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M1611 The Intracellular Tyrosine Kinase PTK6 Regulates Protein Phosphatase 2a in Intestinal Epithelial Cells
AGA Abstracts
phosphorylation sites by ICK. Results: Knockdown of ICK expression in colorectal carcinoma cells altered gene expression of several key regulators of G1-S transition, including downregulation of cyclin D1, c-myc and up-regulation of p21Cip/WAF1, consistent with the G1 cell cycle delay induced by ICK shRNA. Given that the expression of cyclin D1 and c-myc is tightly controlled at the protein translational level by p70 S6 kinase (S6K1) and eukaryotic translation initiation factor 4E (eIF4E) of the mTOR pathway, we investigated the expression and activity of the key components of the mTOR pathway in ICK knockdown cells. ICK deficiency led to a significant decrease in the expression levels of S6K1 and eIF4E. The activity of S6K1 was also drastically reduced in association with a significant decrease in the expression level of its upstream activating kinase mTOR. Furthermore, ICK associated with the mTOR complex In Vivo and phosphorylated the mTOR regulatory protein Raptor, suggesting that ICK may target the mTOR pathway to regulate protein translation and cell proliferation. Reduced ICK expression in colonic epithelial cells also triggered a significant increase in the expression level of Cdx2, a homeobox transcription factor required for intestinal differentiation, associated with the up-regulated expression of p21 and Cadherin 17, both of which are downstream transcriptional targets of Cdx2. Conclusion: ICK may impact the balance between proliferation and differentiation of intestinal epithelial cells by targeting critical regulators within the mTOR and the Cdx2 pathways.
M1610 Constitutive Activation of MEK/ERK Pathway Inhibits Intestinal Epithelial Cell Differentiation Etienne Lemieux, Francois Boudreau, Nathalie Rivard Strong evidences support the implication of Ras/Raf/MEK/ERK cascade in intestinal epithelial proliferation. The activated forms of ERK1/2 are mostly detected in the nucleus of undifferentiated crypt cells in human intestine while a barely detectable ERK activity is observed in differentiated cells of the villi. We and others have reported that ERKs are selectively inactivated during enterocyte differentiation. However, whether inactivation of the ERK pathway is necessary for inhibition of proliferation and induction of differentiation of intestinal epithelial cells is unknown. Methods: The undifferentiated crypt cells IEC-6 engineered to conditionally express Cdx3 transcription factor and known to exhibit primitive cellular differentiation after several days of post-confluency (Suh and Traber, 1996) were used. Retrovirus encoding the HA-tagged MEK1wild type (wtMEK) or constitutively active mutant of MEK1 (MEK1-S218D/S222D, caMEK) were used to infect IEC-6/CDX3 cells. Expression of specific genes and proteins was assessed by western blotting and RT-PCR. Morphology was analyzed by electron microscopy. Results: 1- IEC-6/Cdx3 cells grew in multicellular layers after reaching confluence and many cells presented differentiation features after 30 days in culture including polarization, microvilli formation and expression of differentiation markers. 2- Interestingly, down-regulation of ERK1/2 activities was also observed during differentiation of IEC-6/Cdx3 cells. 3- Co-overexpression of caMEK, but not wtMEK, prevented ERK inhibition resulting in the maintenance of high ERK1/2 activities during postconfluency. 4- Interestingly, IEC-6/Cdx3 cells expressing caMEK exhibited altered multicellular structures organization and poorly defined tight junctions; in addition, strong reduction in the number of microvilli on the apical surface of the cells was observed in comparison to cells expressing wtMEK. 5- The expression of caMEK in IEC-6/Cdx3 cells caused a marked reduction in the expression of enterocyte differentiation markers namely Apo4A, FABP, Calbindine-3, MUC2, alkaline phosphatase and sucrase-isomaltase. 6- Cdx3 was highly phosphorylated on serine 60 (S60) in IEC-6/Cdx3 expressing caMEK; S60-phosphorylation decreases Cdx2 transactivation in intestinal epithelial cells. 7- Finally, S60-phosphorylation on endogenous Cdx2 was also observed in poorly differentiated cancer cells lines having activating mutation in K-Ras and high ERK1/2 activities. Conclusion: These results indicate that inactivation of the ERK pathway may be required for induction of Cdx2/3 transcriptional activity necessary for terminal differentiation of intestinal epithelial cells.
