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Human BAMBI Cooperates with Smad7 to Inhibit Transforming Growth Factor-β Signaling

2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês

10.1074/jbc.m109.049304

1083-351X

Xiaohua Yan, Zhenghong Lin, Feng Chen, Xingang Zhao, Hua Chen, Yuanheng Ning, Ye‐Guang Chen,

Heterotopic Ossification and Related Conditions

Transforming growth factor β (TGF-β) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-β/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-β- and BMP-mediated transcriptional responses as well as TGF-β-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-β-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-β signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-β type I receptor ALK5/TβRI and inhibits the interaction between ALK5/TβRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-β signaling. Transforming growth factor β (TGF-β) and related growth factors are essential regulators of embryogenesis and tissue homeostasis. The signaling pathways mediated by their receptors and Smad proteins are precisely modulated by various means. Xenopus BAMBI (bone morphogenic protein (BMP) and activin membrane-bound inhibitor) has been shown to function as a general negative regulator of TGF-β/BMP/activin signaling. Here, we provide evidence that human BAMBI (hBAMBI), like its Xenopus homolog, inhibits TGF-β- and BMP-mediated transcriptional responses as well as TGF-β-induced R-Smad phosphorylation and cell growth arrest, whereas knockdown of endogenous BAMBI enhances the TGF-β-induced reporter expression. Mechanistically, in addition to interfering with the complex formation between the type I and type II receptors, hBAMBI cooperates with Smad7 to inhibit TGF-β signaling. hBAMBI forms a ternary complex with Smad7 and the TGF-β type I receptor ALK5/TβRI and inhibits the interaction between ALK5/TβRI and Smad3, thus impairing Smad3 activation. These findings provide a novel insight to understand the molecular mechanism underlying the inhibitory effect of BAMBI on TGF-β signaling. Transforming growth factor-β (TGF-β) 3The abbreviations used are: TGFtransforming growth factorBMPbone morphogenic proteinALK5activin receptor-like kinase 5 (TβRI)shRNAsmall hairpin RNAcaconstitutively activeICDintracellular domainHAhemagglutininAREactivin-responsive element. 3The abbreviations used are: TGFtransforming growth factorBMPbone morphogenic proteinALK5activin receptor-like kinase 5 (TβRI)shRNAsmall hairpin RNAcaconstitutively activeICDintracellular domainHAhemagglutininAREactivin-responsive element. and related growth factors regulate various aspects of cellular events, including cell growth, differentiation, migration, and death and play pivotal roles in many physiological and pathological processes (1Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4201) Google Scholar, 2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 3Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 4Miyazono K. Suzuki H. Imamura T. Cancer Sci. 2003; 94: 230-234Crossref PubMed Scopus (161) Google Scholar, 5Goumans M.J. Liu Z. ten Dijke P. Cell Res. 2009; 19: 116-127Crossref PubMed Scopus (392) Google Scholar, 6Padua D. Massagué J. Cell Res. 2009; 19: 89-102Crossref PubMed Scopus (666) Google Scholar, 7Watabe T. Miyazono K. Cell Res. 2009; 19: 103-115Crossref PubMed Scopus (338) Google Scholar). TGF-β signaling is initiated by binding of ligands to two types of transmembrane receptors, both of which possess Ser/Thr kinase activity in their intracellular domains. Ligand binding induces the heterocomplex formation between the type I (ALK5/TβRI) and the type II (TβRII) receptors and thus the TβRII-mediated activation of ALK5. Then the activated ALK5 recruits and phosphorylates the downstream signal mediators Smad2 or Smad3 proteins, which subsequently associates with Smad4, accumulates in the nucleus, and modulates target gene expression (8Feng X.H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1519) Google Scholar, 9Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1900) Google Scholar, 10Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 11Schmierer B. Hill C.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 970-982Crossref PubMed Scopus (949) Google Scholar, 12ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 13Guo X. Wang X.F. Cell Res. 2009; 19: 71-88Crossref PubMed Scopus (727) Google Scholar, 14Hill C.S. Cell Res. 2009; 19: 36-46Crossref PubMed Scopus (180) Google Scholar, 15Lönn P. Morén A. Raja E. Dahl M. Moustakas A. Cell Res. 2009; 19: 21-35Crossref PubMed Scopus (147) Google Scholar). TGF-β signaling is tightly regulated temporally and spatially through multiple mechanisms at different levels: from the extracellular environment, the plasma membrane, and the cytoplasm to the nucleus. The regulation can take place in either positive or negative manners. Deregulation of TGF-β signaling might be linked to pathogenesis of various clinical disorders such