Regulation of Biglycan Gene Expression by Transforming Growth Factor-β Requires MKK6-p38 Mitogen-activated Protein Kinase Signaling Downstream of Smad Signaling

Several signaling pathways have been implicated in mediating TGF-β1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-β1 depended on a functional Smad pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W., Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem.277, 36118–36128). Here, we present evidence that the ability of TGF-β1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-β effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-β type I receptor or Smad2 phosphorylation at concentrations up to 10 μm, 2) the up-regulation of BGN mRNA was preceded by a delayed increase in the phosphorylation of p38 and its upstream activator MKK6 in TGF-β1-treated PANC-1 cells, 3) inhibition of the p38 pathway by stable retroviral transduction with a dominant negative mutant of either p38 or MKK6 reduced TGF-β1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-β1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-β1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-β-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-β1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-β-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-β induction of BGN expression in pancreatic cells requires activation of MKK6-p38 MAPK signaling downstream of Smad signaling and provide a mechanistic clue to the up-regulation of BGN seen in inflammatory response-related fibrosis and desmoplasia. Several signaling pathways have been implicated in mediating TGF-β1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-β1 depended on a functional Smad pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W., Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem.277, 36118–36128). Here, we present evidence that the ability of TGF-β1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-β effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-β type I receptor or Smad2 phosphorylation at concentrations up to 10 μm, 2) the up-regulation of BGN mRNA was preceded by a delayed increase in the phosphorylation of p38 and its upstream activator MKK6 in TGF-β1-treated PANC-1 cells, 3) inhibition of the p38 pathway by stable retroviral transduction with a dominant negative mutant of either p38 or MKK6 reduced TGF-β1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-β1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-β1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-β-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-β1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-β-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-β induction of BGN expression in pancreatic cells requires activation of MKK6-p38 MAPK signaling downstream of Smad signaling and provide a mechanistic clue to the up-regulation of BGN seen in inflammatory response-related fibrosis and desmoplasia. transforming growth factor activin receptor-like kinase 5 biglycan fetal calf serum glyceraldehyde-3-phosphate dehydrogenase mitogen-activated protein MAP kinase plasminogen activator inhibitor-1 epithelial to mesenchymal transdifferentiation extracellular signal-regulated kinase c-Jun N-terminal kinase mitogen-activated protein kinase kinase kinase MAPK/ERK kinase stress-activated protein kinase reverse transcriptase phosphate-buffered saline TGF-β1 has emerged as a multifunctional cytokine involved in autocrine and paracrine regulation of proliferation, differentiation, wound healing, apoptosis, and immunomodulation (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology. Springer Verlag, Heidelberg, Germany1990: 419-472Google Scholar, 2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Google Scholar). TGF-β1, one of three mammalian TGF-β isoforms, TGF-β1–3, is a potent inducer of extracellular matrix formation and has been implicated as the key mediator of fibrogenesis and desmoplasia in a variety of tissues (3Border W.A. Noble N.A. N. Engl. J. Med. 1994; 331: 1286-1292Google Scholar). In epithelial cells, TGF-β, besides its powerful antiproliferative function, induces morphological and biochemical changes toward a mesenchymal phenotype designated epithelial to mesenchymal transdifferentiation (EMT) (4Piek E. Moustakas A. Kurisaki A. Heldin C.H. ten Dijke P. J. Cell Sci. 1999; 112: 4557-4568Google Scholar). A hallmark of both fibrogenesis and EMT is the TGF-β-induced up-regulation of matrix-associated proteins, such as certain integrins and their extracellular matrix ligands (collagens, fibronectin, proteoglycans). The TGF-β responses are initiated by the interaction of the TGF-β ligand with cell surface receptors that form a heterotetrameric complex of two type I and two type II serine/threonine kinase receptors (5Ebner R. Chen R.H. Shum L. Lawler S. Zioncheck T.F. Lee A. Lopez A.R. Derynck R. Science. 