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• W2044399712 abstract "Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) β1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected withfur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21ras (RasN17) inhibited TGFβ1-inducedfur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGFβ induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGFβ1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGFβ1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGFβ1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions. Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) β1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected withfur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21ras (RasN17) inhibited TGFβ1-inducedfur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGFβ induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGFβ1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGFβ1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGFβ1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions. transforming growth factor bone morphogenic protein extracellular signal-regulated kinase mitogen-activated protein kinase kinase kinase-1 mitogen-activated protein kinase MAPK/ERK kinase phosphate-buffered saline Furin is a mammalian subtilisin/Kex2p-like Ca2+-dependant endoprotease involved in the processing of various types of higher molecular mass precursor substrates, containing the minimal basic amino acid RXXR recognition motif. This prototype of the pro-protein convertase family (for reviews, see Refs.1Seidah N.G. Day R. Marcinkiewicz M. Chrétien M. Ann. N. Y. Acad. Sci. 1998; 839: 9-24Crossref PubMed Scopus (168) Google Scholar and 2Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar) is primarily located in the trans-Golgi apparatus (3Vey M. Schafer W. Berghofer S. Klenk H.D. Garten W. J. Cell Biol. 1994; 127: 1829-1842Crossref PubMed Scopus (136) Google Scholar), although some proportion of the furin molecules can recycle from the cell membrane to endosomes (4Molloy S.S. Anderson E.D. Jean F. Thomas G. Trends Cell Biol. 1999; 9: 28-35Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). The biological importance of this convertase stems from the large number and variety of bioactive proteins and peptides that can be generated through its activity. These include key molecules involved in normal and physiopathological conditions including growth/differentiation processes. Furin cleaves C-terminal to an unique processing site (RX(K/R)R) found in many growth/differentiation-related peptides and proteins including TGFβ,1 activin A, BMP family members, Nodal, lefty-1, and lefty-2 as well as growth factor pro-receptors such as insulin-like growth factor receptor and the hepatocyte growth factor receptor (c-Met) (2Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar, 5Constam D.B. Robertson E.J. J. Cell Biol. 1999; 144: 139-149Crossref PubMed Scopus (259) Google Scholar, 6Constam D.B. Robertson E.J. Development. 2000; 127: 245-254Crossref PubMed Google Scholar). Silencing of the expression of mouse furin results in embryonic lethality because of hemodynamic insufficiency associated with several development defects including disruption of development of the heart and vascular system and failure to undergo axial rotation (7Roebroek A.J. Umans L. Pauli I.G. Robertson E.J. van Leuven F. Van de Ven W.J. Constam D.B. Development. 1998; 125: 4863-4876Crossref PubMed Google Scholar). These findings highlight the role of furin in growth and development and in the physiological maturation of substrates involved in these processes including members of the TGFβ family.The results from previous studies have demonstrated that pro-TGFβ1 is efficiently processed by furin releasing the genuine mature growth factor (8Dubois C.M. Laprise M.H. Blanchette F. Gentry L.E. Leduc R. J. Biol. Chem. 1995; 270: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and that among the pro-protein convertase members, furin more closely satisfies the requirements needed for an authentic TGFβ1 converting enzyme (9Dubois C.M. Blanchette F. Laprise M.H. Leduc R. Grondin F. Seidah N.G. Am. J. Pathol. 2001; 158: 305-316Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In fibroblastic and synovial cells, the furin cleavage product, TGFβ1, up-regulated gene expression of its own converting enzyme, resulting in an increase in endogenous TGFβ1 processing activity and release of the bioactive peptide (10Blanchette F. Day R. Dong W. Laprise M.H. Dubois C.M. J. Clin. Invest. 