Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model

BackgroundThe TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation.ResultsThe expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes.ConclusionOur results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells.

[1]  A. Dignass,et al.  Peptide growth factors in the intestine. , 2001, European journal of gastroenterology & hepatology.

[2]  M. Goumans,et al.  Regulation of cell proliferation by Smad proteins , 2002, Journal of cellular physiology.

[3]  A. Merrill,et al.  Modulation of intracellular beta-catenin localization and intestinal tumorigenesis in vivo and in vitro by sphingolipids. , 2001, Cancer research.

[4]  E. Miska,et al.  Acetylation of β-Catenin by CREB-binding Protein (CBP)* , 2002, The Journal of Biological Chemistry.

[5]  M. Kedinger,et al.  Cellular and molecular partners involved in gut morphogenesis and differentiation. , 1998, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[6]  J. Romashkova,et al.  NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. , 1999, Nature.

[7]  M. Ashburner.  A Laboratory manual , 1989 .

[8]  K. Barrett,et al.  p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells. , 2003, American journal of physiology. Cell physiology.

[9]  Antony W Burgess,et al.  Epidermal growth factor receptor: mechanisms of activation and signalling. , 2003, Experimental cell research.

[10]  M. Mäki,et al.  TGF-β induces the expression of SAP30L, a novel nuclear protein , 2003, BMC Genomics.

[11]  Hans Clevers,et al.  Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. , 2003, Genes & development.

[12]  B. Craig,et al.  Gene expression profiling of Caco-2 BBe cells suggests a role for specific signaling pathways during intestinal differentiation. , 2003, Physiological genomics.

[13]  J. Seidelin,et al.  Transcriptome changes during intestinal cell differentiation. , 2002, Biochimica et biophysica acta.

[14]  T. Visakorpi,et al.  Identification of novel transcription factor-like gene from human intestinal cells. , 2000, Biochemical and biophysical research communications.

[15]  K. Barrett,et al.  Phosphatidylinositol 3-Kinase Mediates the Inhibitory Effect of Epidermal Growth Factor on Calcium-dependent Chloride Secretion* , 1996, The Journal of Biological Chemistry.

[16]  L. Attisano,et al.  TGFβ and Wnt pathway cross-talk , 2004, Cancer and Metastasis Reviews.

[17]  R. Derynck,et al.  Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor , 1995, Nature.

[18]  Andrew J. Wilson,et al.  Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis. , 2005, Gastroenterology.

[19]  A. Mauviel,et al.  TGF-β-induced SMAD signaling and gene regulation : consequences for extracellular matrix remodeling and wound healing , 2004 .

[20]  Hans Clevers,et al.  Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors , 2002, Molecular and Cellular Biology.

[21]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[22]  J. Mariadason,et al.  Quantitative rather than qualitative differences in gene expression predominate in intestinal cell maturation along distinct cell lineages. , 2005, Experimental cell research.

[23]  J. Behrens,et al.  Biochemical interactions in the wnt pathway. , 2000, Biochimica et biophysica acta.

[24]  J. Romashkova,et al.  NF-κB is a target of AKT in anti-apoptotic PDGF signalling , 1999, Nature.

[25]  C. Hill,et al.  New insights into TGF-beta-Smad signalling. , 2004, Trends in biochemical sciences.

[26]  J. Massagué,et al.  A self-enabling TGFbeta response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells. , 2003, Molecular cell.

[27]  J. Massagué,et al.  E2F4/5 and p107 as Smad Cofactors Linking the TGFβ Receptor to c-myc Repression , 2002, Cell.

[28]  E. Miska,et al.  Acetylation of beta-catenin by CREB-binding protein (CBP). , 2002, The Journal of biological chemistry.

[29]  J. Massagué.  How cells read TGF-beta signals. , 2000, Nature reviews. Molecular cell biology.

[30]  M. Mäki,et al.  Serum immunoglobulin A from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation. , 1999, Gastroenterology.