as tumors, vascular diseases, and tissue fibrosis (4Miyazono K. Suzuki H. Imamura T. Cancer Sci. 2003; 94: 230-234Crossref PubMed Scopus (161) Google Scholar, 5Goumans M.J. Liu Z. ten Dijke P. Cell Res. 2009; 19: 116-127Crossref PubMed Scopus (392) Google Scholar, 16Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar, 17Massagué J. Chen Y.G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar, 18Miyazono K. J. Cell Sci. 2000; 113: 1101-1109Crossref PubMed Google Scholar). Inhibitory Smads, including Smad7 and Smad6, are key regulators of TGF-β family signaling through a negative feedback circuit (19Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1547) Google Scholar, 20Park S.H. J. Biochem. Mol. Biol. 2005; 38: 9-16Crossref PubMed Google Scholar, 21Yan X. Liu Z. Chen Y. Acta. Biochim. Biophys Sin. 2009; 41: 263-272Crossref Scopus (302) Google Scholar).BAMBI (BMP and activin membrane-bound inhibitor), a 260-amino acid transmembrane glycoprotein that is evolutionally conserved in vertebrates from fish to humans, is closely related to the type I receptors of the TGF-β family in the extracellular domain but has a shorter intracellular domain that exhibits no enzymatic activity (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar, 23Loveland K.L. Bakker M. Meehan T. Christy E. von Schönfeldt V. Drummond A. de Kretser D. Endocrinology. 2003; 144: 4180-4186Crossref PubMed Scopus (38) Google Scholar, 24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). It has been documented that Xenopus BAMBI functions as a general antagonist of TGF-β family members by acting as a pseudoreceptor to block the interaction between signaling type I and type II receptors (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). BAMBI is tightly co-expressed with BMP4 during the embryo development of zebrafish, Xenopus, bird, or mouse, and its expression is also induced by BMP4 (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar, 24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar, 25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar, 26Higashihori N. Song Y. Richman J.M. Dev. Dyn. 2008; 237: 1500-1508Crossref PubMed Scopus (15) Google Scholar, 27Karaulanov E. Knöchel W. Niehrs C. EMBO J. 2004; 23: 844-856Crossref PubMed Scopus (115) Google Scholar). Therefore, BAMBI is believed to act as a negative feedback regulator of BMP signaling during embryo development (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar, 25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar), although a recent gene target study indicated that BAMBI is dispensable for mouse embryo development and postnatal survival (28Chen J. Bush J.O. Ovitt C.E. Lan Y. Jiang R. Genesis. 2007; 45: 482-486Crossref PubMed Scopus (38) Google Scholar). BAMBI expression is also induced by TGF-β (29Xi Q. He W. Zhang X.H. Le H.V. Massagué J. J. Biol. Chem. 2008; 283: 1146-1155Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30Sekiya T. Oda T. Matsuura K. Akiyama T. Biochem. Biophys. Res. Commun. 2004; 320: 680-684Crossref PubMed Scopus (79) Google Scholar) and Wnt signaling (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Our recent work showed that human BAMBI (hBAMBI) can promote Wnt signaling by enhancing the interaction of the Wnt receptor Frizzled5 and the downstream mediator Dishevelled2 (32Lin Z. Gao C. Ning Y. He X. Wu W. Chen Y.G. J. Biol. Chem. 2008; 283: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), implicating that BAMBI might integrate different cellular signaling pathways.Several studies have suggested that BAMBI is involved in pathogenesis of human diseases. Human BAMBI, initially named nma, is down-regulated in metastatic melanoma cell lines (33Degen W.G. Weterman M.A. van Groningen J.J. Cornelissen I.M. Lemmers J.P. Agterbos M.A. Geurts van Kessel A. Swart G.W. Bloemers H.P. Int. J. Cancer. 1996; 65: 460-465Crossref PubMed Scopus (60) Google Scholar) and in a subset of high grade bladder cancer (34Khin S.S. Kitazawa R. Win N. Aye T.T. Mori K. Kondo T. Kitazawa S. Int. J. Cancer. 2009; 125: 328-338Crossref PubMed Scopus (40) Google Scholar). Its elevated expression was suggested to attenuate the TGF-β-mediated growth arrest in colorectal and hepatocellular carcinomas (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and to induce cell growth and invasion of human gastric carcinoma cells (35Sasaki T. Sasahira T. Shimura H. Ikeda S. Kuniyasu H. Oncol. Rep. 2004; 11: 1219-1223PubMed Google Scholar). A recent study has also suggested that BAMBI is involved in Toll-like receptor 4- and lipopolysaccharide-mediated hepatic fibrosis (36Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. Nat. Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1440) Google Scholar).Although BAMBI acts as a critical regulator of TGF-β/BMP signaling, how the regulation takes place is not fully understood. In this study, we demonstrated that hBAMBI, like its Xenopus homolog, inhibits TGF-β- and BMP-mediated transcriptional responses, TGF-β-induced phosphorylation of R-Smads, and cell growth arrest. In addition to its interference with receptor complex formation, we found that hBAMBI can synergize with Smad7 to inhibit TGF-β signaling by forming a ternary complex with ALK5 and Smad7 and inhibiting the interaction between ALK5 and R-Smads. These findings together suggest that the function of BAMBI is evolutionally conserved as a negative regulator of TGF-β signaling.DISCUSSIONThe TGF-β family members play pivotal roles in embryo development and tissue homeostasis. Although the canonical Smad-mediated signaling pathway is relatively simple, it is precisely controlled at different levels from the availability and activation of extracellular ligands, the activity and stability of membrane receptors, to the activity, location, and stability of Smad proteins in the cytoplasm and in the nucleus (14Hill C.S. Cell Res. 2009; 19: 36-46Crossref PubMed Scopus (180) Google Scholar, 15Lönn P. Morén A. Raja E. Dahl M. Moustakas A. Cell Res. 2009; 19: 21-35Crossref PubMed Scopus (147) Google Scholar, 21Yan X. Liu Z. Chen Y. Acta. Biochim. Biophys Sin. 2009; 41: 263-272Crossref Scopus (302) Google Scholar, 45Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar, 46Deheuninck J. Luo K. Cell Res. 2009; 19: 47-57Crossref PubMed Scopus (196) Google Scholar, 47Itoh S. ten Dijke P. Curr. Opin Cell Biol. 2007; 19: 176-184Crossref PubMed Scopus (337) Google Scholar). Here, we demonstrated that hBAMBI, like its Xenopus homolog, functions as a general antagonist to attenuate the transcriptional activity of TGF-β/BMPs and inhibit TGF-β-induced R-Smad phosphorylation and cell growth arrest, indicating that the inhibitory activity of BAMBI on TGF-β signaling is evolutionally conserved.Although the possible function of BAMBI in TGF-β signaling has been examined in different animal models such as Xenopus (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar), zebrafish (25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar), rat (23Loveland K.L. Bakker M. Meehan T. Christy E. von Schönfeldt V. Drummond A. de Kretser D. Endocrinology. 2003; 144: 4180-4186Crossref PubMed Scopus (38) Google Scholar), and mouse (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar), our understanding of the molecular mechanism underlying its function is still limited. It has been shown that Xenopus BAMBI can bind to TGF-β receptors and interfere with the heterocomplex formation between the type I and type II receptors (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). Here, we confirmed this mechanism with hBAMBI. Moreover, this study has discovered an additional mechanism whereby BAMBI cooperates with Smad7 to inhibit TGF-β signaling.Smad7 is a well characterized antagonist of TGF-β signaling. It was found to directly interact with the active type I receptors in competition with R-Smads and thus inhibit the activation of R-Smads (19Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1547) Google Scholar, 42Hayashi 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 (1149) Google Scholar), to recruit the ubiquitin E3 ligases Smurf1 or Smurf2 and lead to the ubiquitination and degradation of ALK5 (48Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 49Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 50Suzuki C. Murakami G. Fukuchi M. Shimanuki T. Shikauchi Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; 277: 39919-39925Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), to promote receptor dephosphorylation via protein phosphatase 1c (51Shi W. Sun C. He B. Xiong W. Shi X. Yao D. Cao X. J. Cell Biol. 2004; 164: 291-300Crossref PubMed Scopus (206) Google Scholar), or to block functional Smad-DNA complex formation in the nucleus (37Zhang S. Fei T. Zhang L. Zhang R. Chen F. Ning Y. Han Y. Feng X.H. Meng A. Chen Y.G. Mol. Cell. Biol. 2007; 27: 4488-4499Crossref PubMed Scopus (205) Google Scholar). In this study, we found that hBAMBI also interacts with inhibitory Smads Smad6 and Smad7, and the interaction between hBAMBI and Smad7 was enhanced by TGF-β treatment. TGF-β promoted the association of hBAMBI with Smad7, and hBAMBI exhibited a higher binding affinity to active ALK5. Our study further demonstrated that hBAMBI, Smad7, and ALK5 form a ternary complex. Interestingly, unlike Smad7 and Smurfs, hBAMBI does not affect ALK5 turnover nor further promote Smad7/Smurf1-induced degradation of ALK5. Rather hBMABI inhibits the interaction between Smad3 and ALK5 and interferes with Smad3 phosphorylation. Therefore, our data suggest that BAMBI could exploit dual mechanisms to interfere with TGF-β signaling, and these two mechanisms may work cooperatively. Because BAMBI can also interact with Smad6, and both of them are negative regulators of BMP signaling, it is possible that BAMBI and Smad6 might cooperatively inhibit BMP signaling in similar manners. Indeed, overexpression of BAMBI inhibited the binding of Smad1(3A) with the constitutive active BMP type I receptor ca-ALK6, suggesting that BAMBI utilizes similar mechanisms to function as a general antagonist of the TGF-β family members.Despite a recent study reporting that BAMBI is dispensable for mouse embryo development and postnatal survival (28Chen J. Bush J.O. Ovitt C.E. Lan Y. Jiang R. Genesis. 