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132 BMP Signaling Promotes Basal Cell Differentiation Through Activation of NRF2 Signaling in the Adult Mouse Esophagus
AGA Abstracts
increasing intestinal surface area. In mice, a large increase in surface area is initiated with villus development at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a regularly patterned field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found that a unique type of cell division occurs only at E14.5, which we have named an e-division (lumen extending division). Functionally and cell biologically, e-divisions are distinct from cell divisions that occur before E14.5, called g-divisions (girth increasing divisions). Midbodies (spindle remnants) are bent in g-divisions and the cytokinetic plane is perpendicular to the lumen. In e-divisions, the midbody is straight and nascent apical surface separates the two daughters; the cytokinetic plane is parallel to the extending lumen. These e-divisions are both temporally and spatially patterned, and this pattern is perturbed in Wnt5a-/- mice, which have notoriously short guts. Our working model is that g-divisions promote proliferation of epithelial progenitors and drive the growth of intestinal girth, while e-divisions function to carve out the villi at E14.5. Mice lacking Ezrin appear to exhibit stochastic failure of the e-division. This is being modeled in an in vitro organoid assay. Further understanding of this process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding a new avenue for treatment of intestinal failure. 129 Figure 1 GSI treatment reduces proliferation and partially rescues Mtgr1-/- secretory lineage depletion. Quantification of A) PH3 B) Goblet C) Enterendocrine and D) Paneth cells per ileal crypt. One way anova statistical analysis: # WT Control vs WT GSI P ,0.05; Φ WT Control vs Mtgr1-/- Control P,0.05; *** Mtgr1-/- Control vs Mtgr1-/- GSI P,0.001.
Cell Division Cycle 42 Is Essential for Intestinal Crypt Homeostasis and Brush Border Morphogenesis Ryotaro Sakamori, Nan Gao BACKGROUND & AIMS: Malabsorptive and maldigestive diseases are the major causes of morbidity and mortality on a global scale, causing millions of death per year. Abnormal morphogenesis of the intestinal enterocytes can cause severe nutrient deprivation particularly in infants in the first year of life. The mammalian intestinal villus mucosa is continuously replenished by stem cells in the crypts, following a well-staged program composed of transient amplification, polarization, and terminal differentiation. We aimed to investigate the hypothesis that intestinal stem cell defects may contribute to abnormal enterocyte morphogenesis and function that underlie the ontogenesis of microvillus diseases. METHODS: A major regulator of cell polarity, cell division cycle 42 (Cdc42), was inactivated globally in the mouse intestinal epithelium, or specifically in the intestinal stem cell population. The resulting phenotypes were characterized at histological and molecular levels. RESULTS: Intestine-specific Cdc42 inactivation in mice led to severe accumulations of microvillus bodies within the mutant enterocytes. The inner surfaces of these inclusion bodies were lined by typical brush borders that normally appear at apical surfaces of the enterocytes. This phenotype recapitulates the microvillus inclusion human disease counterpart. Cdc42deficient intestinal stem cells underwent defective cell division and polarization, and did not give rise to Paneth cells. The affected mice demonstrated severely reduced epithelial uptake of glucose, amino acids and peptides. CONCLUSIONS: Stem cell division and polarization are tightly coupled to enterocyte development and brush border morphogenesis. Molecular defects targeting the stem cell compartment may contribute to the ontogenesis of microvillus diseases.
