Jean‐Baptiste Demoulin

For about four decades, platelet-derived growth factors (PDGF) and their receptors have been the subject of intense research, revealing their roles in embryo development and human diseases. Drugs such as imatinib, which selectively inhibit the tyrosine kinase activity of these receptors, have been approved for the treatment of cancers such as gastrointestinal stromal tumors and chronic eosinophilic leukemia. Today, the interest in these factors is still increasing in relationship with new potential clinical applications in cancer, stroke, fibrosis and infectious diseases. This review focuses on the mechanisms of PDGF receptor signaling, with an emphasis on pathways that are important for disease development. Of particular interest, recent studies revealed significant differences between normal and cancer cells regarding signal transduction by these growth factors.

FOXO (Forkhead box O) transcription factors induce cell growth arrest and apoptosis, which can be prevented by FOXO phosphorylation by AKT in response to growth factors such as platelet-derived growth factors (PDGF) and insulin-like growth factor I (IGF-I). In addition to this well characterized post-translational modification, we showed that FOXO1, FOXO3, and FOXO4 were also regulated at the transcriptional level. PDGF, fibroblast growth factors (FGF), and IGF-I repressed the expression of FOXO genes in human fibroblasts. This process was sensitive to phosphatidylinositol 3-kinase inhibition by LY294002. FOXO1-specific shRNA decreased FOXO1 mRNA expression and enhanced fibroblast proliferation, mimicking the effects of growth factors. Conversely, ectopic FOXO3 activation blocked the proliferation of fibroblasts and induced the expression of FOXO1, FOXO4, and p27-KIP1. Using luciferase reporter assays and chromatin immunoprecipitations, we identified a conserved FOXO-binding site in the promoter of the FOXO1 gene, which was required for regulation by PDGF, and mediated the up-regulation of FOXO1 by itself and by FOXO3. Altogether, our results suggest that the expression of FOXO1 and FOXO4 genes is stimulated by FOXO3 and possibly by other FOXO factors in a positive feedback loop, which is disrupted by growth factors. FOXO (Forkhead box O) transcription factors induce cell growth arrest and apoptosis, which can be prevented by FOXO phosphorylation by AKT in response to growth factors such as platelet-derived growth factors (PDGF) and insulin-like growth factor I (IGF-I). In addition to this well characterized post-translational modification, we showed that FOXO1, FOXO3, and FOXO4 were also regulated at the transcriptional level. PDGF, fibroblast growth factors (FGF), and IGF-I repressed the expression of FOXO genes in human fibroblasts. This process was sensitive to phosphatidylinositol 3-kinase inhibition by LY294002. FOXO1-specific shRNA decreased FOXO1 mRNA expression and enhanced fibroblast proliferation, mimicking the effects of growth factors. Conversely, ectopic FOXO3 activation blocked the proliferation of fibroblasts and induced the expression of FOXO1, FOXO4, and p27-KIP1. Using luciferase reporter assays and chromatin immunoprecipitations, we identified a conserved FOXO-binding site in the promoter of the FOXO1 gene, which was required for regulation by PDGF, and mediated the up-regulation of FOXO1 by itself and by FOXO3. Altogether, our results suggest that the expression of FOXO1 and FOXO4 genes is stimulated by FOXO3 and possibly by other FOXO factors in a positive feedback loop, which is disrupted by growth factors. Forkhead transcription factors, which were initially described in Drosophila melanogaster, constitute a family of transcription factors that share a conserved DNA-binding domain, the so-called forkhead box (1Huang H. Tindall D.J. J. Cell Sci... 2007; 120: 2479-2487Google Scholar, 2Van Der Heide L.P. Hoekman M.F. Smidt M.P. Biochem. J... 2004; 380: 297-309Google Scholar). The FOXO (forkhead box O) group comprises four homologous mammalian proteins, namely FOXO1 (also called FKHR), FOXO3 (FKHR-L1), FOXO4 (AFX), and FOXO6. Activation of these factors induces cell cycle arrest, DNA damage repair, differentiation, and apoptosis. They also increase the resistance to oxidative stress, which was shown to be particularly important in hematopoietic stem cells, and regulate glucose metabolism in various organs. In Caenorhabditis elegans, the FOXO orthologue DAF-16 prolongs life span. The activity of FOXO proteins is tightly controlled by multiple post-translational modifications (3Obsil T. Obsilova V. Oncogene.. 