2007; 45: 482-486Crossref PubMed Scopus (38) Google Scholar), other studies have underlined the possible important roles of hBAMBI in several pathological processes such as tumorigenesis and fibrogenesis. BAMBI was firstly reported to be a gene whose expression was reversely correlated with metastasis progression of human melanomas (33Degen W.G. Weterman M.A. van Groningen J.J. Cornelissen I.M. Lemmers J.P. Agterbos M.A. Geurts van Kessel A. Swart G.W. Bloemers H.P. Int. J. Cancer. 1996; 65: 460-465Crossref PubMed Scopus (60) Google Scholar). BAMBI was also found to counteract the effect of TGF-β on cell growth and invasion of human gastric carcinoma cell lines (35Sasaki T. Sasahira T. Shimura H. Ikeda S. Kuniyasu H. Oncol. Rep. 2004; 11: 1219-1223PubMed Google Scholar) and bladder cancer cells (34Khin S.S. Kitazawa R. Win N. Aye T.T. Mori K. Kondo T. Kitazawa S. Int. J. Cancer. 2009; 125: 328-338Crossref PubMed Scopus (40) Google Scholar). The expression of BAMBI is significantly up-regulated in most colorectal and hepatocellular carcinomas, and this up-regulation is mediated by β-catenin (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Furthermore, overexpression of BAMBI attenuated TGF-β signaling in these tumor cells, suggesting that β-catenin can promote the formation of colorectal and hepatocellular tumors by interfering with TGF-β-mediated growth arrest via BAMBI. A recent report also indicated that BAMBI might be involved in hepatic fibrogenesis (36Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. Nat. Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1440) Google Scholar). Toll-like receptor 4 and lipopolysaccharide sensitize hepatic stellate cells to TGF-β signaling by down-regulating the expression of BAMBI via a MyD88-NF-κB-dependent pathway. These studies suggest that the appropriate negative regulation of TGF-β by BAMBI is important for tissue homeostasis. Transforming growth factor-β (TGF-β) 3The abbreviations used are: TGFtransforming growth factorBMPbone morphogenic proteinALK5activin receptor-like kinase 5 (TβRI)shRNAsmall hairpin RNAcaconstitutively activeICDintracellular domainHAhemagglutininAREactivin-responsive element. 3The abbreviations used are: TGFtransforming growth factorBMPbone morphogenic proteinALK5activin receptor-like kinase 5 (TβRI)shRNAsmall hairpin RNAcaconstitutively activeICDintracellular domainHAhemagglutininAREactivin-responsive element. and related growth factors regulate various aspects of cellular events, including cell growth, differentiation, migration, and death and play pivotal roles in many physiological and pathological processes (1Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4201) Google Scholar, 2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 3Massagué J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 4Miyazono K. Suzuki H. Imamura T. Cancer Sci. 2003; 94: 230-234Crossref PubMed Scopus (161) Google Scholar, 5Goumans M.J. Liu Z. ten Dijke P. Cell Res. 2009; 19: 116-127Crossref PubMed Scopus (392) Google Scholar, 6Padua D. Massagué J. Cell Res. 2009; 19: 89-102Crossref PubMed Scopus (666) Google Scholar, 7Watabe T. Miyazono K. Cell Res. 2009; 19: 103-115Crossref PubMed Scopus (338) Google Scholar). TGF-β signaling is initiated by binding of ligands to two types of transmembrane receptors, both of which possess Ser/Thr kinase activity in their intracellular domains. Ligand binding induces the heterocomplex formation between the type I (ALK5/TβRI) and the type II (TβRII) receptors and thus the TβRII-mediated activation of ALK5. Then the activated ALK5 recruits and phosphorylates the downstream signal mediators Smad2 or Smad3 proteins, which subsequently associates with Smad4, accumulates in the nucleus, and modulates target gene expression (8Feng X.H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1519) Google Scholar, 9Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1900) Google Scholar, 10Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 11Schmierer B. Hill C.S. Nat. Rev. Mol. Cell Biol. 2007; 8: 970-982Crossref PubMed Scopus (949) Google Scholar, 12ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 13Guo X. Wang X.F. Cell Res. 2009; 19: 71-88Crossref PubMed Scopus (727) Google Scholar, 14Hill C.S. Cell Res. 2009; 19: 36-46Crossref PubMed Scopus (180) Google Scholar, 15Lönn P. Morén A. Raja E. Dahl M. Moustakas A. Cell Res. 2009; 19: 21-35Crossref PubMed Scopus (147) Google Scholar). TGF-β signaling is tightly regulated temporally and spatially through multiple mechanisms at different levels: from the extracellular environment, the plasma membrane, and the cytoplasm to the nucleus. The regulation can take place in either positive or negative