the time points. P-β-catenin activation was significantly more predominant in the small bowel at 7 and 14 days. There were no significant differences in P-β-catenin in the small bowel and colon at 21 days. Conclusions: P-β-catenin activation in the developing intestinal tract involves signaling besides Akt.
Su1684 Expression Profiling of the Proximal Small Intestine in Mature Mice With Intestine-Specific PDX1 Inactivation Chin Chen, Eric Sibley
Su1687 Genetically-Altered Enteric Nervous System Results in an Amplified Adaptation Response to Massive Small Bowel Resection Meredith C. Hitch, Jennifer A. Leinicke, Jun Guo, Christopher R. Erwin, Robert O. Heuckeroth, Brad W. Warner
Background: Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, less is known about the function of Pdx1 in the small intestine. Objective: We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated following inactivation of Pdx1 specifically in the intestinal epithelium. Methods: Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1flox/flox;VilCre mice, by crossing mutant mice homozygous for loxP site-flanked Pdx1 alleles with transgenic mice expressing Cre recombinase under the control of the mouse villin 1 gene promoter. Total RNA was isolated from the first five centimeters of the small intestine from adult Pdx1flox/ flox ;VilCre and littermate control mice. Microarray analysis was performed to investigate
Introduction: Intestinal adaptation following massive small bowel resection (SBR) is a crucial compensatory response to enhance absorptive and digestive surface area within the remnant bowel. Adaptation is characterized by increased villus height and crypt depth as a consequence of a mitogenic stimulus to the intestinal crypt cells via unknown mechanisms. A few prior studies suggested that the enteric nervous system (ENS) might regulate enterocyte proliferation. The purpose of the present study was to elucidate the contribution of the
S-473
AGA Abstracts
AGA Abstracts
population. The presence of autophagy in this specific cell population could be essential for the maintenance of homeostasis and for the prevention of malignant transformation via regulation of the Wnt/β-catenin pathway.