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• W2016087279 abstract "OASIS is a basic leucine zipper transmembrane transcription factor localized in the endoplasmic reticulum (ER) that is cleaved in its transmembrane region in response to ER stress. This novel ER stress transducer has been demonstrated to express in osteoblasts and astrocytes and promote terminal maturation of these cells. Additionally, OASIS is highly expressed in goblet cells of the large intestine. In this study, we investigated the roles of OASIS in goblet cell differentiation in the large intestine. To analyze the functions of OASIS in goblet cells, we examined morphological changes and the expression of goblet cell differentiation markers in the large intestine of Oasis−/− mice. By disrupting the Oasis gene, the number of goblet cells and production of mucus were decreased in the large intestine. Oasis−/− goblet cells showed abnormal morphology of mucous vesicles and rough ER. The expression levels of mature goblet cell markers were lower, and conversely those of early goblet cell markers were higher in Oasis−/− mice, indicating that differentiation from early to mature goblet cells is impaired in Oasis−/− mice. To determine the association of OASIS with other factors involved in goblet cell differentiation, in vitro experiments using a cell culture model were performed. We found that OASIS was activated in response to mild ER stress that is induced in differentiating goblet cells. Knockdown of the Oasis transcript perturbed goblet cell terminal differentiation. Together, our data indicate that OASIS plays crucial roles in promoting the differentiation of early goblet cells to mature goblet cells in the large intestine. OASIS is a basic leucine zipper transmembrane transcription factor localized in the endoplasmic reticulum (ER) that is cleaved in its transmembrane region in response to ER stress. This novel ER stress transducer has been demonstrated to express in osteoblasts and astrocytes and promote terminal maturation of these cells. Additionally, OASIS is highly expressed in goblet cells of the large intestine. In this study, we investigated the roles of OASIS in goblet cell differentiation in the large intestine. To analyze the functions of OASIS in goblet cells, we examined morphological changes and the expression of goblet cell differentiation markers in the large intestine of Oasis−/− mice. By disrupting the Oasis gene, the number of goblet cells and production of mucus were decreased in the large intestine. Oasis−/− goblet cells showed abnormal morphology of mucous vesicles and rough ER. The expression levels of mature goblet cell markers were lower, and conversely those of early goblet cell markers were higher in Oasis−/− mice, indicating that differentiation from early to mature goblet cells is impaired in Oasis−/− mice. To determine the association of OASIS with other factors involved in goblet cell differentiation, in vitro experiments using a cell culture model were performed. We found that OASIS was activated in response to mild ER stress that is induced in differentiating goblet cells. Knockdown of the Oasis transcript perturbed goblet cell terminal differentiation. Together, our data indicate that OASIS plays crucial roles in promoting the differentiation of early goblet cells to mature goblet cells in the large intestine. The endoplasmic reticulum (ER) 2The abbreviations used are: ERendoplasmic reticulumUPRunfolded protein responseOASISold astrocyte specifically induced substancebZIPbasic leucine zipperCREcAMP response elementATFactivating transcription factorETSE26 transformation-specificSPDEFSAM-pointed domain-containing ETS-like factorPASperiodic acid-SchiffCREBcyclic AMP-response element-binding protein. is a central cellular organelle responsible for the synthesis, folding, and posttranslational modifications of proteins destined for the secretory pathway. A number of cellular stress conditions lead to the accumulation of unfolded or misfolded proteins in the ER lumen. These conditions, which are collectively termed ER stress, have the potential to induce cellular damage (1.Rutkowski D.T. Kaufman R.J. A trip to the ER. Coping with stress.Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar, 2.Zhang K. Kaufman R.J. From endoplasmic reticulum stress to the inflammatory response.Nature. 2008; 454: 455-462Crossref PubMed Scopus (1541) Google Scholar). The ER responds to these perturbations by activating an integrated signal transduction pathway through the ER stress transducers, which is called the unfolded protein response (UPR) (3.Schröder M. Kaufman R.J. ER stress and the unfolded protein response.Mutat. Res. 2005; 569: 29-63Crossref PubMed Scopus (1414) Google Scholar, 4.Ron D. Translational control in the endoplasmic reticulum stress response.J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (745) Google Scholar, 5.Kaufman R.J. Orchestrating the unfolded protein response in health and disease.J. Clin. Invest. 2002; 110: 1389-1398Crossref PubMed Scopus (1105) Google Scholar). The UPR involves at least three distinct components: translational attenuation to decrease the demands made on the organelle (6.Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar), transcriptional induction of genes encoding ER-resident chaperones to facilitate protein folding (7.Li M. Baumeister P. Roy B. Phan T. Foti D. Luo S. Lee A.S. ATF6 as a transcription activator of the endoplasmic reticulum stress element. Thapsigargin stress-induced changes and synergistic interactions with NF-Y and YY1.Mol. Cell. Biol. 2000; 20: 5096-5106Crossref PubMed Scopus (275) Google Scholar, 8.Yoshida H. Haze K. Yanagi H. Yura T. Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors.J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar), and ER-associated degradation to degrade the unfolded proteins accumulated in the ER (9.Ng D.T. Spear E.D. Walter P. The unfolded protein response regulates multiple aspects of secretory and membrane protein biogenesis and endoplasmic reticulum quality control.J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (276) Google Scholar, 10.Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation.Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar). If these strategies fail, cells undergo ER stress-induced apoptosis (11.Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β.Nature. 2000; 403: 98-103Crossref PubMed Scopus (2966) Google Scholar, 12.Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.Science. 2000; 287: 664-666Crossref PubMed Scopus (2345) Google Scholar). The UPR was originally described as a system by which cells evade damage in response to acute ER perturbation. However, recent advances have revealed that the UPR also provides important signals for regulating cell differentiation and maturation or the maintenance of basal cellular homeostasis (13.Rutkowski D.T. Hegde R.S. Regulation of basal cellular physiology by the homeostatic unfolded protein response.J. Cell Biol. 2010; 189: 783-794Crossref PubMed Scopus (294) Google Scholar, 14.Hotamisligil G.S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease.Cell. 2010; 140: 900-917Abstract Full Text Full Text PDF PubMed Scopus (2119) Google Scholar, 15.Kondo S. Saito A. Asada R. Kanemoto S. Imaizumi K. Physiological unfolded protein response regulated by OASIS family members, transmembrane bZIP transcription factors.IUBMB Life. 2011; 63: 233-239Crossref PubMed Scopus (61) Google Scholar, 16.Asada R. Kanemoto S. Kondo S. Saito A. Imaizumi K. The signalling from endoplasmic reticulum-resident bZIP transcription factors involved in diverse cellular physiology.J Biochem. 