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Intestinal Epithelial Cell Differentiation Involves Activation of p38 Mitogen-activated Protein Kinase That Regulates the Homeobox Transcription Factor CDX2

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m100236200

ISSN

1083-351X

Autores

Mathieu Houde, Patrick Laprise, Dominique Jean, Mylène Blais, Claude Asselin, Nathalie Rivard,

Tópico(s)

Cancer Cells and Metastasis

Resumo

The intracellular signaling pathways responsible for cell cycle arrest and differentiation along the crypt-villus axis of the human small intestine remain largely unknown. p38 mitogen-activated protein kinases (MAPKs) have recently emerged as key modulators of various vertebrate cell differentiation processes. In order to elucidate further the mechanism(s) responsible for the loss of proliferative potential once committed intestinal cells begin to differentiate, the role and regulation of p38 MAPK with regard to differentiation were analyzed in both intact epithelium as well as in well established intestinal cell models recapitulating the crypt-villus axis in vitro. Results show that phosphorylated and active forms of p38 were detected primarily in the nuclei of differentiated villus cells. Inhibition of p38 MAPK signaling by 2–20 μm SB203580 did not affect E2F-dependent transcriptional activity in subconfluent Caco-2/15 or HIEC cells. p38 MAPK activity dramatically increased as soon as Caco-2/15 cells reached confluence, whereas addition of SB203580 during differentiation of Caco-2/15 cells strongly attenuated sucrase-isomaltase gene and protein expression as well as protein expression of villin and alkaline phosphatase. The binding of CDX2 to the sucrase-isomaltase promoter and its transcriptional activity were significantly reduced by SB203580. Pull-down glutathione S-transferase and immunoprecipitation experiments demonstrated a direct interaction of CDX3 with p38. Finally, p38-dependent phosphorylation of CDX3 was observed in differentiating Caco-2/15 cells. Taken together, our results indicate that p38 MAPK may be involved in the regulation of CDX2/3 function and intestinal cell differentiation. The intracellular signaling pathways responsible for cell cycle arrest and differentiation along the crypt-villus axis of the human small intestine remain largely unknown. p38 mitogen-activated protein kinases (MAPKs) have recently emerged as key modulators of various vertebrate cell differentiation processes. In order to elucidate further the mechanism(s) responsible for the loss of proliferative potential once committed intestinal cells begin to differentiate, the role and regulation of p38 MAPK with regard to differentiation were analyzed in both intact epithelium as well as in well established intestinal cell models recapitulating the crypt-villus axis in vitro. Results show that phosphorylated and active forms of p38 were detected primarily in the nuclei of differentiated villus cells. Inhibition of p38 MAPK signaling by 2–20 μm SB203580 did not affect E2F-dependent transcriptional activity in subconfluent Caco-2/15 or HIEC cells. p38 MAPK activity dramatically increased as soon as Caco-2/15 cells reached confluence, whereas addition of SB203580 during differentiation of Caco-2/15 cells strongly attenuated sucrase-isomaltase gene and protein expression as well as protein expression of villin and alkaline phosphatase. The binding of CDX2 to the sucrase-isomaltase promoter and its transcriptional activity were significantly reduced by SB203580. Pull-down glutathione S-transferase and immunoprecipitation experiments demonstrated a direct interaction of CDX3 with p38. Finally, p38-dependent phosphorylation of CDX3 was observed in differentiating Caco-2/15 cells. Taken together, our results indicate that p38 MAPK may be involved in the regulation of CDX2/3 function and intestinal cell differentiation. epidermal growth factor glutathione S-transferase mitogen-activated protein kinase c-Jun NH2-terminal kinase MAPK/extracellular signal-regulated kinase kinase Dulbecco's modified Eagle's medium human intestinal