M1608 Zebrafish Pekin Mutants Demonstrate the Importance of Intracellular Trafficking in Hepatobiliary Development Steven F. EauClaire, Liyuan Ma, Randolph P. Matthews Zebrafish mutagenesis screens allow identification of novel genes and pathways involved in important developmental and physiological processes. We have used the fluorescent lipid reporter PED-6 to identify mutant fish in which there is diminished gallbladder uptake of PED-6, a finding suggestive of defects in hepatic lipid processing and/or intrahepatic bile duct development. We have identified a mutant, pekin, in which there is decreased gallbladder PED-6 uptake and generalized hypopigmentation. Development of intrahepatic bile ducts in pekin is abnormal, and there appears to be apoptosis of bile duct cells. Examination of pekin livers demonstrates severely abnormal bile duct cells, with accumulation of abnormal intracellular vesicles. We have identified the causative gene of pekin as atp6ap2, which encodes a protein that interacts with the vacuolar H+-ATPase, a critical enzyme in establishing acidic pH in intracellular compartments. The biliary defects in pekin are likely to be caused by defects in intracellular sorting that lead to cell death. Preliminary evidence implicates abnormalities in copper metabolism as contributory to this phenotype. Interestingly, Atp6ap2 has also been shown to act as an intracellular renin receptor. Renin activation is a fibrogenic stimulus, and as such our mutant appears to be “anti-fibrogenic”, as there is a decrease in expression of several fibrogenic genes. Our results demonstrate the importance of intracellular trafficking in biliary development and bile duct cell survival, consistent with the known role of intracellular trafficking in biliary disorders such as arthrogryposis-renal dysfunctioncholestasis (ARC) syndrome. Furthermore, these results suggest that renin may be involved in vertebrate biliary development, and may contribute to hepatic fibrosis. These results demonstrate an intriguing connection between renin activity, intracellular trafficking, fibrogenesis and hepatobiliary development.
M1611 The Intracellular Tyrosine Kinase PTK6 Regulates Protein Phosphatase 2a in Intestinal Epithelial Cells Jessica Gierut, Ansu O. Perekatt, Angela L. Tyner Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is distantly related to members of the Src family of tyrosine kinases. PTK6 is expressed in differentiated cells in the linings of the gastrointestinal tract and skin. Disruption of the mouse Ptk6 gene leads to increased growth and impaired differentiation, and increased AKT phosphorylation and activity in the intestine. Protein Phosphatase 2A (PP2A) is a ubiquitously expressed serine/ threonine phosphatase that negatively regulates AKT. Although evidence has suggested that PP2A might be a tumor suppressor protein, it has been difficult to pin down its role in tumor suppression because it acts on a wide variety of substrates and signaling pathways. Inhibiting PP2A activity has been shown to enhance phosphorylation of proteins involved in controlling cell growth, including AKT and its downstream signaling events. To determine if PTK6 can modulate PP2A activity in intestinal epithelial cells, we transfected full-length wild-type mouse PTK6, PTK6 YF (constitutively active) and PTK6 KM (kinase dead) expression constructs in the pcDNA3 vector into the IEC-18 nontransformed rat intestinal crypt cell line that expresses low levels of PTK6. PP2A activity was then determined using a PP2A immunoprecipitation phosphatase assay. Expression of wild type and constitutively active forms of PTK6 led to a kinase dependent increase in PP2A activity in IEC-18 cells. Next, we wanted to determine if knockdown of PTK6 would influence endogenous PP2A activity. Using a lentivirus vector to express shRNAs that target PTK6, PTK6 was stably knocked down in the HCT-116 human colon carcinoma cell line. Phosphatase assays were performed and significantly higher levels of PP2A activity were detected in the vector control cell population compared with two different PTK6 knockdown cell populations. Using immunoblotting we found that PTK6 does not regulate the expression of the PP2A catalytic subunit. Our data indicate that expression of active PTK6 promotes PP2A activity. The ability of PTK6 to positively regulate PP2A could contribute to its negative regulation of AKT In Vivo, and account for some of its impact on growth and differentiation in the intestine.