manners. Deregulation of TGF-β signaling might be linked to pathogenesis of various clinical disorders such as tumors, vascular diseases, and tissue fibrosis (4Miyazono K. Suzuki H. Imamura T. Cancer Sci. 2003; 94: 230-234Crossref PubMed Scopus (161) Google Scholar, 5Goumans M.J. Liu Z. ten Dijke P. Cell Res. 2009; 19: 116-127Crossref PubMed Scopus (392) Google Scholar, 16Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar, 17Massagué J. Chen Y.G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar, 18Miyazono K. J. Cell Sci. 2000; 113: 1101-1109Crossref PubMed Google Scholar). Inhibitory Smads, including Smad7 and Smad6, are key regulators of TGF-β family signaling through a negative feedback circuit (19Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1547) Google Scholar, 20Park S.H. J. Biochem. Mol. Biol. 2005; 38: 9-16Crossref PubMed Google Scholar, 21Yan X. Liu Z. Chen Y. Acta. Biochim. Biophys Sin. 2009; 41: 263-272Crossref Scopus (302) Google Scholar). transforming growth factor bone morphogenic protein activin receptor-like kinase 5 (TβRI) small hairpin RNA constitutively active intracellular domain hemagglutinin activin-responsive element. transforming growth factor bone morphogenic protein activin receptor-like kinase 5 (TβRI) small hairpin RNA constitutively active intracellular domain hemagglutinin activin-responsive element. BAMBI (BMP and activin membrane-bound inhibitor), a 260-amino acid transmembrane glycoprotein that is evolutionally conserved in vertebrates from fish to humans, is closely related to the type I receptors of the TGF-β family in the extracellular domain but has a shorter intracellular domain that exhibits no enzymatic activity (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar, 23Loveland K.L. Bakker M. Meehan T. Christy E. von Schönfeldt V. Drummond A. de Kretser D. Endocrinology. 2003; 144: 4180-4186Crossref PubMed Scopus (38) Google Scholar, 24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). It has been documented that Xenopus BAMBI functions as a general antagonist of TGF-β family members by acting as a pseudoreceptor to block the interaction between signaling type I and type II receptors (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). BAMBI is tightly co-expressed with BMP4 during the embryo development of zebrafish, Xenopus, bird, or mouse, and its expression is also induced by BMP4 (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar, 24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar, 25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar, 26Higashihori N. Song Y. Richman J.M. Dev. Dyn. 2008; 237: 1500-1508Crossref PubMed Scopus (15) Google Scholar, 27Karaulanov E. Knöchel W. Niehrs C. EMBO J. 2004; 23: 844-856Crossref PubMed Scopus (115) Google Scholar). Therefore, BAMBI is believed to act as a negative feedback regulator of BMP signaling during embryo development (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar, 25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar), although a recent gene target study indicated that BAMBI is dispensable for mouse embryo development and postnatal survival (28Chen J. Bush J.O. Ovitt C.E. Lan Y. Jiang R. Genesis. 2007; 45: 482-486Crossref PubMed Scopus (38) Google Scholar). BAMBI expression is also induced by TGF-β (29Xi Q. He W. Zhang X.H. Le H.V. Massagué J. J. Biol. Chem. 2008; 283: 1146-1155Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30Sekiya T. Oda T. Matsuura K. Akiyama T. Biochem. Biophys. Res. Commun. 2004; 320: 680-684Crossref PubMed Scopus (79) Google Scholar) and Wnt signaling (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Our recent work showed that human BAMBI (hBAMBI) can promote Wnt signaling by enhancing the interaction of the Wnt receptor Frizzled5 and the downstream mediator Dishevelled2 (32Lin Z. Gao C. Ning Y. He X. Wu W. Chen Y.G. J. Biol. Chem. 2008; 283: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), implicating that BAMBI might integrate different cellular signaling pathways. Several studies have suggested that BAMBI is involved in pathogenesis of human diseases. Human BAMBI, initially named nma, is down-regulated in metastatic melanoma cell lines (33Degen W.G. Weterman M.A. van Groningen J.J. Cornelissen I.M. Lemmers J.P. Agterbos M.A. Geurts van Kessel A. Swart G.W. Bloemers H.P. Int. J. Cancer. 1996; 65: 460-465Crossref PubMed Scopus (60) Google Scholar) and in a subset of high grade bladder cancer (34Khin S.S. Kitazawa R. Win N. Aye T.T. Mori K. Kondo T. Kitazawa S. Int. J. Cancer. 2009; 125: 328-338Crossref PubMed Scopus (40) Google Scholar). Its elevated expression was suggested to attenuate the TGF-β-mediated growth arrest in colorectal and hepatocellular carcinomas (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and to induce cell growth and invasion of human gastric carcinoma cells (35Sasaki T. Sasahira T. Shimura H. Ikeda S. Kuniyasu H. Oncol. Rep. 2004; 11: 1219-1223PubMed Google Scholar). A recent study has also suggested that BAMBI is involved in Toll-like receptor 4- and lipopolysaccharide-mediated hepatic fibrosis (36Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. Nat. Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1440) Google Scholar). Although BAMBI acts as a critical regulator of TGF-β/BMP signaling, how the regulation takes place is not fully understood. In this study, we demonstrated that hBAMBI, like its Xenopus homolog, inhibits TGF-β- and BMP-mediated transcriptional responses, TGF-β-induced phosphorylation of R-Smads, and cell growth arrest. In addition to its interference with receptor complex formation, we found that hBAMBI can synergize with Smad7 to inhibit TGF-β signaling by forming a ternary complex with ALK5 and Smad7 and inhibiting the interaction between ALK5 and R-Smads. These findings together suggest that the function of BAMBI is evolutionally conserved as a negative regulator of TGF-β signaling. DISCUSSIONThe TGF-β family members play pivotal roles in embryo development and tissue homeostasis. Although the canonical Smad-mediated signaling pathway is relatively simple, it is precisely controlled at different levels from the availability and activation of extracellular ligands, the activity and stability of membrane receptors, to the activity, location, and stability of Smad proteins in the cytoplasm and in the nucleus (14Hill C.S. Cell Res. 2009; 19: 36-46Crossref PubMed Scopus (180) Google Scholar, 15Lönn P. Morén A. Raja E. Dahl M. Moustakas A. Cell Res. 2009; 19: 21-35Crossref PubMed Scopus (147) Google Scholar, 21Yan X. Liu Z. Chen Y. Acta. Biochim. Biophys Sin. 2009; 41: 263-272Crossref Scopus (302) Google Scholar, 45Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar, 46Deheuninck J. Luo K. Cell Res. 2009; 19: 47-57Crossref PubMed Scopus (196) Google Scholar, 47Itoh S. ten Dijke P. Curr. Opin Cell Biol. 2007; 19: 176-184Crossref PubMed Scopus (337) Google Scholar). Here, we demonstrated that hBAMBI, like its Xenopus homolog, functions as a general antagonist to attenuate the transcriptional activity of TGF-β/BMPs and inhibit TGF-β-induced R-Smad phosphorylation and cell growth arrest, indicating that the inhibitory activity of BAMBI on TGF-β signaling is evolutionally conserved.Although the possible function of BAMBI in TGF-β signaling has been examined in different animal models such as Xenopus (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar), zebrafish (25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar), rat (23Loveland K.L. Bakker M. Meehan T. Christy E. von Schönfeldt V. Drummond A. de Kretser D. Endocrinology. 2003; 144: 4180-4186Crossref PubMed Scopus (38) Google Scholar), and mouse (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar), our understanding of the molecular mechanism underlying its function is still limited. It has been shown that Xenopus BAMBI can bind to TGF-β receptors and interfere with the heterocomplex formation between the type I and type II receptors (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). Here, we confirmed this mechanism with hBAMBI. Moreover, this study has discovered an additional mechanism whereby BAMBI cooperates with Smad7 to inhibit TGF-β signaling.Smad7 is a well characterized antagonist of TGF-β signaling. It was found to directly interact with the active type I receptors in competition with R-Smads and thus inhibit the activation of R-Smads (19Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1547) Google Scholar, 42Hayashi 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 (1149) Google Scholar), to recruit the ubiquitin E3 ligases Smurf1 or Smurf2 and lead to the ubiquitination and degradation of ALK5 (48Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 49Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 50Suzuki C. Murakami G. Fukuchi M. Shimanuki T. Shikauchi Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; 277: 39919-39925Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), to promote receptor dephosphorylation via protein phosphatase 1c (51Shi W. Sun C. He B. Xiong W. Shi X. Yao D. Cao X. J. Cell Biol. 2004; 164: 291-300Crossref PubMed Scopus (206) Google Scholar), or to block functional Smad-DNA complex formation in the nucleus (37Zhang S. Fei T. Zhang L. Zhang R. Chen F. Ning Y. Han Y. Feng X.H. Meng A. Chen Y.G. Mol. Cell. Biol. 2007; 27: 4488-4499Crossref PubMed Scopus (205) Google Scholar). In this study, we found that hBAMBI also interacts with inhibitory Smads Smad6 and Smad7, and the interaction between hBAMBI and Smad7 was enhanced by TGF-β treatment. TGF-β promoted the association of hBAMBI with Smad7, and hBAMBI exhibited a higher binding affinity to active ALK5. Our study further demonstrated that hBAMBI, Smad7, and ALK5 form a ternary complex. Interestingly, unlike Smad7 and Smurfs, hBAMBI does not affect ALK5 turnover nor further promote Smad7/Smurf1-induced degradation of ALK5. Rather hBMABI inhibits the interaction between Smad3 and ALK5 and interferes with Smad3 