epithelial cells fetal bovine serum phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis myelin basic protein dihydrofolate reductase polymerase chain reaction dipeptidyl peptidase IV poly(ADP-ribose) polymerase hemagglutinin primary cultures of differentiated enterocytes retinoblastoma protein The epithelium of the small intestine is a highly dynamic system continuously renewed by a process involving cell generation and migration from the stem cell population located at the bottom of the crypt to the extrusion of the terminally differentiated cells at the tip of the villus (1Babyatsky M.W. Podolsky D.K. Growth and Development of the Gastrointestinal Tract (Yamada, T., ed). 3rd Ed. J. B. Lippincott, Philadelphia1999: 547-584Google Scholar, 2Karam S.M. Front. Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar). The crypt-villus functional axis unit, which develops relatively early during human ontogeny (being established by mid-pregnancy), can be defined by typical morphological and functional properties displayed by the mature villus enterocytes that distinguish them from crypt cells (1Babyatsky M.W. Podolsky D.K. Growth and Development of the Gastrointestinal Tract (Yamada, T., ed). 3rd Ed. J. B. Lippincott, Philadelphia1999: 547-584Google Scholar, 2Karam S.M. Front. Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar, 3Ménard D. Lebenthal E. Growth-promoting Factors and the Development of the Human Gut. Raven Press, Ltd., New York1989: 123-150Google Scholar). Indeed, the villi are mainly lined by functional absorptive, goblet, and endocrine cells, whereas the crypts contain stem cells, proliferative and poorly differentiated cells, as well as a subset of differentiated secretory cells, namely Paneth cells (3Ménard D. Lebenthal E. Growth-promoting Factors and the Development of the Human Gut. Raven Press, Ltd., New York1989: 123-150Google Scholar). The differentiation of each cell type takes place as the cells move either upward toward the villus (absorptive, mucus and endocrine cells) or downward to concentrate at the bottom of the crypt (Paneth cells) (2Karam S.M. Front. Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar). The basic mechanisms responsible for induction of cell differentiation are little understood. The decision to differentiate is taken by the committed crypt cells abruptly, while in their most rapid state of proliferation (4Cairnie A.B. Lamerton L.F. Steel G.G. Exp. Cell Res. 1965; 39: 539-553Crossref PubMed Scopus (104) Google Scholar). The newly differentiated cells acquire their distinctive ultrastructural features and cell surface markers after leaving the proliferative cell cycle, at the top of the crypts or the base of the villi (2Karam S.M. Front. Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar, 5Doell R.G. Rosen G. Kretchmer N. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1268-1273Crossref PubMed Scopus (69) Google Scholar, 6Quaroni A. Dev. Biol. 1985; 111: 280-292Crossref PubMed Scopus (27) Google Scholar, 7Quaroni A Isselbacher K.J. Dev. Biol. 1985; 111: 267-279Crossref PubMed Scopus (78) Google Scholar). It is noteworthy that in all species studied, the crypt-villus axis junction represents a physical limit from which enterocytes acquire their final functional characteristics (1Babyatsky M.W. Podolsky D.K. Growth and Development of the Gastrointestinal Tract (Yamada, T., ed). 3rd Ed. J. B. Lippincott, Philadelphia1999: 547-584Google Scholar, 2Karam S.M. Front. Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar, 3Ménard D. Lebenthal E. Growth-promoting Factors and the Development of the Human Gut. Raven Press, Ltd., New York1989: 123-150Google Scholar, 4Cairnie A.B. Lamerton L.F. Steel G.G. Exp. Cell Res. 1965; 39: 539-553Crossref PubMed Scopus (104) Google Scholar, 5Doell R.G. Rosen G. Kretchmer N. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1268-1273Crossref PubMed Scopus (69) Google Scholar, 6Quaroni A. Dev. Biol. 1985; 111: 280-292Crossref PubMed Scopus (27) Google Scholar, 7Quaroni A Isselbacher K.J. Dev. Biol. 1985; 111: 267-279Crossref PubMed Scopus (78) Google Scholar). The process of cell differentiation in the intestinal epithelium has been the subject of extensive studies, for which the morphological and functional characteristics of the intestinal mucosa (8Gordon J.I. Hermiston M.L. Curr. Opin. Cell Biol. 1994; 6: 795-803Crossref PubMed Scopus (212) Google Scholar, 9Pageot L.P. Perreault N. Basora N. Francoeur C. Magny P. Beaulieu J.F. Microsc. Res. Tech. 2000; 49: 394-406Crossref PubMed Scopus (109) Google Scholar), the growth kinetics of the epithelial cells (10Cheng H. Bjerknes M. Anat. Rec. 1985; 211: 420-426Crossref PubMed Scopus (109) Google Scholar), and the chronological changes that affect brush border enzyme activities during pre- and postnatal development (11Traber P.G. Silberg D.J. Annu. Rev. Physiol. 1996; 58: 275-297Crossref PubMed Scopus (134) Google Scholar, 12Ménard D. Bkaily Membrane Physiopathology. 6. Kluwer Academic Publishers, Norwell, MA1994: 319-341Google Scholar) have all been well documented. However, the basic mechanisms involved in the induction and the modulation of cell differentiation in the upper portion of the crypts, and the cellular interactions responsible for the orderly arrangement of the relative numbers of proliferative, maturing, and functional epithelial cells are still largely unknown. Hormones, such as glucocorticoids, and growth factors, such as epidermal growth factor (EGF),1 have been implicated in the regulation of intestinal growth and development (12Ménard D. Bkaily Membrane Physiopathology. 6. Kluwer Academic Publishers, Norwell, MA1994: 319-341Google Scholar, 13Chailler P. Ménard D. Front. Biosci. 1999; 4: 87-101Crossref PubMed Google Scholar). However, little is known about the molecular signals responsible for the ontogenic changes in intestinal gene expression. Several lines of evidence suggest that the intestinal specific, caudal-related cdx1 and cdx2/3 homeobox genes encode nuclear transcription factors that play critical roles in intestinal cell proliferation and differentiation. CDX1 is mainly expressed in the crypt compartment although not restricted to proliferative cells (14Silberg D.G. Furth E.E. Taylor J.K. Schuk T. Chiou T. Traber P.G. Gastroenterology. 1997; 113: 478-486Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), and its inhibition by antisense RNA reduces cell proliferation in vitro (15Lorentz O. Duluc I. Arcangelis A.D. Simon-Assmann P. Kedinger M. Freund J.N. J. Cell Biol. 1997; 139: 1553-1565Crossref PubMed Scopus (252) Google Scholar). The CDX2/3 homeoproteins (the protein designated CDX3 in the hamster and CDX2 in the mouse and humans) are mainly expressed in differentiating enterocytes (16James R. Erler T. Kazenwadel J. J. Biol. 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Boll W. Levy T. Suh E.H. Siang S. Mantei N. Traber P.G. DNA Cell Biol. 1997; 16: 1419-1428Crossref PubMed Scopus (60) Google Scholar), carbonic anhydrase (21Lambert M. Colnot S. Suh E.H. L'Horset F. Blin C. Calliot M.E. Raymondjean M. Thomasset M. Traber P.G. Perret C. Eur. J. Biochem. 1996; 236: 778-788Crossref PubMed Scopus (78) Google Scholar), and lactase (22Fang R. Santiago N.A. Olds L.C. Sibley E. Gastroenterology. 2000; 118: 115-127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar)). However, little is known about the intracellular signaling pathways that positively regulate the activities of CDX transcription factors, especially those involved in receiving and transducing extracellular cues. In eukaryotic cells, the mitogen-activated protein kinase (MAPK) family has been shown to play various important roles in regulating gene expression via transcription factor phosphorylation (23Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1156) Google Scholar, 24Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3570) Google Scholar, 25Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.T. Nature. 