M1609 Intestinal Microbiota Matures the Colonic Epithelium Through a Well Synchronized Induction of Cell-Cycle Related Proteins Claire Cherbuy, Edith Honvo-Houeto, Marie-Louise Noordine, Camille Mayeur, Aurélia Bruneau, Chantal Bridonneau, Philippe Langella, Muriel Thomas Previous studies suggest that the intestinal microbiota modulates colonic epithelium renewal. Thus, a reduced proliferative activity and a prolonged cell cycle have been reported in colonic epithelium of germ-free (GF) rats [1]. The objective of our work was to study the effects of microbiota colonization on colonic epithelium structure and cell cycle-related proteins by using gnotobiotic rats. Colonic epithelium structure was compared between GF and conventionalized rats by histological analysis. The level and localization of cell-cycle related proteins were studied by immunohistochemistry and Western blot: two markers of proliferation (Ki67 and PCNA) and two markers of cell cycle arrest (p21cip1 and p27Kip1) were targeted. We show here that the colonization of microbiota leads to 30% increase of colonic crypt length that we correlated to an induction of proteins involved in proliferation process. Indeed, we observed 40% increase of the proliferative area 2 days after inoculation of GF rats with a complex microbiota. The rapid recruitment of proliferative cells enhanced by microbiota may be involved in the increase of crypt length. This proliferation increase induced by microbiota was then counterbalanced by an increase in proteins involved in cell cycle arrest. Indeed, 4-fold increase of p21cip1 and p27 Kip1 levels was observed 14 days after microbiota inoculation. To define whether these effects could be also observed with a single strain, colonic cell-cycle related proteins were also studied in Bacteroides thetaiotaomicron and Ruminococcus gnavus mono-associated rats. In mono-associated rats, P21cip1 and PCNA amounts were differentially restored: both of strains can partially restore the conventional pattern on p21cip1, but had no effect on PCNA. To conclude, we show that the colonisation with a complex microbiota leads to rapid commitment of colon cells into proliferation process allowing the increase of crypt length. The microbiota-induced hyperplasia is then compensated by an induction of p21cip1 and p27Kip1 proteins restraining proliferation. Up to now, p21cip1 and p27Kip1 were known to be targeted by toxins produced by pathogenic bacteria [2]. Here, we demonstrated that intestinal microbiota can also modulate similar cell-cycle related pathways as pathogenic bacteria. 1. Alam, M., Midtvedt, T. & Uribe, A. (1994) Scand J Gastroenterol. 29, 445-51. 2. Samba-Louaka, A., Nougayrede, J. P., Watrin, C., Jubelin, G., Oswald, E. & Taieb, F. (2008) Cell Microbiol.