1997; 99: 1974-1983Crossref PubMed Scopus (118) Google Scholar). TGFβ1 did not increase furin mRNA stability and treatment of synovial cells with actinomycin D, before TGFβ1 addition prevented the increase in fur gene expression. This observation suggested that furin concentrations could be regulated at the transcriptional level, resulting in the increase in local concentrations of bioactive growth factors. However, the molecular mechanisms by which TGFβ1 exerts its effects have not been fully elucidated.In the last few years significant progress has been made in the signaling mechanisms utilized by TGFβ1. A major discovery comes from the recent uncovering and cloning of specific Smad signaling proteins consisting of pathway-restricted Smads (Smad2 and Smad3), the common mediator Smad4, and the inhibitory Smads (Smad6 and Smad7) (for reviews see Refs. 11Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (177) Google Scholar, 12Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3963) Google Scholar, 13Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar) that directly inhibit the TGFβ type I receptor serine-threonine kinase and the transcriptional machinery. TGFβ signals through sequential activation of two cell surface serine-threonine kinase receptors, which phosphorylate Smad2 and Smad3 within their conserved C-terminal SSXS motif (14Macias-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 15Souchelnytskyi S. Tamaki K. Engstrom U. Wernstedt C. ten Dijke P. Heldin C.H. J. Biol. Chem. 1997; 272: 28107-28115Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). These activated Smads, together with Smad4, translocate to the nucleus and, in association with other transcription factors, activate the transcription of target genes (16Zhang Y. Feng X. We R. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (757) Google Scholar, 17Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (900) Google Scholar). Among the participating transcription factors are the winged helix factor FAST (now known as FoxH1) (18Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar) in the case of Smad2 (19Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (624) Google Scholar) or AP-1 in the case of Smad3 (reviewed in Refs. 20Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 21Zhang Y. Feng X.H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 22Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar). Naturally occurring inhibitors, Smad6 and Smad7, block and hence control TGFβ superfamily signaling by competitively interacting with the activated type I receptors (23Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar,24Hayashi H. Abdollah S. Qiu Y. Cai J. Xu Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar).As increasing information is collected regarding the detailed molecular mechanism of Smad protein signaling, a number of functional interactions between these proteins and other signaling pathways have been reported. For example, recent work has demonstrated that the linker region of BMP pathway-restricted Smad1 was phosphorylated by ERK2 (25Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar), a member of the classic ERK-activated protein kinase pathway, leading to the inhibition of nuclear translocation of the Smad1-Smad4. In contrast, other studies have demonstrated positive functional interaction between the two stress-activated protein kinase pathways and Smads. For instance, it has been demonstrated that the mitogen-activated protein kinase kinase kinase-1 (MEKK-1), an upstream activator of the stress-activated protein kinase/c-Jun N-terminal kinase pathway, enhances Smad protein transcriptional co-activator interactions in endothelial cells (26Brown J.D. DiChiara M.R. Anderson K.R. Gimbrone Jr., M.A. Topper J.N. J. Biol. Chem. 1999; 274: 8797-8805Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Also, other groups have demonstrated that TGFβ1 activates Smad and TAK1 pathways, resulting in the formation of an active transcription complex composed of Smad3-Smad4 and the p38 nuclear target ATF-2 (27Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 28Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar).In a recent study, we provided evidence for the central role of Smads in the transcriptional activation of the TGFβ1-induciblefur P1 promoter activity (29Blanchette F. Rudd P. Grondin F. Attisano L. Dubois C.M. J. Cell Physiol. 2001; 188: 264-273Crossref PubMed Scopus (43) Google Scholar). Using HepG2 cells transfected with LUC (luciferase) promoter constructs, we observed that among the three furin promoters, the P1 promoter was the strongest and the most sensitive to TGFβ1 and that Smads were essential for mediating such responsiveness. We also observed that the proximal P1 promoter region (positions −8734 to −7925) that contains one SBE (Smad binding element) and one ARE (activin-responsive element) binding site carries most of the Smad responsiveness. These results highlight the central role of Smads in the expression of furin, an important gear of the complex TGFβ1 maturation/activation machinery.In light of the emerging evidence for the interactions between the Smad and the MAPK pathways and the role of furin in growth and differentiation events, it