1997; 386: 296-299Crossref PubMed Scopus (676) Google Scholar, 26Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar). In mammals, two distinct classes have been identified to date as follows: p42–p44 (extracellular signal-regulated kinase) MAPKs inducible by growth factors, and SAPKs (stress-activated protein kinases), which include p38 MAPKs and p46-p54 JNKs, inducible by cytokines and cellular stress (27Robinson M.J. Cobbs M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2266) Google Scholar). Unique structural features, specific activation pathways, and varying substrate specificities support the contention that different MAPKs are independently regulated and control different cellular responses to extracellular stimuli (28Su B. Karin M. Curr. Opin. Immunol. 1996; 8: 402-411Crossref PubMed Scopus (712) Google Scholar, 29Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 795-805Crossref Scopus (532) Google Scholar). We (30Aliaga J.C. Deschênes C. Beaulieu J.F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar) and others (31Taupin D. Podolsky D.K. Gastroenterology. 1999; 116: 1072-1080Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) recently analyzed the role and regulation of p42/p44 MAPKs in the process of proliferation and differentiation of human intestinal cells. Our results demonstrated that elevated p42/p44 MAPK activities stimulated cell cycle progression of intestinal epithelial cells, whereas low sustained levels were correlated with G1 arrest and differentiation. However, the intracellular pathways responsible for establishment of differentiated cells occupying specific positions along the gut axis still remain largely unknown. Several recent studies have demonstrated that p38 MAPK is involved in various vertebrate cell differentiation processes, namely adipocytic (32Engelman J.A. Lisanti M.P. Scherer P.E. J. Biol. Chem. 1998; 273: 32111-32120Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) and myogenic differentiation (33Wu Z. Woodring P.J. Bhakta K.S. Tamura K. Wen F. Feramisco J.R. Karin M. Wang Y.J. Puri P.L. Mol. Cell. Biol. 2000; 20: 3951-3964Crossref PubMed Scopus (390) Google Scholar). The role of p38 MAPK in intestinal cell differentiation is, however, not known. In the present work, the role and regulation of p38 MAPK were analyzed in relation to human intestinal cell proliferation and differentiation in the intact epithelium as well as in well established intestinal cell models that allow the recapitulation of the crypt-villus axis in vitroas follows: Caco-2/15 cells, which have the ability to differentiate into fully functional villus-like enterocytes (34Beaulieu J.F. Quaroni A. Biochem. J. 1991; 280: 599-608Crossref PubMed Scopus (133) Google Scholar, 35Pinto M. Robine-Leon S. Appay M.D. Kedinger M. Triadou N. Bussaulx N. Lacroix B. Simon-Assmann P. Haffen K. Fogh J. Zweibaum A. Biol. Cell. 1983; 47: 323-330Google Scholar, 36Vachon P.H. Beaulieu J.F. Am. J. Physiol. 1995; 268: G857-G867PubMed Google Scholar, 37Vachon P.H. Beaulieu J.F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (186) Google Scholar); normal crypt-like HIEC cells, which are proliferative and undifferentiated (38Perreault N. Beaulieu J.F. Exp. Cell Res. 1996; 224: 354-364Crossref PubMed Scopus (132) Google Scholar); and finally PCDE cells, which are primary cultures of differentiated and non-proliferative villus enterocytes (39Perreault N. Beaulieu J.F. Exp. Cell Res. 1998; 254: 34-42Crossref Scopus (117) Google Scholar). By using a combination of different approaches, p38 MAPK was found to be activated rapidly in intestinal cells induced to differentiate. Specific inhibition of p38 significantly reduced the expression of several differentiation markers including sucrase-isomaltase, alkaline phosphatase, lactase, and villin. Finally, p38 exerted its stimulatory effect on intestinal differentiation by directly interacting with CDX2/3 and enhancing its transcriptional activity. [γ-32P]ATP and the enhanced chemiluminescence (ECL) immunodetection system were obtained fromAmersham Pharmacia Biotech. Antiserum that specifically recognizes p38α on Western blots (40Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar) was a kind gift from Dr. J. Landry (Laval University, Québec, Canada). Rabbit polyclonal antibodies against phosphorylated and active forms of