1993; 260: 1344-1348Google Scholar, 6Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Google Scholar). The activated ligand-receptor complex, in turn, activates one or more downstream signaling pathway, the most prominent one being the Smad pathway (7Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Google Scholar, 8Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Google Scholar). This pathway is initiated with the phosphorylation of Smad2 or Smad3, which then heterodimerize with the co-Smad, Smad4, followed by translocation of the Smad2/3-Smad4 complex to the nucleus; here, it binds directly or via other DNA-binding proteins to the promoters of TGF-β-responsive genes to stimulate or repress their transcription (7Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Google Scholar, 8Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Google Scholar). Activation of Smad signaling is blocked by Smad7, which inhibits the phosphorylation of Smad2/3 by the type I receptor thereby preventing their association with Smad4 (9Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 549-551Google Scholar, 10Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Google Scholar). Evidence over the past few years suggests that TGF-β may also stimulate other downstream pathways, involving RhoA (11Edlund S. Landstrom M. Heldin C.-H. Aspenstrom P. Mol. Biol. Cell. 2002; 13: 902-914Google Scholar), as well as the MAP kinases, extracellular signal-regulated kinases (ERKs) (12Hartsough M.T. Mulder K.M. J. Biol. Chem. 1995; 270: 7117-7124Google Scholar), c-Jun N-terminal kinases (JNKs) (13Engel M.E. McDonnell M.A. Law B.K. Moses H.L. J. Biol. Chem. 1999; 274: 37413-37420Google Scholar, 14Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Google Scholar), and p38 MAPKs (15Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Google Scholar, 16Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Google Scholar, 17Bhowmick N.A. Ghiassi M. Bakin A. Aakre M. Lundquist C.A. Engel M.E. Arteaga C.L. Moses H.L. Mol. Biol. Cell. 2001; 12: 27-36Google Scholar). However, the question whether MAP kinases depend on a functional Smad pathway and, if so, how both pathways interact, has only been addressed in a few cases (13Engel M.E. McDonnell M.A. Law B.K. Moses H.L. J. Biol. Chem. 1999; 274: 37413-37420Google Scholar, 14Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Google Scholar, 15Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Google Scholar, 18Yu L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 14: 3749-3759Google Scholar, 19Watanabe H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Google Scholar, 20Takekawa M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google Scholar). Nevertheless, is evidence to a functional the signaling pathways J. Rev. Mol. Cell. Biol. 2000; Scholar). Smads, but not p38 to TGF-β-induced growth inhibition of epithelial cells their ability to the promoters of and J. P. M. M. Massagué J. Cell Biol. 2001; Scholar, K. Kurisaki A. Moren A. ten Dijke P. D. Moustakas A. J. Biol. Chem. 2000; Scholar, K. M. H. D. H. K. Miyazono K. M. J. Biol. Chem. 2002; Scholar). In TGF-β-induced in epithelial cells is but requires activation of p38 L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 14: 3749-3759Google Scholar). other to activation of both pathways L. Hebert M.C. Zhang Y.E. EMBO J. 2002; 14: 3749-3759Google Scholar). the that are by TGF-β in is biglycan a of the in J. Scholar, Rev. Biochem. Mol. Biol. 1997; Scholar, T. Biol. 1998; Scholar). BGN a TGF-β activity that is by the and association of both proteins and BGN has been implicated in the regulation of matrix L. E. S. M. M. K. M. H. J. Biol. Chem. 1997; Scholar), J. Biol. Chem. 1997; Scholar), and growth TGF-β, activity E. Y. Cell. Scholar). BGN has been shown to directly the growth of cells in a P. H. M. F. 2001; Scholar), a effect that it with its A. M. A. A. J. Biol. Chem. Scholar). In with that pancreatic cells and BGN in to TGF-β in and that a functional Smad pathway is to K. H. H. J. Biol. Chem. 2002; Scholar), we have a growth inhibition via In the pancreatic cell PANC-1 the to TGF-β-induced increase in BGN mRNA and a in and in a BGN and by the transcriptional activation of a in cells K. H. H. J. Biol. Chem. 2002; Scholar). This the of signaling that to PANC-1 cells EMT in to TGF-β T. A. 2001; Scholar), which is by of a a of epithelial and up-regulation of mesenchymal EMT requires activation of both Smad and and it is that may at the of genes involved in formation of with BGN production results a inflammatory to with p38 MAPK signaling K. J. Cell. 2000; 12: Scholar). to