AGA Abstracts
M1612 CDx2 Establishes Intestinal Identity and Epithelial-Mesenchymal Interaction Nan Gao, Klaus H. Kaestner The molecular mechanisms that control the positional identity of the gastrointestinal tract have been elucidated only partially. We demonstrate that conditional ablation of the homeobox transcription factor Cdx2 from early endoderm led to complete loss of intestinal identity and colon dysgenesis. The mutant posterior prospective intestinal epithelium was replaced by keratinocyte, a dramatic cell fate conversion propagated by genome-wide ectopic activation of esophageal differentiation program. The early anterior homeotic transformation of Cdx2deficient gut first became evident as crucial foregut regulators caudally extended their expression domain. Meanwhile, the initial activation or/and maintenance of a group of prointestinal transcription factors are strictly dependent on Cdx2, exhibiting a feed-forward mechanism in directing the posterior endoderm towards intestinal fates. In contrast to the severe alteration of intestinal and esophageal transcriptome, Cdx2-deficiency transiently modified selected posterior Hox genes while the primary enteric Hox code was maintained. Further, we demonstrate that Cdx2-directed intestinal cell fate adoption plays an important role in the establishment of normal epithelial-mesenchymal interaction, as multiple signaling
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phosphorylation sites by ICK. Results: Knockdown of ICK expression in colorectal carcinoma cells altered gene expression of several key regulators of G1-S transition, including downregulation of cyclin D1, c-myc and up-regulation of p21Cip/WAF1, consistent with the G1 cell cycle delay induced by ICK shRNA. Given that the expression of cyclin D1 and c-myc is tightly controlled at the protein translational level by p70 S6 kinase (S6K1) and eukaryotic translation initiation factor 4E (eIF4E) of the mTOR pathway, we investigated the expression and activity of the key components of the mTOR pathway in ICK knockdown cells. ICK deficiency led to a significant decrease in the expression levels of S6K1 and eIF4E. The activity of S6K1 was also drastically reduced in association with a significant decrease in the expression level of its upstream activating kinase mTOR. Furthermore, ICK associated with the mTOR complex In Vivo and phosphorylated the mTOR regulatory protein Raptor, suggesting that ICK may target the mTOR pathway to regulate protein translation and cell proliferation. Reduced ICK expression in colonic epithelial cells also triggered a significant increase in the expression level of Cdx2, a homeobox transcription factor required for intestinal differentiation, associated with the up-regulated expression of p21 and Cadherin 17, both of which are downstream transcriptional targets of Cdx2. Conclusion: ICK may impact the balance between proliferation and differentiation of intestinal epithelial cells by targeting critical regulators within the mTOR and the Cdx2 pathways.
M1610 Constitutive Activation of MEK/ERK Pathway Inhibits Intestinal Epithelial Cell Differentiation Etienne Lemieux, Francois Boudreau, Nathalie Rivard Strong evidences support the implication of Ras/Raf/MEK/ERK cascade in intestinal epithelial proliferation. The activated forms of ERK1/2 are mostly detected in the nucleus of undifferentiated crypt cells in human intestine while a barely detectable ERK activity is observed in differentiated cells of the villi. We and others have reported that ERKs are selectively inactivated during enterocyte differentiation. However, whether inactivation of the ERK pathway is necessary for inhibition of proliferation and induction of differentiation of intestinal epithelial cells is unknown. Methods: The undifferentiated crypt cells IEC-6 engineered to conditionally express Cdx3 transcription factor and known to exhibit primitive cellular differentiation after several days of post-confluency (Suh and Traber, 1996) were used. Retrovirus encoding the HA-tagged MEK1wild type (wtMEK) or constitutively active mutant of MEK1 (MEK1-S218D/S222D, caMEK) were used to infect IEC-6/CDX3 cells. Expression of specific genes and proteins was assessed by western blotting and RT-PCR. Morphology was analyzed by electron microscopy. Results: 1- IEC-6/Cdx3 cells grew in multicellular layers after reaching confluence and many cells presented differentiation features after 30 days in culture including polarization, microvilli formation and expression of differentiation markers. 2- Interestingly, down-regulation of ERK1/2 activities was also observed during differentiation of IEC-6/Cdx3 cells. 3- Co-overexpression of caMEK, but not wtMEK, prevented ERK inhibition resulting in the maintenance of high ERK1/2 activities during postconfluency. 4- Interestingly, IEC-6/Cdx3 cells expressing caMEK exhibited altered multicellular structures organization and poorly defined tight junctions; in addition, strong reduction in the number of microvilli on the apical surface of the cells was observed in comparison to cells expressing wtMEK. 5- The expression of caMEK in IEC-6/Cdx3 cells caused a marked reduction in the expression of enterocyte differentiation markers namely Apo4A, FABP, Calbindine-3, MUC2, alkaline phosphatase and sucrase-isomaltase. 6- Cdx3 was highly phosphorylated on serine 60 (S60) in IEC-6/Cdx3 expressing caMEK; S60-phosphorylation decreases Cdx2 transactivation in intestinal epithelial cells. 7- Finally, S60-phosphorylation on endogenous Cdx2 was also observed in poorly differentiated cancer cells lines having activating mutation in K-Ras and high ERK1/2 activities. Conclusion: These results indicate that inactivation of the ERK pathway may be required for induction of Cdx2/3 transcriptional activity necessary for terminal differentiation of intestinal epithelial cells.