2008; 27: 2263-2275Google Scholar, 4Vogt P.K. Jiang H. Aoki M. Cell Cycle.. 2005; 4: 908-913Google Scholar). Growth factors, insulin, and other cell stimuli induce FOXO phosphorylation and inactivation by AKT (also called protein kinase B), a serine/threonine kinase that is activated via the phosphatidylinositol (PI) 3The abbreviations used are: PI, phosphatidylinositol; PDGF, platelet-derived growth factors; FGF, fibroblast growth factor; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; shRNA, small hairpin RNA. 3-kinase pathway (1Huang H. Tindall D.J. J. Cell Sci... 2007; 120: 2479-2487Google Scholar, 3Obsil T. Obsilova V. Oncogene.. 2008; 27: 2263-2275Google Scholar). All FOXO proteins are substrates of AKT, which phosphorylates three conserved sites, resulting in the exclusion of FOXOs from the nucleus and in their subsequent ubiquitination and degradation. Phosphorylation by AKT may also regulate FOXO ability to bind to DNA (3Obsil T. Obsilova V. Oncogene.. 2008; 27: 2263-2275Google Scholar). In addition, the mitogen-activated protein kinases ERK and p38, as well as serum- and glucocorticoid-inducible kinase, DIRK1, and IKKβ, also inactivate FOXO1 and/or FOXO3 by direct phosphorylation (1Huang H. Tindall D.J. J. Cell Sci... 2007; 120: 2479-2487Google Scholar, 5Yang J.Y. Zong C.S. Xia W. Yamaguchi H. Ding Q. Xie X. Lang J.Y. Lai C.C. Chang C.J. Huang W.C. Huang H. Kuo H.P. Lee D.F. Li L.Y. Lien H.C. Cheng X. Chang K.J. Hsiao C.D. Tsai F.J. Tsai C.H. Sahin A.A. Muller W.J. Mills G.B. Yu D. Hortobagyi G.N. Hung M.C. Nat. Cell Biol... 2008; 10: 138-148Google Scholar, 6Asada S. Daitoku H. Matsuzaki H. Saito T. Sudo T. Mukai H. Iwashita S. Kako K. Kishi T. Kasuya Y. Fukamizu A. Cell Signal... 2007; 19: 519-527Google Scholar). By contrast, phosphorylation by c-Jun N-terminal kinase (JNK) kinases upon cell stress activates FOXO4 (7Essers M.A. Weijzen S. de Vries-Smits A.M. Saarloos I. de Ruiter N.D. Bos J.L. Burgering B.M. EMBO J... 2004; 23: 4802-4812Google Scholar). In the absence of growth factors, FOXOs reside in the nucleus and up-regulate genes that inhibit the cell cycle (p27 KIP1 and p21 WAF1), promote apoptosis (Fas ligand, Bim, and TRAIL), and decrease oxidative stress (superoxide dismutase and catalase) (1Huang H. Tindall D.J. J. Cell Sci... 2007; 120: 2479-2487Google Scholar). A number of genes are also repressed by activated FOXOs, including cyclin D1 and D2. The different members of the FOXO family share a common DNA-binding site and regulate overlapping sets of target genes. FOXO transcriptional activity is further regulated by acetylation in the nucleus (3Obsil T. Obsilova V. Oncogene.. 2008; 27: 2263-2275Google Scholar). In line with their ability to block cell growth and to induce apoptosis, FOXO genes act as tumor suppressors. Deletion of all FOXO1, FOXO3, and FOXO4 alleles in adult mice induces a cancer prone condition, characterized by hemangiomas and thymic lymphomas (8Paik J.H. Kollipara R. Chu G. Ji H. Xiao Y. Ding Z. Miao L. Tothova Z. Horner J.W. Carrasco D.R. Jiang S. Gilliland D.G. Chin L. Wong W.H. Castrillon D.H. DePinho R.A. Cell.. 2007; 128: 309-323Google Scholar). In human cancers, several chromosomal translocations disrupt FOXO genes, producing hybrid proteins in which the forkhead DNA-binding domain and the AKT phosphorylation sites are lost (9Fu Z. Tindall D.J. Oncogene.. 2008; 27: 2312-2319Google Scholar). Pax3-FOXO1 and Pax6-FOXO1 were described in alveolar rhabdomyosarcomas, and the fusion of MLL with FOXO3 or FOXO4 was found in acute myeloid leukemias. In many tumor types, the constitutive activation of PI 3-kinase, AKT, and ERK is expected to inhibit FOXO proteins (9Fu Z. Tindall D.J. Oncogene.. 