that BGN induction by TGF-β, in addition to the well Smad pathway, requires activation of p38 a of as well as overexpression of wild-type proteins the p38 MAPK and their dominant negative we present evidence that the p38 pathway is induction of BGN and that it with the Smad We further demonstrate that activation of p38 is to and on activation of Smad This is the the of the p38 MAPK pathway in the TGF-β of BGN and The p38 MAPK pathway inhibitors and and the inhibitors and and in or The p38 MAPK was TGF-β1 was and was The pancreatic cell PANC-1 and CFPAC-1 the or of cell in with and PANC-1 and CFPAC-1 cells with in the of or active or The MAP kinase MAP Cell Heidelberg, proteins with was cells with Heidelberg, to the The and the of BGN and in K. H. H. J. Biol. Chem. 2002; Scholar, H. J. Biol. Chem. 276: Scholar). of and we a K. H. H. J. Biol. Chem. 2002; Scholar). mRNA in of to of but concentrations of the was and in the the of with of was to BGN and of on and with and the TGF-β induction of BGN and mRNA was that of and The of mRNA in was to and the to of at two the on a a BGN and mRNA concentrations to mRNA at two the retroviral of and and MKK6 in by S. with and and in the of the retroviral K. H. H. J. Biol. Chem. 2002; Scholar). The wild-type and by with and and and and PANC-1 as The and in In both cases with the mRNA The of a Smad4 was in K. H. H. J. Biol. Chem. 2002; Scholar). A Smad7 was by the Smad7 C.-H. followed by by and of the cells, with retroviral as L. D. H. 2000; Scholar). by cells to PANC-1 and CFPAC-1 or by of cells by with or and expression of the proteins by and In the of Smad7 cells without followed by of BGN mRNA, PANC-1 cells in on and on with expression MKK6 a of S. to the a of in growth to expression of proteins the cells and with TGF-β1 in the cells and of PANC-1 cells in at the cells with the TGF-β and a a constitutively active mutant of TGF-β receptor type both by J. of the cells in growth of the effect of the MAPK inhibitors on of then to the of in with the in the The and in that the results not by to activity was cells with phosphate-buffered saline and in 5 and to followed by with protein then in in and to cells either in and in of proteins, directly in of protein with protein or of by and on to blocked with with and then with the of proteins, saline serum was and was with the and by or the of activated MAP kinases with the and then and with the protein or to Several that in PANC-1 cells pathway is activated by TGF-β with the Smad pathway, to the TGF-β the BGN mRNA up-regulation whether p38 MAP kinases or are involved in the TGF-β effect on BGN expression, we pharmacological inhibitors that The and SB202190 the activity of p38 its but not its of the cells with or SB202190 the TGF-β effect on BGN mRNA and not in a inhibition of p38 was SB202190 by reduced phosphorylation of a of the p38 pathway SB202190 inhibition of TGF-β-induced BGN mRNA was also seen in other cell cells not of PANC-1 cells with or two inhibitors of the upstream kinase that activates not TGF-β induction of BGN mRNA. the inhibitors the TGF-β effect which with effect of the on TGF-β-induced BGN expression and SB202190 shown recently M. N. P. M. Chem. Biol. 1998; Scholar, A. A. S. J. J. R. J. L. B.A. Mol. 2002; to at concentrations because of in the kinase of and p38 further the that the effect of on TGF-β-induced up-regulation of BGN mRNA was because of inhibition of we to the functional effect of on more we PANC-1 cells with a constitutively active with the TGF-β and activity in the or of shown in concentrations of up to 10 not significantly transcriptional we the which is to p38 A. S. S. Lee J.L. J. 2001; Scholar). and inhibited the TGF-β effect on BGN with of only TGF-β induction of mRNA that is not a of TGF-β results we that the inhibition of TGF-β1-induced BGN mRNA expression by and SB202190 was not by inhibition of the that TGF-β of its effect on BGN expression via the p38 MAPK Several MAPK pathways have been shown to activated in to TGF-β in PANC-1 cells K. P. A. 2000; 19: Scholar). pharmacological inhibitors of the p38 MAPK the TGF-β we whether p38 was activated in to TGF-β1 by the phosphorylation of p38 by a it is that TGF-β1 increase in p38 activation that was TGF-β1 at and to of the stress-activated protein kinases, was not activated TGF-β in PANC-1 cells, it was activated by a activator both and p38 p38 is activated by the two upstream MAPK kinases, and MKK6 J. T. Mol. Cell. Biol. Scholar), we that activated by TGF-β in a to that of This was by a two kinases In with activation of p38 but not the upstream kinase of was not activated in to TGF-β1 not more demonstrate the of p38 MAPK in of we inhibited p38 by stable expression of a mutant of that has been shown to in a dominant negative