p38 MAPK were from New England Biolabs (Mississauga, Ontario, Canada). Mouse monoclonal antibody against pRb (14001A) was purchased from PharMingen (Mississauga, Ontario, Canada). Monoclonal antibody HSI-14 (41Beaulieu J.F. Nichols B. Quaroni A. J. Biol. Chem. 1989; 264: 20000-20011Abstract Full Text PDF PubMed Google Scholar) against sucrase-isomaltase was kindly provided by Dr. A. Quaroni (Cornell University, Ithaca, NY). Monoclonal antibody CII10 recognizing the 89-kDa apoptotic fragment and the 113-kDa non-cleaved fragment of poly(ADP-ribose) polymerase (PARP) was a kind gift from Dr. G. G. Poirier (Laval University, Québec, Canada). Polyclonal antibodies against CDX2/3 protein were provided by Dr. D. J. Drucker (University of Toronto, Ontario, Canada) (42Trinh K.Y. Jin T. Drucker D.J. J. Biol. Chem. 1999; 274: 6011-6019Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The monoclonal HA antibody raised against a peptide from influenza hemagglutinin HA1 protein was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Goat anti-rabbit IgG-fluorescein isothiocyanate (FITC) and goat anti-mouse IgG-fluorescein isothiocyanate were from Roche Molecular Biochemicals. The specific inhibitors of MEK1/2 (PD98059) and of p38α/β (SB203580) were purchased from Calbiochem. EGF was obtained from Collaborative Biomedicals (Bedford, MA), and insulin was from Connaught Novo Laboratories (Willowdale, Ontario, Canada). All other materials were obtained from Sigma-Aldrich unless stated otherwise. Tissues from five fetuses of 20 weeks of gestation (post-fertilization fetal ages were estimated according to Streeter (43Streeter G.L. Contributions Embryology. 1920; 11: 143-179Google Scholar)) were obtained from normal elective pregnancy terminations. No tissue was collected from cases associated with known fetal abnormalities or fetal death. All studies were approved by the Institutional Human Subject Review Board. Segments of fetal small intestine were rinsed with 0.15 m NaCl, sectioned into small fragments, embedded in optimum cutting temperature compound, and quickly frozen in liquid nitrogen (36Vachon P.H. Beaulieu J.F. Am. J. Physiol. 1995; 268: G857-G867PubMed Google Scholar). Frozen sections 2–3 μm thick were spread on silane-coated glass slides and air-dried for 1 h at room temperature before storage at −80 °C. For indirect immunofluorescence, sections were fixed with 2% formaldehyde in phosphate-buffered saline (pH 7.4; 45 min, 4 °C), before immunostaining as described previously (37Vachon P.H. Beaulieu J.F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Negative controls (no primary antibody) were included in all experiments. Nuclei were stained with propidium iodide as per instructions of the manufacturer (Molecular Probes, Eugene, OR). The Caco-2/15 cell line was obtained from A. Quaroni (Cornell University, Ithaca, NY). This clone of the parent Caco-2 cell line (HTB 37; American Type Culture Collection, Manassas, VA) has been characterized extensively elsewhere (30Aliaga J.C. Deschênes C. Beaulieu J.F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar, 34Beaulieu J.F. Quaroni A. Biochem. J. 1991; 280: 599-608Crossref PubMed Scopus (133) Google Scholar, 36Vachon P.H. Beaulieu J.F. Am. J. Physiol. 1995; 268: G857-G867PubMed Google Scholar, 37Vachon P.H. Beaulieu J.F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (186) Google Scholar) and was selected originally as expressing the highest level of sucrase-isomaltase among 16 clones obtained by random cloning. This cell line was cultured in plastic dishes in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum (FBS), as described previously. Caco-2/15 cells were used between passages 53 and 78. Studies were performed on cultures at subconfluence (50–70% confluence), confluence, and between 2 and 40 days post-confluence. Human intestinal epithelial cells (HIEC) were cultured as described (38Perreault N. Beaulieu J.F. Exp. Cell Res. 1996; 224: 354-364Crossref PubMed Scopus (132) Google Scholar) in DMEM supplemented with 4 mmglutamine, 20 mm HEPES, 50 units/ml penicillin, 50 μg/ml streptomycin, 5 