M1608 Zebrafish Pekin Mutants Demonstrate the Importance of Intracellular Trafficking in Hepatobiliary Development Steven F. EauClaire, Liyuan Ma, Randolph P. Matthews Zebrafish mutagenesis screens allow identification of novel genes and pathways involved in important developmental and physiological processes. We have used the fluorescent lipid reporter PED-6 to identify mutant fish in which there is diminished gallbladder uptake of PED-6, a finding suggestive of defects in hepatic lipid processing and/or intrahepatic bile duct development. We have identified a mutant, pekin, in which there is decreased gallbladder PED-6 uptake and generalized hypopigmentation. Development of intrahepatic bile ducts in pekin is abnormal, and there appears to be apoptosis of bile duct cells. Examination of pekin livers demonstrates severely abnormal bile duct cells, with accumulation of abnormal intracellular vesicles. We have identified the causative gene of pekin as atp6ap2, which encodes a protein that interacts with the vacuolar H+-ATPase, a critical enzyme in establishing acidic pH in intracellular compartments. The biliary defects in pekin are likely to be caused by defects in intracellular sorting that lead to cell death. Preliminary evidence implicates abnormalities in copper metabolism as contributory to this phenotype. Interestingly, Atp6ap2 has also been shown to act as an intracellular renin receptor. Renin activation is a fibrogenic stimulus, and as such our mutant appears to be “anti-fibrogenic”, as there is a decrease in expression of several fibrogenic genes. Our results demonstrate the importance of intracellular trafficking in biliary development and bile duct cell survival, consistent with the known role of intracellular trafficking in biliary disorders such as arthrogryposis-renal dysfunctioncholestasis (ARC) syndrome. Furthermore, these results suggest that renin may be involved in vertebrate biliary development, and may contribute to hepatic fibrosis. These results demonstrate an intriguing connection between renin activity, intracellular trafficking, fibrogenesis and hepatobiliary development.
M1611 The Intracellular Tyrosine Kinase PTK6 Regulates Protein Phosphatase 2a in Intestinal Epithelial Cells Jessica Gierut, Ansu O. Perekatt, Angela L. Tyner Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is distantly related to members of the Src family of tyrosine kinases. PTK6 is expressed in differentiated cells in the linings of the gastrointestinal tract and skin. Disruption of the mouse Ptk6 gene leads to increased growth and impaired differentiation, and increased AKT phosphorylation and activity in the intestine. Protein Phosphatase 2A (PP2A) is a ubiquitously expressed serine/ threonine phosphatase that negatively regulates AKT. Although evidence has suggested that PP2A might be a tumor suppressor protein, it has been difficult to pin down its role in tumor suppression because it acts on a wide variety of substrates and signaling pathways. Inhibiting PP2A activity has been shown to enhance phosphorylation of proteins involved in controlling cell growth, including AKT and its downstream signaling events. To determine if PTK6 can modulate PP2A activity in intestinal epithelial cells, we transfected full-length wild-type mouse PTK6, PTK6 YF (constitutively active) and PTK6 KM (kinase dead) expression constructs in the pcDNA3 vector into the IEC-18 nontransformed rat intestinal crypt cell line that expresses low levels of PTK6. PP2A activity was then determined using a PP2A immunoprecipitation phosphatase assay. Expression of wild type and constitutively active forms of PTK6 led to a kinase dependent increase in PP2A activity in IEC-18 cells. Next, we wanted to determine if knockdown of PTK6 would influence endogenous PP2A activity. Using a lentivirus vector to express shRNAs that target PTK6, PTK6 was stably knocked down in the HCT-116 human colon carcinoma cell line. Phosphatase assays were performed and significantly higher levels of PP2A activity were detected in the vector control cell population compared with two different PTK6 knockdown cell populations. Using immunoblotting we found that PTK6 does not regulate the expression of the PP2A catalytic subunit. Our data indicate that expression of active PTK6 promotes PP2A activity. The ability of PTK6 to positively regulate PP2A could contribute to its negative regulation of AKT In Vivo, and account for some of its impact on growth and differentiation in the intestine.