2008; 27: 2312-2319Google Scholar). Not much is known about the regulation of FOXO mRNA expression. The transcription factor E2F1 was shown to induce FOXO1 and FOXO3 expression (10Nowak K. Killmer K. Gessner C. Lutz W. Biochim. Biophys. Acta.. 2007; 1769: 244-252Google Scholar), and FoxC1 up-regulates FOXO1 in the eye (11Berry F.B. Skarie J.M. Mirzayans F. Fortin Y. Hudson T.J. Raymond V. Link B.A. Walter M.A. Hum. Mol. Genet... 2008; 17: 490-505Google Scholar). In the present report, we observed that the RNA expression of FOXO1, FOXO3, and FOXO4 was repressed by growth factors such as platelet-derived growth factors (PDGF) and fibroblast growth factors (FGF), which induce cell proliferation. These growth factors play a key role in the development of the embryo and in various human diseases, including cancer (12Andrae J. Gallini R. Betsholtz C. Genes Dev... 2008; 22: 1276-1312Google Scholar, 13Pietras K. Sjoblom T. Rubin K. Heldin C.H. Ostman A. Cancer Cell.. 2003; 3: 439-443Google Scholar-14Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev... 2005; 16: 139-149Google Scholar). PDGFs are dimeric ligands that bind to specific receptor tyrosine kinases, PDGF receptors α and β, forming homo- or heterodimers (15Heldin C.H. Östman A. Rönnstrand L. Biochim. Biophys. Acta.. 1998; 1378: 79-113Google Scholar). They activate multiple signaling effectors, including PI 3-kinase, AKT, ERK, and phospholipase Cγ (15Heldin C.H. Östman A. Rönnstrand L. Biochim. Biophys. Acta.. 1998; 1378: 79-113Google Scholar, 16Tallquist M. Kazlauskas A. Cytokine Growth Factor Rev... 2004; 15: 205-213Google Scholar, 17Kallin A. Demoulin J.B. Nishida K. Hirano T. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 17897-17904Google Scholar-18Demoulin J.B. Seo J.K. Ekman S. Grapengiesser E. Hellman U. Ronnstrand L. Heldin C.H. Biochem. J... 2003; 376: 505-510Google Scholar). These signal transduction pathways regulate the activity of numerous transcription factors, controlling the expression of more than one hundred target genes (19Demoulin J.B. Ericsson J. Kallin A. Rorsman C. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 35392-35402Google Scholar, 20Demoulin J.B. Enarsson M. Larsson J. Essaghir A. Heldin C.H. Forsberg-Nilsson K. Growth Factors.. 2006; 24: 184-196Google Scholar, 21Kallin A. Johannessen L.E. Cani P.D. Marbehant C.Y. Essaghir A. Foufelle F. Ferre P. Heldin C.H. Delzenne N.M. Demoulin J.B. J. Lipid Res... 2007; 48: 1628-1636Google Scholar-22Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell.. 1999; 97: 727-741Google Scholar). PDGF was reported to induce the phosphorylation and inactivation of FOXO1 protein in hepatic stellate cells, in fibroblasts, and in vascular smooth muscle cells (23Adachi M. Osawa Y. Uchinami H. Kitamura T. Accili D. Brenner D.A. Gastroenterology.. 2007; 132: 1434-1446Google Scholar, 24Abid M.R. Yano K. Guo S. Patel V.I. Shrikhande G. Spokes K.C. Ferran C. Aird W.C. J. Biol. Chem... 2005; 280: 29864-29873Google Scholar, 25Aoki M. Jiang H. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 13613-13617Google Scholar, 26Vantler M. Caglayan E. Zimmermann W.H. Baumer A.T. Rosenkranz S. J. Biol. Chem... 2005; 280: 14168-14176Google Scholar-27Vantler M. Huntgeburth M. Caglayan E. Ten Freyhaus H. Schnabel P. Rosenkranz S. FEBS Lett... 2006; 580: 6769-6776Google Scholar). Here, we suggest that the inactivation of FOXO3 protein by PDGF disrupts a positive feedback mechanism, whereby FOXO3 stimulates the expression of FOXO genes. Cells and Reagents—AG01518 human foreskin fibroblasts were grown in Quantum 333 fibroblast growth medium (PAA laboratories, Pasching, Austria). PDGF-AA, -BB, FGF-2 (basic-FGF), FGF-4, and insulin-like growth factor I were obtained from Peprotech (London, UK). Antibodies FOXO and AKT were from Cell and and were from was from human fibroblasts, and were as described (19Demoulin J.B. Ericsson J. Kallin A. Rorsman C. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 35392-35402Google Scholar). assays were as described with modifications J.B. C. E. de D. J. Mol. Biol... 16: Scholar). were in in a in Quantum 333 the cells were and in medium for Growth factors were with for were a with a cell The of was a fibroblasts were for in medium with or were or RNA was the The RNA was in a further the expression of known target genes and was by (19Demoulin J.B. Ericsson J. Kallin A. Rorsman C. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 35392-35402Google Scholar). were the The results were and the as a line to J. Essaghir A. M. K. K. R. G. Demoulin J.B. V. 2008; 22: Scholar). In the the factor all sets was to and the factor was to The were to number sets that were all in the were was as described (19Demoulin J.B. Ericsson J. Kallin A. Rorsman C. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 35392-35402Google Scholar, 20Demoulin J.B. Enarsson M. Larsson J. Essaghir A. Heldin C.H. Forsberg-Nilsson K. Growth Factors.. 2006; 24: 184-196Google Scholar, J. Essaghir A. M. K. K. R. G. Demoulin J.B. V. 2008; 22: and the are shown in shRNA of for human FOXO1 was obtained from All of were for and and for target and were used for further A was obtained from number The and were with the and the and D. H. Yu W.C. R.A. C.D. 2003; cells were at and and used to fibroblasts in the of the cells were with growth medium for to cell fibroblasts were grown for in a cells were with and or number J. L. J. Cell.. 2004; or the cells were with and in medium in the of for The cells were stimulated with for with PBS, and in in for The cells were and for at in and The cells were at with or in the of The cells were and with to the cells were and a cells were identified by FOXO1 promoter was by from the as for The promoter was and and The from with the FOXO1 and the The was by with and The and were by and and The was obtained by All of the were The were by and fibroblasts were with of and of in the of of the cells were with and in medium with for and stimulated with for cells were with of of and FOXO1, FOXO3, or C. L. Burgering B.M. J.W. L. Mol. Biol... Scholar). The cells were for with PBS, and stimulated with or The luciferase activity was and activity as described (19Demoulin J.B. Ericsson J. Kallin A. Rorsman C. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 35392-35402Google Scholar, 21Kallin A. Johannessen L.E. Cani P.D. Marbehant C.Y. Essaghir A. Foufelle F. Ferre P. Heldin C.H. Delzenne N.M. Demoulin J.B. J. Lipid Res... 2007; 48: 1628-1636Google Scholar). was with and was by the addition of to a of The cells were with was by was used to and to the of chromatin was for with protein the were at with or or used as a The were to protein for at and in a and and and and The proteins were in the of at DNA was in and used as for with the shown in were was the in the of of specific promoter was as the of the one of at three is shown with was to Growth the of FOXO1, FOXO3, and FOXO4 a we the regulation of expression by PDGF and in human fibroblasts. The results be described that the expression of the genes FOXO1, FOXO3, and FOXO4 was decreased growth factor this we the RNA of FOXO genes by that the expression of FOXO1 is repressed of fibroblasts with or FOXO4 expression was also FOXO3 was regulated to a by which was described as a was in these cells results were obtained in fibroblasts with growth factor I also repressed FOXO1 expression in a In the expression of FOXO1 and FOXO4 was decreased the addition of PDGF FOXO expression the of FOXO1 protein was by in cells stimulated for with The the of FOXO1 in PDGF activity of FOXO proteins is by phosphorylation upon growth factor the phosphorylation of FOXO1, FOXO3, and FOXO4 in fibroblasts stimulated with PDGF by of FOXO1 with or of FOXO3 This conserved site is by AKT Der Heide L.P. Hoekman M.F. Smidt M.P. Biochem. J... 2004; 380: 297-309Google Scholar, 3Obsil T. Obsilova V. Oncogene.. 