131 RNA-Binding Protein HuR Regulates MicroRNA Processing by Altering Drosha Expression in Intestinal Epithelial Cells Ran Zhuang, Rao N. Jaladanki, Tongtong Zou, Lan Xiao, Natasha Z. Hansraj, Myriam Gorospe, Jian-Ying Wang microRNAs (miRNAs) are small noncoding RNAs and regulate gene expression by triggering translational repression or mRNA degradation. miRNAs have emerged as master regulators of GI mucosal growth and are linked to several GI diseases recently. Tissue miRNA levels are tightly regulated and their maturation is controlled by Drosha that is responsible for the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is the first step of miRNA maturation and influences cellular levels of mature miRNAs. However, the exact mechanisms that control Drosha expression remain largely unknown. The RNAbinding protein HuR primarily binds to AU-rich elements located in the 3'-untranslated regions (UTRs) of target transcripts and regulates mRNA stability and translation. Since there are several predicted HuR-hit motifs in the Drosha mRNA, this study determined if HuR regulates miRNA processing by modulating Drosha expression. Methods: Studies were conducted in IEC-6 cells derived from rat intestinal crypts. miRNA Levels were examined by real time-PCR analysis; HuR interaction with the Drosha mRNA was detected by ribonucleoprotein/IP and biotin pull-down assays. HuR function was investigated by HuR silencing and its ectopic overexpression. Drosha translation was measured by polysomal profile analysis and chimeric Drosha 3'-UTR luciferase reporter assays. Results: HuR silencing by transfection with specific siRNA targeting HuR (siHuR) inhibited Drosha expression and repressed maturation of several miRNAs, including miR-195, miR-542, and miR-34c, as indicated by decreased levels of mature miRNAs, along with increased content of primary miRNAs. HuR was found to bind the Drosha mRNA via its 3'-UTR rather than 5'-UTR and coding region. HuR silencing by siHuR reduced [HuR/Drosha mRNA] complex, inhibited Drosha translation, and decreased its protein levels. In contrast, ectopic HuR overexpression enhanced HuR association with the Drosha mRNA and stimulated Drosha translation. Interestingly, HuR silencing also reduced Drosha mRNA levels, whereas HuR overexpression increased Drosha mRNA abundance; neither intervention changed the stability of Drosha mRNA. Further studies showed that increased HuR also enhanced Drosha gene transcription as indicated by an increase in Drosha-promoter activity. This induction in Drosha transcription by HuR resulted predominantly from induced c-Myc transcription factor, since HuR enhanced c-Myc expression and because increased Drosha transcription was prevented by c-Myc silencing in cells overexpressing HuR. Conclusions: These results indicate that 1) HuR regulates miRNA maturation by altering Drosha levels and 2) the mechanism underlying Drosha expression by HuR operates at the levels of transcription and translation.
130 MTGR1 Is Required for Gsi-Induced Paneth Cell Differentiation Bobak Parang, Vishruth K. Reddy, Daniel Rosenblatt, Amanda Williams, Aubrey Hunt, Frank Revetta, Yuan-hung Lo, Kay Washington, Michael Engel, Scott W. Hiebert, Noah F. Shroyer, Christopher S. Williams Background: The specification of cell fate in the intestinal epithelium is determined by Notch signaling. High Notch activity drives progenitors toward enterocyte differentiation while low Notch expression triggers secretory cell differentiation. Myeloid translocation gene related-1 (Mtgr1) is a transcriptional co-repressor in the MTG/ETO family. Mtgr1-/- mice display loss of intestinal epithelial secretory cells and MTG family members interact with Notch family intracellular domains (ICD). We hypothesized that Mtgr1 suppresses Notch signaling and that Notch inhibition could rescue the lineage allocation deficit seen in Mtgr1-/small intestine. Methods: 8-12 week old Wild-type (WT) and Mtgr1-/- mice were treated intraperitoneally with vehicle or γ-secretase inhibitor-20 (GSI-20) at 10 umol/L/kg twice a day for 5 consecutive days. Mice were sacrificed, and the small intestine and thymus were harvested. Thymocyte subsets were determined by flow cytometry. Crypt architecture and secretory cells were analyzed by histology and immunohistochemistry (IHC). Proliferation was measured using anti-phospho-histone H3 (PH3) in IHC. Co-immunoprecipitation was performed by transfecting HA-epitope tagged Mtgr1 and Myc-epitope tagged CSL in COS7L cells. Cell lysates were immunoprecipitated with anti-Myc monoclonal antibody 9E10 and immune complexes were collected on Protein-G sepharose beads. Hes1 promoter activity was assessed by transiently expressing Mtgr1 in NIH3T3 cells with or without N1-ICD coexpression, along with the transcriptional reporter Hes1-Lux. Results: As predicted, GSI treatment decreased thymus weight and cellularity and increased thymic CD4-CD8- cells in both WT and Mtgr1-/- mice. GSI increased Goblet cells per crypt (0.97 ±0.3 to 6.1 ±0.8 P,0.001) and EE cells per crypt (0.035 ±0.02 to 0.36 ±0.06 P ,0.001) in Mtgr1-/- mice. Consistent with Notch-inhibition, GSI also reduced the number of PH3 positive cells per crypt in the Mtgr1-/- mice (2.7 ±0.2 to 1.2 ±0.1 P ,0.001). GSI treatment did not increase Paneth cells, suggesting Mtgr1 is required for the effects of Notch-inhibition on Paneth cells. To determine how Mtgr1 modifies Notch signaling, we tested for Mtgr1 associations with Notch signaling family members. We discovered that Mtgr1 immunopurified with CSL, the DNA-binding partner of Notch intracellular domains. Moreover, Mtgr1 expression suppressed N1-ICD transcriptional activation of the Hes1-promoter. Conclusions: Overall, inhibition of Notch signaling using GSI restored Goblet and EE cells and reduced proliferation in the Mtgr1-/-- mice. GSI did not have an effect on Paneth cells in these mice, suggesting that Mtgr1 is required for the effects of Notch inhibition on Paneth cell differentiation. Mtgr1 interacts with CSL and suppresses N1-ICD transcriptional activation of Hes1.