S. A. M. K. H. J. J. Biol. Chem. 1998; Scholar). of a of PANC-1 as well as by with followed by expression of the mutant The of protein well with the of inhibition of the TGF-β effect on BGN mRNA overexpression of wild-type p38 in a of cells augmented the TGF-β effect on BGN These the results of inhibition and the of p38 in TGF-β regulation of p38 is activated by the MAPK kinases or MKK6, which activated in a as p38 in to TGF-β whether MAPK kinases of the TGF-β signaling pathway we which has been to the activity of MKK6 p38 H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Google Scholar). blocked the TGF-β effect on BGN mRNA as as the p38 inhibitors more with MKK6 we a dominant negative MKK6 mutant This blocked the TGF-β effect on BGN mRNA in a overexpression of the wild-type MKK6 protein the TGF-β effect on BGN the that was only with expression not effect of MKK6 on TGF-β signaling if of the cells In overexpression of wild-type had no effect MKK6, MKK3, as the MAPK kinase in the TGF-β of BGN We have shown K. H. H. J. Biol. Chem. 2002; that a functional Smad pathway is the TGF-β effect on BGN expression in pancreatic The question how the Smad and p38 pathway to the in BGN The results are with either of the two In TGF-β its type I but activates Smad complex and p38 which on a Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Google Scholar). In the activated Smad complex induces expression of which activates p38 shown earlier, phosphorylation of Smad2 TGF-β addition K. H. H. J. Biol. Chem. 2002; phosphorylation of p38 was only TGF-β addition that Smad activation activation of p38 In with inhibition of p38 by SB202190 was to TGF-β-induced phosphorylation of Smad2 5 at concentrations as as no in Smad2 phosphorylation was seen with cells that with TGF-β further the that inhibition of the TGF-β effect on BGN A of is that p38 BGN mRNA is on functional Smad4 we the p38 in the pancreatic cell which functional TGF-β by TGF-β to p38 in CFPAC-1 cells 5 as well as in cells 5 a activation of p38 by 5 that was no in the p38 MAPK pathway In in CFPAC-1 with the wild-type Smad4, TGF-β-induced activation of p38 was restored 5 demonstrated the to BGN mRNA in to TGF-β was also restored in cells K. H. H. J. Biol. Chem. 2002; we p38 activation in PANC-1 cells in which Smad signaling has been blocked by expression of the of Smad7 was shown by to TGF-β-induced BGN mRNA K. H. H. J. Biol. Chem. 2002; Scholar). the TGF-β-induced p38 phosphorylation, was inhibited by Smad7 5 that Smad signaling is p38 activation and that activation of the p38 pathway downstream of Smad signaling, that is and and on signaling involved in TGF-β regulation of BGN of Smad and p38 signaling in induction of BGN This on the signaling involved in the TGF-β the receptor complex to the in the of BGN mRNA TGF-β TGF-β receptor is activated and Smad2 or then active complex with Smad4, which is the to induce expression of as protein This protein activates p38 MAPK via activation of the upstream of MKK6 or of a protein further upstream of the p38 to the and induces BGN mRNA mRNA results M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google that the may which binds and activates the kinase that in other have been shown to involved in activation of p38 are by In we have demonstrated that activation of the p38 MAPK pathway, in addition to Smad signaling K. H. H. J. Biol. Chem. 2002; Scholar), is TGF-β regulation of BGN expression in pancreatic This was in which the expression of of the p38 signaling pathway, MAPK and its upstream and MKK6, was either or blocked by or dominant In we have evidence that Smad proteins are p38 activation and that their activation p38 The that the Smad and the p38 pathways are is the most of was by inhibition and in which one pathway was blocked or followed by a of the activation of the inhibition of p38 with SB202190 had no effect on Smad2 inhibition of Smad signaling by expression of Smad7 inhibited TGF-β-induced p38 activation and BGN mRNA up-regulation K. H. H. J. Biol. Chem. 2002; Scholar), and of Smad signaling by re-expression of Smad4 in and CFPAC-1 cells restored the TGF-β of p38 and BGN mRNA induction K. H. H. J. Biol. Chem. 2002; Scholar). This was the that Smad2 is more K. H. H. J. Biol. Chem. 2002; p38 in to to the of p38 activation by TGF-β, two signaling to in cells, activation of p38 is the and restored by (15Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Google Scholar, 16Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Google Scholar, M. L. Y. Biochem. 1998; Scholar). This activation of p38 has been with a of the K. K. Shibuya H. K. N. T. Nishida E. Matsumoto K. Science. 