2008; 27: 2263-2275Google Scholar), which was also activated in these cells upon PDGF as from with the is well that FOXO phosphorylation induces from the nucleus to the this was reported in vascular smooth muscle cells by PDGF M.R. Yano K. Guo S. Patel V.I. Shrikhande G. Spokes K.C. Ferran C. Aird W.C. J. Biol. Chem... 2005; 280: 29864-29873Google Scholar). that PDGF FOXO in fibroblasts, we cells with FOXO1 or FOXO3 and the of the proteins by that FOXO1 and FOXO3 were from the nucleus upon with PDGF for FOXO transcriptional activity in cells by growth factors, we the regulation of known FOXO target genes by and FGF-2 in fibroblasts our we identified genes that were reported to be by at one FOXO and were present in our as well as genes. of the genes known to be induced by FOXO were repressed by of fibroblasts with PDGF and FGF-2 for characterized FOXO target genes, such as and were all repressed by growth factors. Conversely, the of the genes known to be repressed by FOXO were induced by growth factors, including cyclin and and the that this was obtained by we which a of of growth factors was observed of Altogether, these results that the expression of FOXO was by PDGF and FGF-2 in a with FOXO FOXOs a in the of is well that FOXO factors induce cell growth arrest and apoptosis Oncogene.. 2008; 27: Scholar). this also to human fibroblasts, we used a receptor fusion protein which can be activated by C. L. Burgering B.M. J.W. L. Mol. Biol... Scholar, C. J. M. H. Z. J. G. 2005; Scholar). In this the three AKT phosphorylation sites of FOXO3 are to the inactivation of the protein by growth factors. was in a The number of cells was by or by cells in the of Activation of by the of PDGF and FGF-2 fibroblast proliferation. FOXO proteins are tightly regulated by several post-translational was a in FOXO RNA cell this we used the which is to RNA specific FOXO1 shRNA FOXO1 mRNA expression by a fibroblasts were with FOXO1 expression was repressed by this is with the of PDGF FOXO1 expression. of these FOXO1 shRNA or in the proliferation of fibroblasts, as by the shown in PDGF stimulated fibroblast proliferation more than in the of shRNA increase cell growth in the of FOXO1 In these that FOXO1 expression is to fibroblast cell The of FOXO1 by PDGF to PI the of regulation of FOXO expression by PDGF, we de protein was shown in PDGF was to decrease FOXO1 expression in the of a protein the of a direct to which signal transduction pathway was required in this small The PI 3-kinase the regulation of FOXO1 by PDGF in cells, the mitogen-activated protein kinase pathway results were obtained in cells also blocked the of observed that the activation of AKT by PDGF in as expected (19Demoulin J.B. Ericsson J. Kallin A. Rorsman C. Ronnstrand L. Heldin C.H. J. Biol. Chem... 2004; 279: 35392-35402Google Scholar). Y. Marbehant and PI 3-kinase is also required for FOXO phosphorylation and inactivation by PDGF (23Adachi M. Osawa Y. Uchinami H. Kitamura T. Accili D. Brenner D.A. Gastroenterology.. 2007; 132: 1434-1446Google Scholar, M. Huntgeburth M. Caglayan E. Ten Freyhaus H. Schnabel P. Rosenkranz S. FEBS Lett... 2006; 580: 6769-6776Google Scholar), our the that FOXO proteins the expression of their genes, a process that be disrupted upon AKT activation by growth factors. FOXO1 by this we the expression of FOXO genes in fibroblasts with The activation of by induced the expression of FOXO1 and FOXO4, as shown by The gene, a well characterized target of FOXO3, was used as a positive C. L. Burgering B.M. J.W. L. Mol. Biol... Scholar). shown in was regulated to a as FOXO1 and By contrast, we regulation of FOXO3, which was with with the of FOXO3, in the The expression of was controlled FOXO3 that were to the these that FOXO3 activation can induce the expression of FOXO1 and the FOXO1 a of the human FOXO1 promoter which a of with the This was in of a luciferase reporter and with in In the of enhanced FOXO1 promoter activity of the promoter and the of FOXO3 Deletion of the promoter activity or response to FOXO3 activation By contrast, FOXO3 activation a much a promoter and the activity of or The in of the FOXO1 promoter a FOXO-binding site the This is conserved