132 BMP Signaling Promotes Basal Cell Differentiation Through Activation of NRF2 Signaling in the Adult Mouse Esophagus Ming Jiang, Jeffrey H. Peters, Jianwen Que Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells and involves intricate signaling regulatory networks. We previously reported Bone morphogenetic protein (BMP) signaling is important for the epithelial morphogenesis of the embryonic esophagus. However, it remains unclear whether/how this signaling is involved in the maintenance of adult esophagus. Here, we utilize multiple mouse genetic lines to address the issue. We found that BMP signaling is specifically activated in the differentiated suprabasal cells but not in the undifferentiated basal progenitor cells. When treated with Bmp4 basal progenitor cells differentiate towards a squamous cell fate. Consistent with the in vitro assay, ectopic BMP signaling in the esophageal basal cells of the Krt5-CreER; R26RCABMPR1A mice leads to premature differentiation, resulting in epithelial hyperkeratosis. Interestingly, BMP activation induces intracellular oxidative stress and enhances the nuclear accumulation of Nrf2 and its activity. Suppression of Nrf2 by the negative regulator, Keap1 inhibits Bmp4-induced basal cell differentiation. Together, our study demonstrates a novel role of BMP signaling promoting squamous differentiation of basal progenitor cells mediated through activation of intracellular oxidant stress and Nrf2 signaling.
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AGA Abstracts
increasing intestinal surface area. In mice, a large increase in surface area is initiated with villus development at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a regularly patterned field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found that a unique type of cell division occurs only at E14.5, which we have named an e-division (lumen extending division). Functionally and cell biologically, e-divisions are distinct from cell divisions that occur before E14.5, called g-divisions (girth increasing divisions). Midbodies (spindle remnants) are bent in g-divisions and the cytokinetic plane is perpendicular to the lumen. In e-divisions, the midbody is straight and nascent apical surface separates the two daughters; the cytokinetic plane is parallel to the extending lumen. These e-divisions are both temporally and spatially patterned, and this pattern is perturbed in Wnt5a-/- mice, which have notoriously short guts. Our working model is that g-divisions promote proliferation of epithelial progenitors and drive the growth of intestinal girth, while e-divisions function to carve out the villi at E14.5. Mice lacking Ezrin appear to exhibit stochastic failure of the e-division. This is being modeled in an in vitro organoid assay. Further understanding of this process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding a new avenue for treatment of intestinal failure. 129 Figure 1 GSI treatment reduces proliferation and partially rescues Mtgr1-/- secretory lineage depletion. Quantification of A) PH3 B) Goblet C) Enterendocrine and D) Paneth cells per ileal crypt. One way anova statistical analysis: # WT Control vs WT GSI P ,0.05; Φ WT Control vs Mtgr1-/- Control P,0.05; *** Mtgr1-/- Control vs Mtgr1-/- GSI P,0.001.