2011; 149: 507-518Crossref PubMed Scopus (147) Google Scholar). endoplasmic reticulum unfolded protein response old astrocyte specifically induced substance basic leucine zipper cAMP response element activating transcription factor E26 transformation-specific SAM-pointed domain-containing ETS-like factor periodic acid-Schiff cyclic AMP-response element-binding protein. Previously, we identified old astrocyte specifically induced substance (OASIS) as a novel ER stress transducer (17.Kondo S. Murakami T. Tatsumi K. Ogata M. Kanemoto S. Otori K. Iseki K. Wanaka A. Imaizumi K. OASIS, a CREB/ATF-family member, modulates UPR signalling in astrocytes.Nat. Cell Biol. 2005; 7: 186-194Crossref PubMed Scopus (242) Google Scholar). OASIS is a basic leucine zipper (bZIP) transcription factor that belongs to the cAMP-responsive element (CRE)-binding protein/activating transcription factor (ATF) family. Although OASIS is localized to the ER membrane under normal conditions, it is cleaved at the membrane in response to ER stress. Consequently, its cleaved N-terminal cytoplasmic domain, which contains the bZIP domain, translocates into the nucleus, where it activates the transcription of target genes (18.Murakami T. Kondo S. Ogata M. Kanemoto S. Saito A. Wanaka A. Imaizumi K. Cleavage of the membrane-bound transcription factor OASIS in response to endoplasmic reticulum stress.J. Neurochem. 2006; 96: 1090-1100Crossref PubMed Scopus (115) Google Scholar, 19.Saito A. Hino S. Murakami T. Kondo S. Imaizumi K. A novel ER stress transducer, OASIS, expressed in astrocytes.Antioxid. Redox Signal. 2007; 9: 563-571Crossref PubMed Scopus (29) Google Scholar). High expression of OASIS was observed in the osteoblasts of osseous tissues and the astrocytes of the central nervous system (20.Honma Y. Kanazawa K. Mori T. Tanno Y. Tojo M. Kiyosawa H. Takeda J. Nikaido T. Tsukamoto T. Yokoya S. Wanaka A. Identification of a novel gene, OASIS, which encodes for a putative CREB/ATF family transcription factor in the long-term cultured astrocytes and gliotic tissue.Brain Res. Mol. Brain Res. 1999; 69: 93-103Crossref PubMed Scopus (86) Google Scholar, 21.Nikaido T. Yokoya S. Mori T. Hagino S. Iseki K. Zhang Y. Takeuchi M. Takaki H. Kikuchi S. Wanaka A. Expression of the novel transcription factor OASIS, which belongs to the CREB/ATF family, in mouse embryo with special reference to bone development.Histochem. Cell Biol. 2001; 116: 141-148Crossref PubMed Scopus (40) Google Scholar, 22.Chihara K. Saito A. Murakami T. Hino S. Aoki Y. Sekiya H. Aikawa Y. Wanaka A. Imaizumi K. Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice.J. Neurochem. 2009; 110: 956-965Crossref PubMed Scopus (30) Google Scholar). From the analysis of knockout mice, OASIS has been demonstrated to be involved in terminal differentiation and osteoblasts (23.Murakami T. Saito A. Hino S. Kondo S. Kanemoto S. Chihara K. Sekiya H. Tsumagari K. Ochiai K. Yoshinaga K. Saitoh M. Nishimura R. Yoneda T. Kou I. Furuichi T. Ikegawa S. Ikawa M. Okabe M. Wanaka A. Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.Nat. Cell Biol. 2009; 11: 1205-1211Crossref PubMed Scopus (252) Google Scholar, 24.Murakami T. Hino S. Nishimura R. Yoneda T. Wanaka A. Imaizumi K. Distinct mechanisms are responsible for osteopenia and growth retardation in OASIS-deficient mice.Bone. 2011; 48: 514-523Crossref PubMed Scopus (21) Google Scholar, 25.Funamoto T. Sekimoto T. Murakami T. Kurogi S. Imaizumi K. Chosa E. Roles of the endoplasmic reticulum stress transducer OASIS in fracture healing.Bone. 2011; 49: 724-732Crossref PubMed Scopus (12) Google Scholar) and astrocytes. 3A. Saito, submitted for publication. The intestinal epithelium is composed of four distinct cell types, including the absorptive enterocytes and the goblet, Paneth, and enteroendocrine secretory cell lineages (26.Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types.Am. J. Anat. 1974; 141: 537-561Crossref PubMed Scopus (1120) Google Scholar, 27.Gordon J.I. Intestinal epithelial differentiation: new insights from chimeric and transgenic mice.J. Cell Biol. 1989; 108: 1187-1194Crossref PubMed Scopus (189) Google Scholar). Stem cells are committed to generate these lineages by the Wnt and Notch signaling cascades. Wnt signaling is required for the generation of the secretory lineages, whereas Notch signaling is necessary for the differentiation of enterocytes (28.Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (813) Google Scholar, 29.Nakamura T. Tsuchiya K. Watanabe M. Crosstalk between Wnt and Notch signaling in intestinal epithelial cell fate decision.J. Gastroenterol. 2007; 42: 705-710Crossref PubMed Scopus (139) Google Scholar). However, the molecular mechanisms underlying the differentiation of intestinal epithelial cells are incompletely defined. Recently, the E26 transformation-specific (ETS) domain transcription factor SAM-pointed domain-containing ETS-like factor (SPDEF) (30.Oettgen P. Finger E. Sun Z. Akbarali Y. Thamrongsak U. Boltax J. Grall F. Dube A. Weiss A. Brown L. Quinn G. Kas K. Endress G. Kunsch C. Libermann T.A. PDEF, a novel prostate epithelium-specific ETS transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression.J. Biol. Chem. 2000; 275: 1216-1225Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 31.Ghadersohi A. Sood A.K. Prostate epithelium-derived ETS transcription factor mRNA is overexpressed in human breast tumors and is a candidate breast tumor marker and a breast tumor antigen.Clin. Cancer Res. 2001; 7: 2731-2738PubMed Google Scholar), has been reported to act downstream of ATOH1 (32.Noah T.K. Kazanjian A. Whitsett J. Shroyer N.F. SAM pointed domain ETS factor (SPDEF) regulates terminal differentiation and maturation of intestinal goblet cells.Exp. Cell Res. 2010; 316: 452-465Crossref PubMed Scopus (132) Google Scholar), which is an essential determinant of secretory lineages downstream of β-catenin (33.Yang Q. Bermingham N.A. Finegold M.J. Zoghbi H.Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.Science. 2001; 294: 2155-2158Crossref PubMed Scopus (748) Google Scholar, 34.Shroyer N.F. Helmrath M.A. Wang V.Y. Antalffy B. Henning S.J. Zoghbi H.Y. Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular homeostasis.Gastroenterology. 2007; 132: 2478-2488Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Maturation of Paneth and goblet cells has been shown to be impaired in Spdef-deficient mice, whereas immature secretory progenitors accumulate in the intestine (35.Gregorieff A. Stange D.E. Kujala P. Begthel H. van den Born M. Korving J. Peters P.J. Clevers H. The ETS-domain transcription factor SPDEF promotes maturation of goblet and paneth cells in the intestinal epithelium.Gastroenterology. 2009; 137 (1333–1345.e1-1333–1345.e3)Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). These observations suggest that SPDEF promotes the terminal differentiation of a secretory progenitor pool into Paneth and goblet cells. Interestingly, the expression of CREB4, an ER stress transducer that is structurally similar to OASIS, is completely abolished in Spdef-deficient Paneth and goblet cells (35.Gregorieff A. Stange D.E. Kujala P. Begthel H. van den Born M. Korving J. Peters P.J. Clevers H. The ETS-domain transcription factor SPDEF promotes maturation of goblet and paneth cells in the intestinal epithelium.Gastroenterology. 2009; 137 (1333–1345.e1-1333–1345.e3)Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). These observations suggest that the expression of CREB4 is controlled at downstream of SPDEF and that ER stress response signaling through ER stress transducers such as CREB4 mediates the differentiation and maturation of secretory cell lineages in the intestinal epithelium. In this study, we investigated the roles of the ER stress transducer OASIS, which is expressed in the crypt base of the large intestinal epithelium, in the differentiation of goblet cells. Consequently, we demonstrated that OASIS plays crucial roles in promoting the terminal differentiation of goblet cells in the large intestine. 3-week-old C57BL/6 mice or Oasis−/− mice were used in this study. The Oasis−/− mice were established previously in our laboratory (22.Chihara K. Saito A. Murakami T. Hino S. Aoki Y. Sekiya H. Aikawa Y. Wanaka A. Imaizumi K. Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice.J. Neurochem. 2009; 110: 956-965Crossref PubMed Scopus (30) Google Scholar, 23.Murakami T. Saito A. Hino S. Kondo S. Kanemoto S. Chihara K. Sekiya H. Tsumagari K. Ochiai K. Yoshinaga K. Saitoh M. Nishimura R. Yoneda T. Kou I. Furuichi T. Ikegawa S. Ikawa M. Okabe M. Wanaka A. Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.Nat. Cell Biol. 2009; 11: 1205-1211Crossref PubMed Scopus (252) Google Scholar, 24.Murakami T. Hino S. Nishimura R. Yoneda T. Wanaka A. Imaizumi K. Distinct mechanisms are responsible for osteopenia and growth retardation in OASIS-deficient mice.Bone. 2011; 48: 514-523Crossref PubMed Scopus (21) Google Scholar). In all studies comparing wild-type and Oasis−/− mice, sex-matched littermates derived from the mating of Oasis+/− mice were used. The experimental procedures and housing conditions for animals were approved by the Committee of Animal Experimentation, Hiroshima University. LS174T cells were cultured in Eagle's minimal essential medium supplemented with 10% FCS and 1% non-essential amino acids. LS174T cells were cultured overnight in a 9.2-cm2 dish for RNA or protein isolation and in a Lab-Tek chamber slide (Nalge Nunc) for periodic acid-Schiff (PAS) staining. Subsequently, cells were treated with 2 mm sodium butyrate, which induces LS174T cells to differentiate to mature goblet cells. For Oasis knockdown experiments, LS174T cells were transfected with 1 μg of Oasis siRNA (predesigned siRNA pool targeting Oasis: AAAAGAAGGUGGAGACAUU, GGGACCACCUGCAGCAUGA, GAAGGAGUAUGUGGAGUGU, and CAGGAGAGCCGUCGUAAGA; Thermo Scientific Dermacon, catalog no. M-008579-01-0005) or control siRNA (Silencer Cy3-labeled negative control no.1 siRNA; Invitrogen, catalog no. AM4621). Transfection was performed 12 h before treatment with sodium butyrate using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocols. 24 h after treatment with sodium butyrate, PAS staining was performed as described previously (36.Hatayama H. Iwashita J. Kuwajima A. Abe T. The short chain fatty acid, butyrate, stimulates MUC2 mucin production in the human colon cancer cell line, LS174T.Biochem. Biophys. Res. Commun. 2007; 356: 599-603Crossref PubMed Scopus (137) Google Scholar). The PAS-stained slide was counterstained with hematoxylin solution. Total RNA was isolated from the large intestine of 3-week-old mice or LS174T cells using ISOGEN (Wako) according to the manufacturer's protocol. First-strand cDNA was synthesized in a 20 μl of reaction volume using a random primer (Takara) and Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR was performed using each specific primer set in a total volume of 30 μl containing 0.8 μm of each primer, 0.2 mm dNTPs, 3 units of Taq polymerase, and 10× PCR buffer (Agilent). Primer sequences are summarized in supplemental Table S1. The PCR products were resolved by electrophoresis on a 4.8% acrylamide gel. The density of each band was quantified using the Adobe Photoshop Elements 2.0 program (Adobe Systems Inc.). For Western blotting, proteins were extracted from LS174T cells using cell extraction buffer containing 10% SDS, 0.5 m EDTA (pH 8.0), 100 mm methionine, and a protease inhibitor mixture (MBL International). The lysates were incubated on ice for 45 min. After centrifugation at 16,000 × g for 15 min, the protein concentrations of the supernatants were determined. Protein-equivalent samples were loaded onto sodium dodecyl sulfate-polyacrylamide gels. Anti-β-actin (Sigma, 1:3000) and anti-OASIS (purified from a hybridoma as described previously (22.Chihara K. Saito A. Murakami T. Hino S. Aoki Y. Sekiya H. Aikawa Y. Wanaka A. Imaizumi K. Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice.J. Neurochem. 2009; 110: 956-965Crossref PubMed Scopus (30) Google Scholar, 23.Murakami T. Saito A. Hino S. Kondo S. Kanemoto S. Chihara K. Sekiya H. Tsumagari K. Ochiai K. Yoshinaga K. Saitoh M. Nishimura R. Yoneda T. Kou I. Furuichi T. Ikegawa S. Ikawa M. Okabe M. Wanaka A. Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.Nat. Cell Biol. 2009; 11: 1205-1211Crossref PubMed Scopus (252) Google Scholar)) antibodies were used for Western blotting. The density of each band was quantified using the Adobe Photoshop Elements 2.0 program (Adobe Systems Inc.). Large intestine from 3-week-old mice was fixed overnight in 10% formalin neutral buffer solution (Wako). Samples were then dehydrated with ethanol, embedded in paraffin, and sectioned (5 μm). Hematoxylin-eosin staining and PAS staining were performed according to standard protocols. In situ hybridization was performed using digoxigenin-labeled cRNA probes (supplemental Table S2). Antisense and sense probes were made by in vitro transcription in the presence of digoxigenin-labeled dUTP using various cDNAs subcloned into the pGEM-Teasy vector (Promega) as templates. Large intestine isolated for in situ hybridization was frozen immediately and sectioned (6 μm). The frozen sections were fixed for 20 min with 4% formalin in PBS (pH 7.4). The sections were then washed with PBS and treated with 0.1% proteinase K for 5 min. After washing with PBS, the sections were refixed for 20 min with 4% formalin in PBS and treated with 0.1 m triethanolamine and 2.5% anhydrous acetic acid for 10 min, followed by washing with PBS. Sections were prehybridized for 1 h at 37 °C in hybridization buffer (0.01% dextran sulfate, 0.01 m Tris-HCl (pH 8.0), 0.05 m NaCl, 50% formamide, 0.2% sarcosyl, 1× Denhardt's solution, 0.5 mg/ml yeast tRNA, 0.2 mg/ml salmon testis DNA) and then hybridized overnight at 55 °C in hybridization solution with 100 ng/ml cRNA probe. After washing with 4× saline sodium citrate buffer for 20 min at 60 °C, the sections were further washed in 2× saline sodium citrate buffer and 50% formamide for 30 min at 60 °C. Sections were treated RNaseA in RNase buffer (10 mm Tris-HCl (pH7.4), 1 mm 0.5 m EDTA (pH 8.0), 0.5 m NaCl) for 30 min at 37 °C to remove the unhybridized probe. After RNase treatment, sections were washed with 2× saline sodium citrate buffer and 50% formamide for 30 min at 60 °C and then blocked with 1.5% blocking reagent in 100 mm Tris-HCl (pH 7.5) and 150 mm NaCl for 1 h at room temperature. For detection of digoxigenin-labeled cRNA probes, anti-digoxigenin antibody conjugated to alkaline phosphatase was used at a dilution of 1:500, and color was developed by incubation with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate solution. Large intestine from 3-week-old mice was fixed in 1% glutaraldehyde in PBS for 15 min. After washing with distilled water, the tissues were post-fixed in 0.5% osmium tetroxide in 0.1 m cacodylate buffer for 30 min. Following dehydration, the tissues were embedded in EPON812, and ultra-thin sections were stained with uranyl acetate and lead citrate. Stained sections were visualized using a Hitachi 7100 electron microscope operated at 80 kV. The mean cell area was determined using ImageJ software (National Institutes of Health). Statistical comparisons were made using the unpaired Student's t test. Statistical significance between two samples was determined by a p value of less than 0.05. p values of less than 0.05, 0.01 or 0.001 are described as *, p < 0.05; **, p < 0.01; or ***, p < 0.001, respectively. We reported previously that OASIS is expressed in osteoblasts and astrocytes (17.Kondo S. Murakami T. Tatsumi K. Ogata M. Kanemoto S. Otori K. Iseki K. Wanaka A. Imaizumi K. OASIS, a CREB/ATF-family member, modulates UPR signalling in astrocytes.Nat. Cell Biol. 2005; 7: 186-194Crossref PubMed Scopus (242) Google Scholar, 20.Honma Y. Kanazawa K. Mori T. Tanno Y. Tojo M. Kiyosawa H. Takeda J. Nikaido T. Tsukamoto T. Yokoya S. Wanaka A. Identification of a novel gene, OASIS, which encodes for a putative CREB/ATF family transcription factor in the long-term cultured astrocytes and gliotic tissue.Brain Res. Mol. Brain Res. 1999; 69: 93-103Crossref PubMed Scopus (86) Google Scholar, 21.Nikaido T. Yokoya S. Mori T. Hagino S. Iseki K. Zhang Y. Takeuchi M. Takaki H. Kikuchi S. Wanaka A. Expression of the novel transcription factor OASIS, which belongs to the CREB/ATF family, in mouse embryo with special reference to bone development.Histochem. Cell Biol. 2001; 116: 141-148Crossref PubMed Scopus (40) Google Scholar, 23.Murakami T. Saito A. Hino S. Kondo S. Kanemoto S. Chihara K. Sekiya H. Tsumagari K. Ochiai K. Yoshinaga K. Saitoh M. Nishimura R. Yoneda T. Kou I. Furuichi T. Ikegawa S. Ikawa M. Okabe M. Wanaka A. Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.Nat. Cell Biol. 2009; 11: 1205-1211Crossref PubMed Scopus (252) Google Scholar). To examine the tissue distribution of Oasis mRNA more precisely, we performed RT-PCR using mRNA isolated from various tissues of 3-week-old mice. We detected strong Oasis mRNA signals in the submandibular gland, lung, stomach, and large intestine, where it was most intense (Fig. 1A). Oasis mRNA was expressed highly in all portions of the large intestine, except for the appendix. In contrast, the levels of Oasis mRNA were very low in the small intestine (Fig. 1B). In the digestive tract, there are three distinct cell types, i.e. absorptive enterocytes, enteroendocrine cells, and goblet cells. To identify which of these cells expressed Oasis mRNA, we carried out in situ hybridization using Oasis cRNA probes. The Oasis signals were focally detected in the base of the crypt but not in the apical portion of the crypt (Fig. 1C). In contrast, Gapdh mRNA was observed in all cells (both basal and apical) of the crypt. The cells expressing Oasis mRNA possessed vacuoles in their cytosol, indicating that OASIS is expressed in goblet cells. Moreover, the goblet cells at the base of the crypt are immature cells that are developing from intestinal stem cells (27.Gordon J.I. Intestinal epithelial differentiation: new insights from chimeric and transgenic mice.J. Cell Biol. 1989; 108: 1187-1194Crossref PubMed Scopus (189) Google Scholar, 37.Mills J.C. Gordon J.I. The intestinal stem cell niche. There grows the neighborhood.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 12334-12336Crossref PubMed Scopus (94) Google Scholar, 38.Shaker A. Rubin D.C. Intestinal stem cells and epithelial-mesenchymal interactions in the crypt and stem cell niche.Transl. Res. 2010; 156: 180-187Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Thus, we concluded that the cells expressing OASIS were immature goblet cells. To elucidate the functions of OASIS in immature goblet cells, we first performed histological analysis in the Oasis−/− mice that we generated previously (22.Chihara K. Saito A. Murakami T. Hino S. Aoki Y. Sekiya H. Aikawa Y. Wanaka A. Imaizumi K. Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice.J. Neurochem. 2009; 110: 956-965Crossref PubMed Scopus (30) Google Scholar, 23.Murakami T. Saito A. Hino S. Kondo S. Kanemoto S. Chihara K. Sekiya H. Tsumagari K. Ochiai K. Yoshinaga K. Saitoh M. Nishimura R. Yoneda T. Kou I. Furuichi T. Ikegawa S. Ikawa M. Okabe M. Wanaka A. Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.Nat. Cell Biol. 2009; 11: 1205-1211Crossref PubMed Scopus (252) Google Scholar, 24.Murakami T. Hino S. Nishimura R. Yoneda T. Wanaka A. Imaizumi K. Distinct mechanisms are responsible for osteopenia and growth retardation in OASIS-deficient mice.Bone. 2011; 48: 514-523Crossref PubMed Scopus (21) Google Scholar). The mice were born at the expected Mendelian ratios and were fertile but showed impaired bone formation and growth retardation. We demonstrated previously that these defects are due to impaired differentiation of osteoblasts (23.Murakami T. Saito A. Hino S. Kondo S. Kanemoto S. Chihara K. Sekiya H. Tsumagari K. Ochiai K. Yoshinaga K. Saitoh M. Nishimura R. Yoneda T. Kou I. Furuichi T. Ikegawa S. Ikawa M. Okabe M. Wanaka A. Imaizumi K. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.Nat. Cell Biol. 2009; 11: 1205-1211Crossref PubMed Scopus (252) Google Scholar) and decreased serum levels of growth hormone and insulin-like growth factor (24.Murakami T. Hino S. Nishimura R. Yo" @default.
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• W2016087279 title "The Endoplasmic Reticulum Stress Transducer OASIS Is involved in the Terminal Differentiation of Goblet Cells in the Large Intestine" @default.
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