was of interest to explore the possible integration between these two pathways for the regulation of this convertase. Using HepG2 cells transfected with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant of the small G protein p21ras (RasN17) inhibited TGFβ1-induced fur gene transcription. Furthermore, the role of the p42/p44 MAPK cascade infur gene transcription was emphasized by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that blunted the induction of fur gene transcription by TGFβ1. Conversely, forced expression of a constitutively active form of MEK1 (MEKA) increased unstimulated, TGFβ1-stimulated, and Smad-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Our findings clearly show that activation of the p42/p44 MAPK pathway is involved in fur gene expression and suggest functional interactions between the Smad and the p42/p44 MAPK cascade pathway in this regulation.DISCUSSIONThe findings presented herein clearly show that TGFβ1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Even though other reports such as the one from Hayashida et al. (48Hayashida T. Poncelet A.C. Hubchak S.C. Schnaper H.W. Kidney Int. 1999; 56: 1710-1720Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) have raised the possibility of interactions between the Smad and MAPK pathways for TGFβ-stimulated collagen gene expression, few studies have indeed reported positive cross-talk between the growth/differentiation MAPK pathway and the Smad pathway for the regulation of TGFβ-related functions. For instance, work from de Caestecker et al. (46de Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Gen. Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (252) Google Scholar) indicated that hepatocyte growth factor and epidermal growth factor mediate Smad-dependent reporter gene activation and induce phosphorylation of Smad 2 by kinases downstream of MEK1. More recently, Watanabe et al. (49Watanabe H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) have shown that in differentiated chondrocytes, rapid and sustained activation of both p42/p44 and p38 MAPK is required for high levels of aggrecan gene expression, whereas Smad 2 was also involved in the initial activation of this gene that occurs in undifferentiated cells. In contrast, Kretzschmar et al. (25Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar) have demonstrated that the MAPK/ERK1/2 pathway can negativelyregulate the BMP-Smad-1-dependent transcriptional response. Also, results from Brown et al. (26Brown J.D. DiChiara M.R. Anderson K.R. Gimbrone Jr., M.A. Topper J.N. J. Biol. Chem. 1999; 274: 8797-8805Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) indicated that the stress-activated SAPK/JNK pathway, but not the p42/p44 MAPK pathway, can activate Smad-2-mediated transcriptional activation in bovine endothelial cells. Although Yue and Mulder (50Yue J. Mulder K.M. J. Biol. Chem. 2000; 275: 30765-30773Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) have suggested that the Ras/MAPK pathways are essential for TGFβ3 induction of TGFβ1 in lung and intestinal epithelial cells, they proposed that Smads only contribute to this biological response in an indirect manner. The exact reasons for these discrepancies remain unknown, but it is clear that multiple interactions between MAPK and Smad pathways can occur depending on the cell type and possibly the extent of MAPK activation.To determine whether the classical p42/p44 MAPK cascade is involved in TGFβ1-induced activation of the fur gene, our first initiative was to determine whether the ability of TGFβ1 to stimulatefur gene expression was a p21ras-dependent or p21ras-independent mechanism. Interestingly, constitutive and TGFβ1-induced transcription of the reporter gene was efficiently inhibited by dominant negative RasN17 expression in HepG2 cells. Partial blockage of constitutive P1 promoter activity may reflect the requirement of p42/p44 MAPK cascade or other Ras effectors such as those linked to phosphatidylinositol 3-kinases for basal furtransactivation. Also, it is possible that the inhibition of basal P1 promoter activation by Ras DN is due to interference with basal activation of TGFβ signal transduction pathways. In support of this, recent experiments using TGFβ-neutralizing antibodies have demonstrated that autocrine production of TGFβ accounts for 45% of basal furin P1 promoter activation in the HepG2 cell system (29Blanchette F. Rudd P. Grondin F. Attisano L. Dubois C.M. J. Cell Physiol. 2001; 188: 264-273Crossref PubMed Scopus (43) Google Scholar). The requirement of Ras for TGFβ-induced fur transactivation extends a previous study that identified other GTPases of the Rho family as intermediates of TGFβ1-initiated signaling leading to transcriptional activation of a TGFβ reporter construct (p3TP-Lux) in the same cell line (51Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar).In addition to the fur gene, Ras has been shown to be involved in the expression of other genes/proteins regulated by TGFβ. For example, RasN17 was shown to inhibit the autoinduction of TGFβ1 in lung and intestinal epithelial cells (50Yue J. Mulder K.M. J. Biol. Chem. 2000; 275: 30765-30773Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), the TGFβ-induced cell cycle-related p27Kip1 and p21 Cip1 in intestinal epithelial cells (52Yue J. Buard A. Mulder K.M. Oncogene. 