1995; 270: Scholar, H. K. K. A. Y. N. K. Nishida E. Matsumoto K. Science. Scholar), which is to via proteins and and has been to TGF-β-induced p38 activation without of Smad proteins Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Google Scholar, K. K. Shibuya H. K. N. T. Nishida E. Matsumoto K. Science. 1995; 270: Scholar, S. Roberts A.B. J. Biol. Chem. 2001; 276: Scholar). In other cell and pancreatic p38 activation only TGF-β and L. L. H. M. J. J. Biol. Chem. 1999; 274: Scholar, N. R. R. N.E. J. Cell Sci. 2000; Scholar, M.A. A. J. Biol. Chem. 2001; 276: Scholar, M. M. Roberts ten Dijke P. P. J. 2001; Scholar, N. S. S. A. Mol. Biol. Cell. 2001; 12: Scholar, Zhang L. K. T. J. Cell 2001; Scholar). M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google Scholar), in a that of TGF-β-induced p38 activation in cell pancreatic cells, and their on the of p38 activation are in with These demonstrated that delayed activation of p38 by TGF-β was of but activation of the kinase expression M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google Scholar). expression was to p38 activation in PANC-1 not directly shown in cell inhibition of by a dominant mutant abolished p38 activation by active in by in cells, only but not kinase This is in with PANC-1 cells that a dominant negative which to TGF-β induction of BGN mRNA. Ungefroren, we that the TGF-β effect on BGN expression was and was not via transcriptional activity the BGN K. H. H. J. Biol. Chem. 2002; Scholar). to the of p38 activation and BGN mRNA up-regulation it is that the in BGN mRNA and TGF-β addition P. H. M. F. 2001; Scholar), which is to that M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google and the that activation of p38 was at TGF-β These are with a in which the TGF-β-induced activation of Smads results in the transcriptional induction of protein that activates the p38 pathway The results M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google and by as a protein activation of most via activation of are to whether and are involved in TGF-β regulation of that may activation of p38 by and signaling was N.A. Ghiassi M. Bakin A. Aakre M. Lundquist C.A. Engel M.E. Arteaga C.L. Moses H.L. Mol. Biol. Cell. 2001; 12: 27-36Google and J. D. J. S. J. Biol. Chem. 2002; p38 MAPK p38 activation and plasminogen activator up-regulation but not the has been that p38 activation may by J. D. J. S. J. Biol. Chem. 2002; Scholar). we that overexpression of wild-type MKK6, but not wild-type MKK3, was of TGF-β-induced BGN up-regulation p38 the reverse a of integrins in TGF-β of BGN In with blocking in PANC-1 cells not BGN mRNA up-regulation not and MKK6 are to other and, in the p38 MAPK have been to in certain p38 activation J. D. J. S. J. Biol. Chem. 2002; Scholar), MKK6, MKK3, is and in and cells, S. Y. Nishida E. P. S. J. 1997; Scholar, F. Sci. S. A. 1999; Scholar). MKK6 is p38 and activation in and cells R. M. M. Chen 2000; Scholar). However, MKK3, MKK6, and p38 MAPK activation and cytokine expression in both and M. Sci. S. A. 1999; Scholar, M. E. EMBO J. 1999; 18: Scholar). with inhibitors of are the results provide further evidence of and MKK6 p38 activation of p38 MAPK activity has been demonstrated in cell the of pathways to the regulation of expression have only been in a few the genes which TGF-β regulation the p38 MAPK pathway are matrix proteins such as H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Google Scholar), fibronectin, as the of the p38 MAPK A. A. S. J. J. R. J. L. B.A. Mol. 2002; Scholar), and M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Google Scholar). However, TGF-β up-regulation of in of the Smad and both MAPK pathways H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Google Scholar), BGN induction by TGF-β is by as the inhibitors and The effect of inhibitors may effect of on the p38 A reverse negative regulation of by p38 has been recently Biochem. 2001; in PANC-1 cells with to cell TGF-β has been shown to repress activation by in cell type K. P. A. 2000; 19: it that the TGF-β effect on BGN results both activation of p38 and, to a inhibition of TGF-β of PANC-1 and other TGF-β-responsive pancreatic cell cells to a that requires activation of both the Smad and the p38 of on activation of both pathways as shown and may to the changes with EMT increase in cell and T. A. 2001; Scholar). up-regulation of BGN L. E. S. M. M. K. M. H. J. Biol. Chem. 1997; Scholar, J. Biol. Chem. 1997; and p38 activation M.A. J.L. E. J. Biol. Chem. 1999; 274: have been implicated in and The that the of A. A. S. J. J. R. J. L. B.A. Mol. 2002; Scholar), is by TGF-β via p38 may to of in and and may to the effect of TGF-β in of R. A. 2001; Scholar). In we have shown the that TGF-β regulation of the BGN requires activation of the p38 MAPK signaling pathway, in turn, on functional Smad We are to C.-H. S. and J. Massagué expression and