in the and the FOXO-binding we key A promoter this to with FOXO1 regulate the expression of gene, we cells with FOXO1 and with FOXO3, in a luciferase of FOXO3 stimulated FOXO1 promoter the with FOXO1 stimulated the promoter of to a with FOXO3. of the FOXO-binding site of the FOXO1 promoter the of FOXO1 and decreased the of FOXO3. FOXO1 and FOXO3 were to the FOXO1 we chromatin with A DNA that the FOXO site of the promoter was with FOXO1 in cells This was by FOXO1 FOXO3 was also to the promoter in luciferase described the signal was observed in cells with and stimulated with In our suggest that FOXO1 expression is upon of FOXO1 and FOXO3 proteins to the FOXO1 a may to FOXO1 regulation of the FOXO-binding site that we as by the of FOXO3 the PDGF the Activation of the FOXO1 by FOXO this for the regulation of FOXO1 by PDGF, luciferase reporter assays were in fibroblasts. observed that PDGF repressed the transcriptional activity of the FOXO1 promoter The activity of the FOXO1 which the FOXO-binding was stimulated by we PDGF FOXO to the FOXO1 promoter by chromatin observed that FOXO1 and FOXO3 were to the FOXO1 promoter in fibroblasts in with the results that we obtained in with PDGF decreased the of FOXO1 and FOXO3 Altogether, our that PDGF the activation of FOXO1 promoter by FOXO the inactivation of FOXO proteins by growth factors to the phosphorylation of FOXOs and their subsequent from the nucleus to the (1Huang H. Tindall D.J. J. Cell Sci... 2007; 120: 2479-2487Google Scholar, D.R. A. Oncogene.. 2008; 27: Scholar). This well pathway for the inhibition of FOXO proteins by PDGF in the present In addition, Aoki M. Jiang H. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 13613-13617Google showed that PDGF induces the of FOXO1 protein by observed that fibroblasts with the blocked the of FOXO1 mRNA expression by PDGF that the of FOXO by may play a role in our Altogether, these are in with the by Matsuzaki H. Daitoku H. M. K. Fukamizu A. Proc. Natl. Acad. Sci. U. S. A... 2003; Scholar), showed that and of FOXO1 are required for inactivation by the of of at the of FOXO DNA or transcriptional for D.R. A. Oncogene.. 2008; 27: Scholar). that FOXO1 expression is stimulated by activated FOXO3, the activation to FOXO1 expression and FOXO1 promoter FOXO3 also FOXO1 promoter FOXO3 to at one site in FOXO1 and inactivation of FOXO3 by phosphorylation in the of PDGF with FOXO1 FOXO3 may also regulate the expression of FOXO4, expression is induced by activated and decreased by conserved FOXO-binding site be identified in the promoter of In addition, luciferase and chromatin that FOXO1 induces the expression of all FOXO transcription factors bind to the promoter and share a number of target genes, is that all induce the expression of FOXO1 and FOXO4, at to a positive feedback controlling FOXO expression. positive feedback regulation described for other transcription factors such as V. E. M. H. E. Biochem. J... 2006; Scholar). the regulation of the FOXO3 by growth factors such as further In to the decrease in FOXO1 we also observed a decrease in FOXO1 protein expression in cells with PDGF for FOXO1 by may also the decrease in FOXO1 the transcriptional of FOXO1 also protein was to these In line with other cell M. Jiang H. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 13613-13617Google Scholar, Oncogene.. 2008; 27: Scholar, Biochem. 2003; Scholar), we that activation of FOXO3 blocked the proliferation of human fibroblasts in response to growth factors. Conversely, the expression of the FOXO1 with specific shRNA enhanced fibroblast proliferation. This that the of FOXO1 mRNA expression is important of cell proliferation, in addition to the well characterized post-translational modifications of FOXO proteins by AKT and other signaling The regulation of FOXO mRNA expression may or the and effects that are by the direct and of FOXO proteins by phosphorylation in the of growth factors. Conversely, up-regulation of FOXO genes by activated FOXO3 may also to FOXO expression upon growth factor or In a X. R. Chang J.W. Lee