1998; 17: 47-55Crossref PubMed Scopus (35) Google Scholar), and TGFβ-induced urokinase expression in transformed keratinocytes (53Yu S.J. Boudreau F. Désilets A. Houde M. Rivard N. Asselin C. Biochem. Biophys. Res. Commun. 1999; 259: 544-549Crossref PubMed Scopus (15) Google Scholar). In this context, the participation of Ras in the cellular cascade leading to the regulation of the fur gene by TGFβ would be consistent with the view of Ras as an integrator of a wide variety of TGFβ-related growth/differentiation events.Although Ras is involved in the activation of multiple pathways, we next demonstrated that the p42/p44 MAPK pathway is also involved in TGFβ-induced fur activation. In initial experiments, we have observed that TGFβ1 stimulation of HepG2 cells results in a relatively delayed but sustained p42/p44 MAPK phosphorylation. Next, we observed that a biochemical blockade of p42/p44 MAPK activation or mRNA reduction through antisense technology blocked TGFβ1-induced transcriptional activation of furin P1 promoter and activation of p42/p44 MAPKs. In contrast, MEKA increased unstimulated and TGFβ1-stimulated P1 transactivation. Taken together, these observations provide strong evidence that the Ras-MEKp42/p44 MAPK signaling plays an important role in the regulation of furgene expression. In a similar way, the absolute requirement of p42/p44 MAPKs cascade in TGFβ1-induced functions such as the attenuation of haptoglobin gene expression in intestinal epithelial cells has been reported (54Santibanez J.F. Iglesias M. Frontelo P. Martinez J. Quintanilla M. Biochem. Biophys. Res. Commun. 2000; 273: 521-557Crossref PubMed Scopus (54) Google Scholar).In our study, the delayed (starting 30 min) and sustained stimulation of p42/p44 MAPK phosphorylation by TGFβ is consistent with a possible indirect mechanism of activation. In fact, most studies using different cell types including phorbol 12-myristate 13-acetate-differentiated THP-1 cells, epithelial cells, or chondrocytes have reported more rapid activation of p44 MAPK occurring within 5–10 min of TGFβ1 addition (49Watanabe H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 55Hartsough M.T. Mulder K.M. J. Biol. Chem. 1995; 270: 7117-7124Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 56Han J. Hajjar D.P. Tauras J.M. Feng J. Gotto Jr., A.M. Nicholson A.C. J. Biol. Chem. 2000; 275: 1241-1246Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In human mesanglial cells, however, a more delayed (30 min) kinetic of activation has been observed (48Hayashida T. Poncelet A.C. Hubchak S.C. Schnaper H.W. Kidney Int. 1999; 56: 1710-1720Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Although the significance of this difference in timing of activation has not been elucidated, Hayashida et al. (48Hayashida T. Poncelet A.C. Hubchak S.C. Schnaper H.W. Kidney Int. 1999; 56: 1710-1720Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) have ruled out the involvement of new protein synthesis and/or release of platelet-derived growth factor for the delayed activation observed in mesanglial cells. In our system, platelet-derived growth factor would unlikely mediate the observed early p42/p44 activation because the kinetic of platelet-derived growth factor production in cells typically occurs later (i.e. at 2–4-h time points) after TGFβ stimulation (57Soma Y. Grotendorst G.R. J. Cell. Physiol. 1989; 140: 246-253Crossref PubMed Scopus (123) Google Scholar, 58Leof E.B. Proper J.A. Goustin A.S. Shipley G.D. DiCorleto P.E. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2453-2457Crossref PubMed Scopus (404) Google Scholar). However, it would appear logical to propose that autocrine regulation of growth factors by TGFβ accounts for at least some of the signal amplification observed 2–4 h after TGFβ stimulation.The mechanisms regulating fur expression are not fully understood. In previous studies, we found that TGFβ1-increasedfur gene regulation occurs at the level of gene transcription (10Blanchette F. Day R. Dong W. Laprise M.H. Dubois C.M. J. Clin. Invest. 1997; 99: 1974-1983Crossref PubMed Scopus (118) Google Scholar) and that Smad2 possibly with winged helix transcription factor FAST (FoxH1) participate in this transactivation (29Blanchette F. Rudd P. Grondin F. Attisano L. Dubois C.M. J. Cell Physiol. 