[31]  S. Wölfl,et al.  Ranking: a closer look on globalisation methods for normalisation of gene expression arrays. , 2002, Nucleic acids research.

[32]  M. Bjerknes,et al.  Patterns of gene expression along the crypt‐villus axis in mouse jejunal epithelium , 1996, The Anatomical record.

[33]  J. Beaulieu,et al.  Phosphatidylinositol 3-Kinase Controls Human Intestinal Epithelial Cell Differentiation by Promoting Adherens Junction Assembly and p38 MAPK Activation* , 2002, The Journal of Biological Chemistry.

[34]  Joan Massagu,et al.  How cells read TGF-|[beta]| signals , 2000 .

[35]  A. Meng,et al.  Negative regulation of TGF-beta signaling in development. , 2004, Cell research.

[36]  M. Mäki,et al.  Differentially expressed CC3/TIP30 and rab11 along in vivo and in vitro intestinal epithelial cell crypt-villus axis. , 2001, Life sciences.

[37]  T. Halttunen.  Biological Functions of Coeliac Disease Autoantibodies , 2002 .

[38]  M. Mäki,et al.  Fibroblasts and transforming growth factor beta induce organization and differentiation of T84 human epithelial cells. , 1996, Gastroenterology.

[39]  J. Mills,et al.  A new approach for filtering noise from high-density oligonucleotide microarray datasets. , 2001, Nucleic acids research.

[40]  Hans Clevers,et al.  Signaling pathways in intestinal development and cancer. , 2004, Annual review of cell and developmental biology.

[41]  Hans Clevers,et al.  The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. , 2002, Cell.

[42]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[43]  G A Whitmore,et al.  Power and sample size for DNA microarray studies , 2002, Statistics in medicine.

[44]  P. Dijke,et al.  New insights into TGF-β–Smad signalling , 2004 .

[45]  L. Attisano,et al.  Transcriptional Regulation by Smads: Crosstalk between the TGF-&bgr; and Wnt Pathways , 2001, The Journal of bone and joint surgery. American volume.

[46]  M. Plateroti,et al.  Intestinal Epithelial‐Mesenchymal Cell Interactions , 1998, Annals of the New York Academy of Sciences.

[47]  D. Arango,et al.  A gene expression profile that defines colon cell maturation in vitro. , 2002, Cancer research.

[48]  G. Casey,et al.  The AKT/IκB kinase pathway promotes angiogenic/metastatic gene expression in colorectal cancer by activating nuclear factor-κB and β-catenin , 2005, Oncogene.

[49]  G. Casey,et al.  The AKT/I kappa B kinase pathway promotes angiogenic/metastatic gene expression in colorectal cancer by activating nuclear factor-kappa B and beta-catenin. , 2005, Oncogene.

[50]  Jack A. M. Leunissen,et al.  EXProt: a database for proteins with an experimentally verified function , 2002, Nucleic Acids Res..

[51]  M. Mäki,et al.  A novel human glycosyltransferase: primary structure and characterization of the gene and transcripts. , 2003, Biochemical and biophysical research communications.

[52]  T. Visakorpi,et al.  cDNA microarray analysis of gene expression in coeliac disease jejunal biopsy samples. , 2004, Journal of autoimmunity.

[53]  Michael Koutsilieris,et al.  The Akt pathway: molecular targets for anti-cancer drug development. , 2004, Current cancer drug targets.

[54]  Jason C Mills,et al.  Molecular features of adult mouse small intestinal epithelial progenitors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[55]  Tony Pawson,et al.  β-Catenin and TCF Mediate Cell Positioning in the Intestinal Epithelium by Controlling the Expression of EphB/EphrinB , 2002, Cell.

[56]  A. Mauviel,et al.  TGF-beta-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing. , 2004, Journal of dermatological science.

[57]  M. Bienz.  Faculty Opinions recommendation of Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. , 2002 .