D. E. A. J. Z. R. Lee J. D.A. W. S. P. S. J. 2004; that FOXO1 expression was in by several such as and mRNA expression of FOXO1, FOXO3, and FOXO4 also reported in cells upon activation of the which also induced FOXO1 protein phosphorylation J. 2007; Scholar). The decrease in FOXO1, FOXO3, and FOXO4 expression in these cells was sensitive to PI 3-kinase in our FOXO1 was in cells in kinase, a key of be that the were a different may for FOXO in the constitutive activation of growth factor receptors and the PI 3-kinase pathway is a in cancer cells, our that FOXO expression may be decreased in which may to cell a decreased expression of FOXO3 with cancer cell A.M. Mol. Cancer Res... 2005; 3: Scholar). to the mRNA expression of FOXO genes is in tumor In we identified a of regulation of FOXOs at the transcriptional which fibroblast proliferation. R. and J. for of are to the members of the for with

Deciphering transcription factor networks from microarray data remains difficult. This study presents a simple method to infer the regulation of transcription factors from microarray data based on well-characterized target genes. We generated a catalog containing transcription factors associated with 2720 target genes and 6401 experimentally validated regulations. When it was available, a distinction between transcriptional activation and inhibition was included for each regulation. Next, we built a tool (www.tfacts.org) that compares submitted gene lists with target genes in the catalog to detect regulated transcription factors. TFactS was validated with published lists of regulated genes in various models and compared to tools based on in silico promoter analysis. We next analyzed the NCI60 cancer microarray data set and showed the regulation of SOX10, MITF and JUN in melanomas. We then performed microarray experiments comparing gene expression response of human fibroblasts stimulated by different growth factors. TFactS predicted the specific activation of Signal transducer and activator of transcription factors by PDGF-BB, which was confirmed experimentally. Our results show that the expression levels of transcription factor target genes constitute a robust signature for transcription factor regulation, and can be efficiently used for microarray data mining.

The gut microbiota regulates key hepatic functions, notably through the production of bacterial metabolites that are transported via the portal circulation. We evaluated the effects of metabolites produced by the gut microbiota from aromatic amino acids (phenylacetate, benzoate, p-cresol, and indole) on liver inflammation induced by bacterial endotoxin. Precision-cut liver slices prepared from control mice, Kupffer cell (KC)-depleted mice, and obese mice ( ob/ ob) were treated with or without LPS and bacterial metabolites. We observed beneficial effects of indole that dose-dependently reduced the LPS-induced up-regulation of proinflammatory mediators at both mRNA and protein levels in precision-cut liver slices prepared from control or ob/ ob mice. KC depletion partly prevented the antiinflammatory effects of indole, notably through a reduction of nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) pathway activation. In vivo, the oral administration of indole before an LPS injection reduced the expression of key proteins of the NF-κB pathway and downstream proinflammatory gene up-regulation. Indole also prevented LPS-induced alterations of cholesterol metabolism through a transcriptional regulation associated with increased 4β-hydroxycholesterol hepatic levels. In summary, indole appears as a bacterial metabolite produced from tryptophan that is able to counteract the detrimental effects of LPS in the liver. Indole could be a new target to develop innovative strategies to decrease hepatic inflammation.-Beaumont, M., Neyrinck, A. M., Olivares, M., Rodriguez, J., de Rocca Serra, A., Roumain, M., Bindels, L. B., Cani, P. D., Evenepoel, P., Muccioli, G. G., Demoulin, J.-B., Delzenne, N. M. The gut microbiota metabolite indole alleviates liver inflammation in mice.