Cell Division Cycle 42 Is Essential for Intestinal Crypt Homeostasis and Brush Border Morphogenesis Ryotaro Sakamori, Nan Gao BACKGROUND & AIMS: Malabsorptive and maldigestive diseases are the major causes of morbidity and mortality on a global scale, causing millions of death per year. Abnormal morphogenesis of the intestinal enterocytes can cause severe nutrient deprivation particularly in infants in the first year of life. The mammalian intestinal villus mucosa is continuously replenished by stem cells in the crypts, following a well-staged program composed of transient amplification, polarization, and terminal differentiation. We aimed to investigate the hypothesis that intestinal stem cell defects may contribute to abnormal enterocyte morphogenesis and function that underlie the ontogenesis of microvillus diseases. METHODS: A major regulator of cell polarity, cell division cycle 42 (Cdc42), was inactivated globally in the mouse intestinal epithelium, or specifically in the intestinal stem cell population. The resulting phenotypes were characterized at histological and molecular levels. RESULTS: Intestine-specific Cdc42 inactivation in mice led to severe accumulations of microvillus bodies within the mutant enterocytes. The inner surfaces of these inclusion bodies were lined by typical brush borders that normally appear at apical surfaces of the enterocytes. This phenotype recapitulates the microvillus inclusion human disease counterpart. Cdc42deficient intestinal stem cells underwent defective cell division and polarization, and did not give rise to Paneth cells. The affected mice demonstrated severely reduced epithelial uptake of glucose, amino acids and peptides. CONCLUSIONS: Stem cell division and polarization are tightly coupled to enterocyte development and brush border morphogenesis. Molecular defects targeting the stem cell compartment may contribute to the ontogenesis of microvillus diseases.
131 RNA-Binding Protein HuR Regulates MicroRNA Processing by Altering Drosha Expression in Intestinal Epithelial Cells Ran Zhuang, Rao N. Jaladanki, Tongtong Zou, Lan Xiao, Natasha Z. Hansraj, Myriam Gorospe, Jian-Ying Wang microRNAs (miRNAs) are small noncoding RNAs and regulate gene expression by triggering translational repression or mRNA degradation. miRNAs have emerged as master regulators of GI mucosal growth and are linked to several GI diseases recently. Tissue miRNA levels are tightly regulated and their maturation is controlled by Drosha that is responsible for the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is the first step of miRNA maturation and influences cellular levels of mature miRNAs. However, the exact mechanisms that control Drosha expression remain largely unknown. The RNAbinding protein HuR primarily binds to AU-rich elements located in the 3'-untranslated regions (UTRs) of target transcripts and regulates mRNA stability and translation. Since there are several predicted HuR-hit motifs in the Drosha mRNA, this study determined if HuR regulates miRNA processing by modulating Drosha expression. Methods: Studies were conducted in IEC-6 cells derived from rat intestinal crypts. miRNA Levels were examined by real time-PCR analysis; HuR interaction with the Drosha mRNA was detected by ribonucleoprotein/IP and biotin pull-down assays. HuR function was investigated by HuR silencing and its ectopic overexpression. Drosha translation was measured by polysomal profile analysis and chimeric Drosha 3'-UTR luciferase reporter assays. Results: HuR silencing by transfection with specific siRNA targeting HuR (siHuR) inhibited Drosha expression and repressed maturation of several miRNAs, including miR-195, miR-542, and miR-34c, as indicated by decreased levels of mature miRNAs, along with increased content of primary miRNAs. HuR was found to bind the Drosha mRNA via its 3'-UTR rather than 5'-UTR and coding region. HuR silencing by siHuR reduced [HuR/Drosha mRNA] complex, inhibited Drosha translation, and decreased its protein levels. In contrast, ectopic HuR overexpression enhanced HuR association with the Drosha mRNA and stimulated Drosha translation. Interestingly, HuR silencing also reduced Drosha mRNA levels, whereas HuR overexpression increased Drosha mRNA abundance; neither intervention changed the stability of Drosha mRNA. Further studies showed that increased HuR also enhanced Drosha gene transcription as indicated by an increase in Drosha-promoter activity. This induction in Drosha transcription by HuR resulted predominantly from induced c-Myc transcription factor, since HuR enhanced c-Myc expression and because increased Drosha transcription was prevented by c-Myc silencing in cells overexpressing HuR. Conclusions: These results indicate that 1) HuR regulates miRNA maturation by altering Drosha levels and 2) the mechanism underlying Drosha expression by HuR operates at the levels of transcription and translation.