2001; 188: 264-273Crossref PubMed Scopus (43) Google Scholar). Here we demonstrate that forced expression of Smad2-Smad4 or MEKA leads to the activation of the fur P1 promoter, mimicking the effect of TGFβ1. In addition, MEKA-induced transactivation was abrogated in cells co-expressing the Smad inhibitor Smad7, and similarly, Smad2-Smad4-induced transactivation was blocked using chemical MEK inhibitors. This argues that both Smad and p42/p44 MAPK pathways are essential for mediating TGFβ1-induced transactivation of furin. In this regard, several cross-talk interactions are possible between the Smad and MAPK pathways, depending on the cellular environment and the targeted biological function (21Zhang Y. Feng X.H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 25Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar, 26Brown J.D. DiChiara M.R. Anderson K.R. Gimbrone Jr., M.A. Topper J.N. J. Biol. Chem. 1999; 274: 8797-8805Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 46de Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Gen. Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (252) Google Scholar, 49Watanabe H. de Caestecker M.P. Yamada Y. J. Biol. Chem. 2001; 276: 14466-14473Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar,59Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (847) Google Scholar). As more information is being gathered regarding direct involvement of the stress-activated protein kinase/c-Jun N-terminal kinase pathway in Smad activation, little information is available regarding direct involvement of the p42/p44 MAPK pathway. In our study, we observed that inhibition of MEK by PD98059 blocked most of the enhanced Smad-2 nuclear localization induced by TGFβ. In contrast, activation of p42/p44 MAPKs by activated MEK1 resulted in an enhanced nuclear localization of Smad2. One explanation for this cross-talk is a direct interaction between MEK1 or p42/p44 MAPKs and Smad2. It has been recently demonstrated that growth factors, namely hepatocyte growth factor and epidermal growth factor, can also mediate both Smad-dependent 3TP-lux reporter gene activation and nuclear translocation of Smad2 (46de Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Gen. Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (252) Google Scholar). This correlates with an increase of Smad2 phosphorylation that is markedly reduced in the absence of the C-terminal SSXS motif of Smad2, which is the site of TGFβ type I receptor-induced phosphorylation. It has been shown that Smad7 can bind to type I TGFβ1 receptor and inhibits its capacity to phosphorylate Smad2 (22Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar, 23Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar). In our study, the ability of Smad7 to inhibit MEK1-mediated transcriptional activation suggests that phosphorylation at the SSXS motif is needed for MEKA-induced activation of Smad2. This does not rule out the possibility that other potential phosphorylation motifs for the proline-directed kinases MEK/p42/p44 MAPK found within the Smad2 linker region may also participate in Smad activation as demonstrated for Smad1 (25Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar, 59Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (847) Google Scholar).It was surprising to observe an enhanced Smad2 nuclear localization by MEKA in the absence of exogenous TGFβ stimulation. One possible explanation for this is the induction of autocrine TGFβ production by activated MEK. In support of this, a recent study by Yue and Mulder (50Yue J. Mulder K.M. J. Biol. Chem. 2000; 275: 30765-30773Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) indicated a requirement of Ras/MAPK pathway for the induction of TGFβ1 by TGFβ. In this context, p42/p44 MAPK activation by MEKA may result in the induction of TGFβ that in turn activates the Smad pathway for an enhanced Smad2 nuclear localization and increased furin expression. Also, because nuclear translocation of the Smad3 proteins was shown to occur through direct binding to the nuclear transporter importin β (60Xiao Z. Liu X. Lodish H.F. J. Biol. Chem. 2000; 275: 23425-23428Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), it would be tempting to speculate that the increase in Smad nuclear translocation comes from direct or indirect modification by activated MEK/p42/p44 kinases of proteins involved in Smad nuclear transport. In this regard, phosphorylation of the importin 58/97 heterodimer by activated CK2 kinases was shown to increase its affinity for the ligand, leading to enhanced nuclear transport of the complex (61Hubner S. Xiao C.Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract" @default.
• W2044399712 created "2016-06-24" @default.
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• W2044399712 date "2001-09-01" @default.
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• W2044399712 title "Cross-talk between the p42/p44 MAP Kinase and Smad Pathways in Transforming Growth Factor β1-induced Furin Gene Transactivation" @default.
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