ng/ml recombinant human epidermal growth factor, 0.2 IU/ml insulin, and 5% FBS. Primary cultures of human differentiated enterocytes (PCDE) prepared from specimens of small intestine from fetuses ranging from 18 to 20 weeks of age, were cultured in supplemented DMEM as described above for HIEC (39Perreault N. Beaulieu J.F. Exp. Cell Res. 1998; 254: 34-42Crossref Scopus (117) Google Scholar). When tested after 5–7 days, these primary cultures of differentiated enterocytes were well preserved; both goblet and absorptive cells exhibited the main characteristics of intact villus intestinal cells (39Perreault N. Beaulieu J.F. Exp. Cell Res. 1998; 254: 34-42Crossref Scopus (117) Google Scholar). Cells were lysed in SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.005% bromphenol blue, 1 mm phenylmethylsulfonyl fluoride (PMSF)). Proteins (40 μg) from whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 7.5 or 10% gels. Proteins were detected immunologically following electrotransfer onto nitrocellulose membranes (Amersham Pharmacia Biotech). Protein and molecular weight markers (Bio-Rad) were localized by staining with Ponceau Red. Membranes were blocked for 3 h at 25 °C in phosphate-buffered saline containing 10% powdered milk. Membranes were then incubated overnight with primary antibodies in blocking solution and with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit (1:1000) IgG for 1 h. The blots were visualized by the Amersham Pharmacia Biotech ECL system. Protein concentrations were measured using a modified Lowry procedure with bovine serum albumin as standard (44Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7067) Google Scholar). The cells were lysed for 10 min on ice with 1 ml/dish of lysis buffer (150 mm NaCl, 1 mmEDTA, 40 mm Tris, pH 7.6, 1% Triton X-100) supplemented with protease inhibitors (0.1 mm PMSF, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 10 μg/ml aprotinin) and phosphatase inhibitors (0.1 mm orthovanadate, 20 mm para-nitrophenyl phosphate, 40 mmβ-glycerophosphate). Lysates (400 μg) cleared by centrifugation (10 000 × g, 10 min) were incubated for 2 h at 4 °C with protein A-Sepharose (Amersham Pharmacia Biotech) that had been preincubated for 1 h with anti-p38α. Immunocomplexes were then washed four times with ice-cold lysis buffer and three times with ice-cold kinase buffer (20 mm para-nitrophenyl phosphate, 10 mm MgCl2, 1 mmdithiothreitol, 30 mm HEPES, pH 7.4) before performing the kinase assay. The kinase reaction was initiated by incubating the immunocomplexes at 30 °C in the presence of the substrate myelin basic protein (MBP) and [γ-32P]ATP at 20–100 μm, 1–5 μCi/assay. After 30 min, the reaction was stopped by addition of Laemmli's buffer. Radiolabeled substrates were separated from immunocomplexes by SDS-PAGE and autoradiographed. Incorporation of 32P by MBP was linear over the course of the kinase assay. The sucrase-isomaltase reporter construct used for luciferase assays contained the human sucrase-isomaltase promoter from residues −183 to +54 cloned upstream of the luciferase gene of the pGL2 reporter construct as described previously (Dr. P. G. Traber, University of Pennsylvania, Philadelphia) (45Traber P.G. Wu G.D. Wang W. Mol. Cell. Biol. 1992; 12: 3614-3627Crossref PubMed Scopus (113) Google Scholar). Plasmid E2F SV40-luc, which contains a high affinity E2F-binding site from the dihydrofolate reductase (DHFR) promoter coupled to a luciferase gene (46Slansky J.E. Li Y. Kaelin W.G. Farnham P.G. Mol. Cell. Biol. 1993; 13: 1610-1618Crossref PubMed Scopus (257) Google Scholar, 47La Thangue N.B. Curr. Opin. Cell Biol. 1994; 6: 443-450Crossref PubMed Scopus (140) Google Scholar), was a kind gift of Dr. P. Farnham (University of Wisconsin). The expression vectors for wild-type p38α and the dominant-negative mutant p38α (kindly provided by Dr. J. Pouysségur, Université de Nice, Nice, France) were previously cloned into pECE vector. The hamster CDX3 expression vector was a gift from Dr. W. J. Rutter (University of California, San Francisco) (48German M.S. Wang J. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (359) Google Scholar). Total cellular RNAs were prepared from Caco-2/15 cells at subconfluence, confluence, and 3 and 6 days post-confluence by the guanidinium isothiocyanate/phenol method (TRIZOL, Life Technologies, Inc.) as described before (49Lemischka I.R. Farmer S. Racaniello V.R. Sharp P.A. J. Mol. Biol. 1981; 151: 101-120Crossref PubMed Scopus (189) Google Scholar). RNAs were subjected to agarose gel electrophoresis with formaldehyde and transferred to nylon membranes (Nytran, Schleicher & Schuell). Equal RNA loading was confirmed by hybridization to an α-tubulin probe. Hybridizations were performed with a random-primed32P-labeled probe (Amersham Pharmacia Biotech) of a PCR-amplified human CDX2 fragment from nucleotides 1102 to 1706. First, subconfluent Caco-2/15 cells were seeded in 24-well plates and transfected by lipofection (Lipofectin, Life Technologies, Inc.) as described before (30Aliaga J.C. Deschênes C. Beaulieu J.F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar) with 0.1 μg of E2F-SV40-luciferase reporter per well. One day after transfection, cells were exposed to 2–20 μm SB203580 or 20 μm PD98059 for 24 h, and luciferase activity was measured. The increase in luciferase activity was calculated relative to the basal level of E2F-SV40-luciferase set at 1 and corrected for the empty vector effects. Second, 1 day post-confluent Caco-2/15 cells were seeded in 24-well plates and co-transfected by lipofection (LipofectAMINE 2000, Life Technologies, Inc.) as described previously (30Aliaga J.C. Deschênes C. Beaulieu J.F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar) with 0.1 μg of SI-luciferase reporter and 0.1 μg of the relevant expression vector (pECE) containing wild-type or dominant-negative mutant of p38α per well. In some experiments, 0.05 μg of wild-type CDX3 expression vector was co-transfected. One day after transfection, cells were treated with or without 2–20 μm of SB203580 for 24 h, and luciferase activity was measured. The pRL-SV40Renilla luciferase vector (Promega, Madison, WI) was used as a control for transfection efficiency. Two days after transfection, luciferase activity was measured according to the Promega protocol. The 540-base pair sequence encoding the 180-amino acid transactivation domain of CDX3 (42Trinh K.Y. Jin T. Drucker D.J. J. Biol. Chem. 1999; 274: 6011-6019Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) was PCR-amplified and cloned in frame with the DNA-binding domain of GAL4 in the mammalian expression vector pM2 (50Sadowski I. Bell B. Broad P. Hollis M. Gene ( Amst. ). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar). The expression vector was transfected by lipofection (Lipofectin, Life Technologies, Inc.) in Caco-2/15 cells with the pFR-luciferase reporter vector containing five tandem repeats of the GAL4-binding element upstream of a basic promoter element (Stratagene, La Jolla, CA). Luciferase activity was assessed after a 24-h treatment with Me2SO, 20 μmSB203580, or 20 μm PD98059. Caco-2/15 cells, treated with or without 20 μm SB203580, were harvested in water at confluence (day 0) and at 3, 6, and 9 days post-confluence, and sonicated. The disaccharidases sucrase-isomaltase and lactase-phlorizin were assayed using the method of Dahlqvist as modified by Ménard and Arsenault (51Ménard D. Arsenault P. Gastroenterology. 1985; 88: 691-700Abstract Full Text PDF PubMed Scopus (63) Google Scholar). Alkaline phosphatase was assayed by the method of Eichholz (52Eichholz A. Biochim. Biophys. Acta. 1976; 135: 475-482Crossref Scopus (166) Google Scholar). Dipeptidyl peptidase IV (DPPIV) activity was assayed according to the method of Roncari and Zuber (53Roncari G. Zuber H. Int. J. Protein Res. 1969; 1: 45-61Crossref PubMed Scopus (111) Google Scholar), with glycyl-l-proline-p-nitroanilide as substrate. Total homogenate protein content was determined using a modified Lowry procedure with bovine serum albumin as standard (44Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7067) Google Scholar). Data were

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