130 MTGR1 Is Required for Gsi-Induced Paneth Cell Differentiation Bobak Parang, Vishruth K. Reddy, Daniel Rosenblatt, Amanda Williams, Aubrey Hunt, Frank Revetta, Yuan-hung Lo, Kay Washington, Michael Engel, Scott W. Hiebert, Noah F. Shroyer, Christopher S. Williams Background: The specification of cell fate in the intestinal epithelium is determined by Notch signaling. High Notch activity drives progenitors toward enterocyte differentiation while low Notch expression triggers secretory cell differentiation. Myeloid translocation gene related-1 (Mtgr1) is a transcriptional co-repressor in the MTG/ETO family. Mtgr1-/- mice display loss of intestinal epithelial secretory cells and MTG family members interact with Notch family intracellular domains (ICD). We hypothesized that Mtgr1 suppresses Notch signaling and that Notch inhibition could rescue the lineage allocation deficit seen in Mtgr1-/small intestine. Methods: 8-12 week old Wild-type (WT) and Mtgr1-/- mice were treated intraperitoneally with vehicle or γ-secretase inhibitor-20 (GSI-20) at 10 umol/L/kg twice a day for 5 consecutive days. Mice were sacrificed, and the small intestine and thymus were harvested. Thymocyte subsets were determined by flow cytometry. Crypt architecture and secretory cells were analyzed by histology and immunohistochemistry (IHC). Proliferation was measured using anti-phospho-histone H3 (PH3) in IHC. Co-immunoprecipitation was performed by transfecting HA-epitope tagged Mtgr1 and Myc-epitope tagged CSL in COS7L cells. Cell lysates were immunoprecipitated with anti-Myc monoclonal antibody 9E10 and immune complexes were collected on Protein-G sepharose beads. Hes1 promoter activity was assessed by transiently expressing Mtgr1 in NIH3T3 cells with or without N1-ICD coexpression, along with the transcriptional reporter Hes1-Lux. Results: As predicted, GSI treatment decreased thymus weight and cellularity and increased thymic CD4-CD8- cells in both WT and Mtgr1-/- mice. GSI increased Goblet cells per crypt (0.97 ±0.3 to 6.1 ±0.8 P,0.001) and EE cells per crypt (0.035 ±0.02 to 0.36 ±0.06 P ,0.001) in Mtgr1-/- mice. Consistent with Notch-inhibition, GSI also reduced the number of PH3 positive cells per crypt in the Mtgr1-/- mice (2.7 ±0.2 to 1.2 ±0.1 P ,0.001). GSI treatment did not increase Paneth cells, suggesting Mtgr1 is required for the effects of Notch-inhibition on Paneth cells. To determine how Mtgr1 modifies Notch signaling, we tested for Mtgr1 associations with Notch signaling family members. We discovered that Mtgr1 immunopurified with CSL, the DNA-binding partner of Notch intracellular domains. Moreover, Mtgr1 expression suppressed N1-ICD transcriptional activation of the Hes1-promoter. Conclusions: Overall, inhibition of Notch signaling using GSI restored Goblet and EE cells and reduced proliferation in the Mtgr1-/-- mice. GSI did not have an effect on Paneth cells in these mice, suggesting that Mtgr1 is required for the effects of Notch inhibition on Paneth cell differentiation. Mtgr1 interacts with CSL and suppresses N1-ICD transcriptional activation of Hes1.
132 BMP Signaling Promotes Basal Cell Differentiation Through Activation of NRF2 Signaling in the Adult Mouse Esophagus Ming Jiang, Jeffrey H. Peters, Jianwen Que Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells and involves intricate signaling regulatory networks. We previously reported Bone morphogenetic protein (BMP) signaling is important for the epithelial morphogenesis of the embryonic esophagus. However, it remains unclear whether/how this signaling is involved in the maintenance of adult esophagus. Here, we utilize multiple mouse genetic lines to address the issue. We found that BMP signaling is specifically activated in the differentiated suprabasal cells but not in the undifferentiated basal progenitor cells. When treated with Bmp4 basal progenitor cells differentiate towards a squamous cell fate. Consistent with the in vitro assay, ectopic BMP signaling in the esophageal basal cells of the Krt5-CreER; R26RCABMPR1A mice leads to premature differentiation, resulting in epithelial hyperkeratosis. Interestingly, BMP activation induces intracellular oxidative stress and enhances the nuclear accumulation of Nrf2 and its activity. Suppression of Nrf2 by the negative regulator, Keap1 inhibits Bmp4-induced basal cell differentiation. Together, our study demonstrates a novel role of BMP signaling promoting squamous differentiation of basal progenitor cells mediated through activation of intracellular oxidant stress and Nrf2 signaling.
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AGA Abstracts