phosphorylation. Therefore, our data suggest that BAMBI could exploit dual mechanisms to interfere with TGF-β signaling, and these two mechanisms may work cooperatively. Because BAMBI can also interact with Smad6, and both of them are negative regulators of BMP signaling, it is possible that BAMBI and Smad6 might cooperatively inhibit BMP signaling in similar manners. Indeed, overexpression of BAMBI inhibited the binding of Smad1(3A) with the constitutive active BMP type I receptor ca-ALK6, suggesting that BAMBI utilizes similar mechanisms to function as a general antagonist of the TGF-β family members.Despite a recent study reporting that BAMBI is dispensable for mouse embryo development and postnatal survival (28Chen J. Bush J.O. Ovitt C.E. Lan Y. Jiang R. Genesis. 2007; 45: 482-486Crossref PubMed Scopus (38) Google Scholar), other studies have underlined the possible important roles of hBAMBI in several pathological processes such as tumorigenesis and fibrogenesis. BAMBI was firstly reported to be a gene whose expression was reversely correlated with metastasis progression of human melanomas (33Degen W.G. Weterman M.A. van Groningen J.J. Cornelissen I.M. Lemmers J.P. Agterbos M.A. Geurts van Kessel A. Swart G.W. Bloemers H.P. Int. J. Cancer. 1996; 65: 460-465Crossref PubMed Scopus (60) Google Scholar). BAMBI was also found to counteract the effect of TGF-β on cell growth and invasion of human gastric carcinoma cell lines (35Sasaki T. Sasahira T. Shimura H. Ikeda S. Kuniyasu H. Oncol. Rep. 2004; 11: 1219-1223PubMed Google Scholar) and bladder cancer cells (34Khin S.S. Kitazawa R. Win N. Aye T.T. Mori K. Kondo T. Kitazawa S. Int. J. Cancer. 2009; 125: 328-338Crossref PubMed Scopus (40) Google Scholar). The expression of BAMBI is significantly up-regulated in most colorectal and hepatocellular carcinomas, and this up-regulation is mediated by β-catenin (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Furthermore, overexpression of BAMBI attenuated TGF-β signaling in these tumor cells, suggesting that β-catenin can promote the formation of colorectal and hepatocellular tumors by interfering with TGF-β-mediated growth arrest via BAMBI. A recent report also indicated that BAMBI might be involved in hepatic fibrogenesis (36Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. Nat. Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1440) Google Scholar). Toll-like receptor 4 and lipopolysaccharide sensitize hepatic stellate cells to TGF-β signaling by down-regulating the expression of BAMBI via a MyD88-NF-κB-dependent pathway. These studies suggest that the appropriate negative regulation of TGF-β by BAMBI is important for tissue homeostasis. The TGF-β family members play pivotal roles in embryo development and tissue homeostasis. Although the canonical Smad-mediated signaling pathway is relatively simple, it is precisely controlled at different levels from the availability and activation of extracellular ligands, the activity and stability of membrane receptors, to the activity, location, and stability of Smad proteins in the cytoplasm and in the nucleus (14Hill C.S. Cell Res. 2009; 19: 36-46Crossref PubMed Scopus (180) Google Scholar, 15Lönn P. Morén A. Raja E. Dahl M. Moustakas A. Cell Res. 2009; 19: 21-35Crossref PubMed Scopus (147) Google Scholar, 21Yan X. Liu Z. Chen Y. Acta. Biochim. Biophys Sin. 2009; 41: 263-272Crossref Scopus (302) Google Scholar, 45Wrighton K.H. Lin X. Feng X.H. Cell Res. 2009; 19: 8-20Crossref PubMed Scopus (270) Google Scholar, 46Deheuninck J. Luo K. Cell Res. 2009; 19: 47-57Crossref PubMed Scopus (196) Google Scholar, 47Itoh S. ten Dijke P. Curr. Opin Cell Biol. 2007; 19: 176-184Crossref PubMed Scopus (337) Google Scholar). Here, we demonstrated that hBAMBI, like its Xenopus homolog, functions as a general antagonist to attenuate the transcriptional activity of TGF-β/BMPs and inhibit TGF-β-induced R-Smad phosphorylation and cell growth arrest, indicating that the inhibitory activity of BAMBI on TGF-β signaling is evolutionally conserved. Although the possible function of BAMBI in TGF-β signaling has been examined in different animal models such as Xenopus (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar), zebrafish (25Tsang M. Kim R. de Caestecker M.P. Kudoh T. Roberts A.B. Dawid I.B. Genesis. 2000; 28: 47-57Crossref PubMed Scopus (57) Google Scholar), rat (23Loveland K.L. Bakker M. Meehan T. Christy E. von Schönfeldt V. Drummond A. de Kretser D. Endocrinology. 