M1609 Intestinal Microbiota Matures the Colonic Epithelium Through a Well Synchronized Induction of Cell-Cycle Related Proteins Claire Cherbuy, Edith Honvo-Houeto, Marie-Louise Noordine, Camille Mayeur, Aurélia Bruneau, Chantal Bridonneau, Philippe Langella, Muriel Thomas Previous studies suggest that the intestinal microbiota modulates colonic epithelium renewal. Thus, a reduced proliferative activity and a prolonged cell cycle have been reported in colonic epithelium of germ-free (GF) rats [1]. The objective of our work was to study the effects of microbiota colonization on colonic epithelium structure and cell cycle-related proteins by using gnotobiotic rats. Colonic epithelium structure was compared between GF and conventionalized rats by histological analysis. The level and localization of cell-cycle related proteins were studied by immunohistochemistry and Western blot: two markers of proliferation (Ki67 and PCNA) and two markers of cell cycle arrest (p21cip1 and p27Kip1) were targeted. We show here that the colonization of microbiota leads to 30% increase of colonic crypt length that we correlated to an induction of proteins involved in proliferation process. Indeed, we observed 40% increase of the proliferative area 2 days after inoculation of GF rats with a complex microbiota. The rapid recruitment of proliferative cells enhanced by microbiota may be involved in the increase of crypt length. This proliferation increase induced by microbiota was then counterbalanced by an increase in proteins involved in cell cycle arrest. Indeed, 4-fold increase of p21cip1 and p27 Kip1 levels was observed 14 days after microbiota inoculation. To define whether these effects could be also observed with a single strain, colonic cell-cycle related proteins were also studied in Bacteroides thetaiotaomicron and Ruminococcus gnavus mono-associated rats. In mono-associated rats, P21cip1 and PCNA amounts were differentially restored: both of strains can partially restore the conventional pattern on p21cip1, but had no effect on PCNA. To conclude, we show that the colonisation with a complex microbiota leads to rapid commitment of colon cells into proliferation process allowing the increase of crypt length. The microbiota-induced hyperplasia is then compensated by an induction of p21cip1 and p27Kip1 proteins restraining proliferation. Up to now, p21cip1 and p27Kip1 were known to be targeted by toxins produced by pathogenic bacteria [2]. Here, we demonstrated that intestinal microbiota can also modulate similar cell-cycle related pathways as pathogenic bacteria. 1. Alam, M., Midtvedt, T. & Uribe, A. (1994) Scand J Gastroenterol. 29, 445-51. 2. Samba-Louaka, A., Nougayrede, J. P., Watrin, C., Jubelin, G., Oswald, E. & Taieb, F. (2008) Cell Microbiol.
AGA Abstracts
M1612 CDx2 Establishes Intestinal Identity and Epithelial-Mesenchymal Interaction Nan Gao, Klaus H. Kaestner The molecular mechanisms that control the positional identity of the gastrointestinal tract have been elucidated only partially. We demonstrate that conditional ablation of the homeobox transcription factor Cdx2 from early endoderm led to complete loss of intestinal identity and colon dysgenesis. The mutant posterior prospective intestinal epithelium was replaced by keratinocyte, a dramatic cell fate conversion propagated by genome-wide ectopic activation of esophageal differentiation program. The early anterior homeotic transformation of Cdx2deficient gut first became evident as crucial foregut regulators caudally extended their expression domain. Meanwhile, the initial activation or/and maintenance of a group of prointestinal transcription factors are strictly dependent on Cdx2, exhibiting a feed-forward mechanism in directing the posterior endoderm towards intestinal fates. In contrast to the severe alteration of intestinal and esophageal transcriptome, Cdx2-deficiency transiently modified selected posterior Hox genes while the primary enteric Hox code was maintained. Further, we demonstrate that Cdx2-directed intestinal cell fate adoption plays an important role in the establishment of normal epithelial-mesenchymal interaction, as multiple signaling
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