2003; 144: 4180-4186Crossref PubMed Scopus (38) Google Scholar), and mouse (22Grotewold L. Plum M. Dildrop R. Peters T. Rüther U. Mech. Dev. 2001; 100: 327-330Crossref PubMed Scopus (95) Google Scholar), our understanding of the molecular mechanism underlying its function is still limited. It has been shown that Xenopus BAMBI can bind to TGF-β receptors and interfere with the heterocomplex formation between the type I and type II receptors (24Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massagué J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (557) Google Scholar). Here, we confirmed this mechanism with hBAMBI. Moreover, this study has discovered an additional mechanism whereby BAMBI cooperates with Smad7 to inhibit TGF-β signaling. Smad7 is a well characterized antagonist of TGF-β signaling. It was found to directly interact with the active type I receptors in competition with R-Smads and thus inhibit the activation of R-Smads (19Nakao A. Afrakhte M. Morén A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1547) Google Scholar, 42Hayashi 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 (1149) Google Scholar), to recruit the ubiquitin E3 ligases Smurf1 or Smurf2 and lead to the ubiquitination and degradation of ALK5 (48Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 49Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 50Suzuki C. Murakami G. Fukuchi M. Shimanuki T. Shikauchi Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; 277: 39919-39925Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), to promote receptor dephosphorylation via protein phosphatase 1c (51Shi W. Sun C. He B. Xiong W. Shi X. Yao D. Cao X. J. Cell Biol. 2004; 164: 291-300Crossref PubMed Scopus (206) Google Scholar), or to block functional Smad-DNA complex formation in the nucleus (37Zhang S. Fei T. Zhang L. Zhang R. Chen F. Ning Y. Han Y. Feng X.H. Meng A. Chen Y.G. Mol. Cell. Biol. 2007; 27: 4488-4499Crossref PubMed Scopus (205) Google Scholar). In this study, we found that hBAMBI also interacts with inhibitory Smads Smad6 and Smad7, and the interaction between hBAMBI and Smad7 was enhanced by TGF-β treatment. TGF-β promoted the association of hBAMBI with Smad7, and hBAMBI exhibited a higher binding affinity to active ALK5. Our study further demonstrated that hBAMBI, Smad7, and ALK5 form a ternary complex. Interestingly, unlike Smad7 and Smurfs, hBAMBI does not affect ALK5 turnover nor further promote Smad7/Smurf1-induced degradation of ALK5. Rather hBMABI inhibits the interaction between Smad3 and ALK5 and interferes with Smad3 phosphorylation. Therefore, our data suggest that BAMBI could exploit dual mechanisms to interfere with TGF-β signaling, and these two mechanisms may work cooperatively. Because BAMBI can also interact with Smad6, and both of them are negative regulators of BMP signaling, it is possible that BAMBI and Smad6 might cooperatively inhibit BMP signaling in similar manners. Indeed, overexpression of BAMBI inhibited the binding of Smad1(3A) with the constitutive active BMP type I receptor ca-ALK6, suggesting that BAMBI utilizes similar mechanisms to function as a general antagonist of the TGF-β family members. Despite a recent study reporting that BAMBI is dispensable for mouse embryo development and postnatal survival (28Chen J. Bush J.O. Ovitt C.E. Lan Y. Jiang R. Genesis. 2007; 45: 482-486Crossref PubMed Scopus (38) Google Scholar), other studies have underlined the possible important roles of hBAMBI in several pathological processes such as tumorigenesis and fibrogenesis. BAMBI was firstly reported to be a gene whose expression was reversely correlated with metastasis progression of human melanomas (33Degen W.G. Weterman M.A. van Groningen J.J. Cornelissen I.M. Lemmers J.P. Agterbos M.A. Geurts van Kessel A. Swart G.W. Bloemers H.P. Int. J. Cancer. 1996; 65: 460-465Crossref PubMed Scopus (60) Google Scholar). BAMBI was also found to counteract the effect of TGF-β on cell growth and invasion of human gastric carcinoma cell lines (35Sasaki T. Sasahira T. Shimura H. Ikeda S. Kuniyasu H. Oncol. Rep. 2004; 11: 1219-1223PubMed Google Scholar) and bladder cancer cells (34Khin S.S. Kitazawa R. Win N. Aye T.T. Mori K. Kondo T. Kitazawa S. Int. J. Cancer. 2009; 125: 328-338Crossref PubMed Scopus (40) Google Scholar). The expression of BAMBI is significantly up-regulated in most colorectal and hepatocellular carcinomas, and this up-regulation is mediated by β-catenin (31Sekiya T. Adachi S. Kohu K. Yamada T. Higuchi O. Furukawa Y. Nakamura Y. Nakamura T. Tashiro K. Kuhara S. Ohwada S. Akiyama T. J. Biol. Chem. 2004; 279: 6840-6846Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Furthermore, overexpression of BAMBI attenuated TGF-β signaling in these tumor cells, suggesting that β-catenin can promote the formation of colorectal and hepatocellular tumors by interfering with TGF-β-mediated growth arrest via BAMBI. A recent report also indicated that BAMBI might be involved in hepatic fibrogenesis (36Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. Nat. Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1440) Google Scholar). Toll-like receptor 4 and lipopolysaccharide sensitize hepatic stellate cells to TGF-β signaling by down-regulating the expression of BAMBI via a MyD88-NF-κB-dependent pathway. These studies suggest that the appropriate negative regulation of TGF-β by BAMBI is important for tissue homeostasis. We are grateful to Dr. G. W. Swart for hBAMBI/nma cDNA and also to Ziying Liu for critical reading of manuscript.