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• W2022751249 abstract "Smad6 and Smad7, a subgroup of Smad proteins, antagonize the signals elicited by transforming growth factor-β. These two Smads, induced by transforming growth factor-β or bone morphogenetic protein (BMP) stimulation, form stable associations with their activated type I receptors, blocking phosphorylation of receptor-regulated Smads in the cytoplasm. Here we show that Smad6 interacts with homeobox (Hox) c-8 as a transcriptional corepressor, inhibiting BMP signaling in the nucleus. The interaction between Smad6 and Hoxc-8 was identified by a yeast two-hybrid approach and further demonstrated by co-immunoprecipitation assays in cells. Gel shift assays show that Smad6, but not Smad7, interacts with both Hoxc-8 and Hoxa-9 as a heterodimer when binding to DNA. More importantly, the Smad6-Hoxc-8 complex inhibits interaction of Smad1 with Hoxc-8- and Smad1-induced transcription activity. These data indicate that Smad6 interacts with Hox transcription factors as part of the negative feedback circuit in the BMP signaling pathway. Smad6 and Smad7, a subgroup of Smad proteins, antagonize the signals elicited by transforming growth factor-β. These two Smads, induced by transforming growth factor-β or bone morphogenetic protein (BMP) stimulation, form stable associations with their activated type I receptors, blocking phosphorylation of receptor-regulated Smads in the cytoplasm. Here we show that Smad6 interacts with homeobox (Hox) c-8 as a transcriptional corepressor, inhibiting BMP signaling in the nucleus. The interaction between Smad6 and Hoxc-8 was identified by a yeast two-hybrid approach and further demonstrated by co-immunoprecipitation assays in cells. Gel shift assays show that Smad6, but not Smad7, interacts with both Hoxc-8 and Hoxa-9 as a heterodimer when binding to DNA. More importantly, the Smad6-Hoxc-8 complex inhibits interaction of Smad1 with Hoxc-8- and Smad1-induced transcription activity. These data indicate that Smad6 interacts with Hox transcription factors as part of the negative feedback circuit in the BMP signaling pathway. transforming growth factor-β bone morphogenetic proteins glutathione S-transferase forkhead activin signal transducer plasminogen activator inhibitor-1 transcription factor υF3 hemagglutinin mutant Hox-pGL3 green fluorescent protein Members of TGF-β1superfamily transduce their signals into the cell through a family of mediator proteins called Smads. Receptor-regulated Smad1, Smad5, and Smad8 mediate BMP signaling, whereas Smad2 and Smad3 respond to TGF-β (1.Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 2.Nishimura R. Kato Y. Chen D. Harris S.E. Mundy G.R. Yoneda T. J. Biol. Chem. 1998; 273: 1872-1879Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3.Nakayama T. Snyder M.A. Grewal S.S. Tsuneizumi K. Tabata T. Christian J.L. Development. 1998; 125: 857-867Crossref PubMed Google Scholar, 4.Nakao 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 (901) Google Scholar). Upon phosphorylation by their type I receptors, The receptor-regulated Smads interact with the common partner, Smad4, and translocate to the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription (5.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 6.Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar, 7.Heldin C-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar, 8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 9.Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Nature. 1997; 389: 85-89Crossref PubMed Scopus (490) Google Scholar). Smad6 and Smad7 are struturally divergent Smads as antagonists of TGF-β family signaling (5.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 6.Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar). They can associate with activated TGF-β and BMP type I receptors, thereby preventing phosphorylation of receptor-regulated Smads (11.Hayashi H. Abdollah S. Qiu Y. Cai J. Xu 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, 12.Nakao 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: 631-635Crossref PubMed Scopus (1546) Google Scholar, 13.Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar). In addition, Smad6 has also been demonstrated to interact with phosphorylated Smad1 to prevent the formation of an active signaling complex of Smad1 and Smad4, preferentially inhibiting the signaling pathways activated by BMPs (14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar, 15.Ishisaki A. Yamato K. Hashimoto S. Nakao A. Tamaki K. Nonaka K. ten Dijke P. Sugino H. Nishihara T. J. Biol. Chem. 1999; 274: 13637-13642Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar).Studies on the mechanism by which Smads mediate TGF-β-/activin-regulated gene transcription have led to the discovery of several Smad-interacting nuclear transcription factors and their cis-acting DNA elements. In particular, the Xenopus forkhead activin signal transducer-1 (FAST-1) binds to an activin response element upstream of the homeobox gene mix2. The transcription activation requires the presence of activin and assembly of a FAST-1-Smad2-Smad4 complex (9.Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Nature. 1997; 389: 85-89Crossref PubMed Scopus (490) Google Scholar). The mammalian homolog FAST-2 activates the hox gene goosecoid where formation of a higher order complex of FAST-2-Smad2-Smad4 is also essential for transactivation (17.Labbe E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Transcription factor μF3 (TFE3) binds to the E-box of the plasminogen activator inhibitor-1 (PAI-1) promoter, whereas Smad3 and Smad4 bind to a sequence adjacent to the TFE3 binding site to cooperatively activate PAI-1 gene transcription (10.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (257) Google Scholar).We have reported that Smad1 interacts with homeodomain transcription factor Hoxc-8 in response to BMP signaling (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Hoxc-8 belongs to a highly conserved hox gene family and is expressed in limbs, backbone rudiments, the neural tube of mouse mid-gestation embryos, and in the cartilage and skeleton of newborns (19.Simeone A. Mavilio F. Acampora D. Giampaolo A. Faiella A. Zappavigna V. D'Esposito M. Pannese M. Russo G. Boncinelli E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4914-4918Crossref PubMed Scopus (94) Google Scholar, 20.Yueh Y.G. Gardner D.P. Kappen C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9956-9961Crossref PubMed Scopus (91) Google Scholar, 21.Le Mouellic H. Condamine H. Brulet P. Genes Dev. 1988; 2: 125-135Crossref PubMed Scopus (46) Google Scholar). The interaction domains between the two proteins were characterized. Two regions within the amino-terminal 87 amino acid residues of Smad1 were mapped to interact with the homeodomain of Hoxc-8. Stable expression of recombinant cDNAs encoding the Hoxc-8 interaction domains of Smad1 in 2T3 osteoblast precursor cells stimulated osteoblast differentiation-related gene expression and lead to mineralized bone matrix formation. In this communication we show that Smad6 interacts Hoxc-8 as a complex when binding to DNA, thereby inhibiting Smad1-mediated transcriptional activity as negative feedback loop in the nucleus.RESULTS AND DISCUSSIONWe have previously demonstrated that Smad1 interacts with Hoxc-8 in response to BMP stimulation (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Hoxc-8 functions as a transcription repressor in BMP signaling. The interaction of Smad1 with Hoxc-8 dislodges Hoxc-8 binding from its element resulting in initiation of gene transcription (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To characterize the Hoxc-8-mediated transcription mechanism in BMP-induced gene activation, we used a yeast two-hybrid system to identify transcription factors that interact with Hoxc-8. An intact Hoxc-8 cDNA fused with the Gal4 DNA binding domain was used as a bait plasmid to screen a human U2 OS osteoblast-like cell cDNA library constructed in pACT2 plasmid. After two rounds of screening, we obtained 26 positive clones. DNA sequence analysis identified one clone as Smad6 (Table 1) . Smad6 and Smad7 are immunolocalized in the nucleus of rat epiphyseal plate (22.Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar), Xenopus embryo (23.Nakayama T. Gardner H. Berg L.K. Christian J.L. Genes Cells. 1998; 3: 387-394Crossref PubMed Scopus (66) Google Scholar), and Mink lung epithelial (Mv1Lu) cells (24.Itoh S. Landstrom M. Hermansson A. Itoh F. Heldin C.H. Heldin N.E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The interaction of Smad6 with Hoxc-8 suggests that Smad6 may have a novel antagonistic function in the nucleus.Table ISpecific interaction of Smad6 with Hoxc-8 in a yeast two-hybrid systemGroupBaitPreyβ-Gal activity1pGBT9pACT212pGBT9pACT2-Smad633pGBT9pACT2-Smad6c84pGBT9-Hoxc-8pACT225pGBT9-Smad1pACT2-Hoxc-8596pGBT9-Hoxc-8pACT2-Smad62867pGBT9-Hoxc-8pACT2-Smad6c1957The interaction was assayed in liquid culture of mutant yeast strain Y190, which requires His, Leu, and Trp to grow. pGBT9-Hox c-8 and pACT2-Smad6 plasmids carry Trp and Leu as their selective markers, respectively. The interaction between Smad6 and Hoxc-8 enables the yeast to synthesize His and induces β-gal expression. The arbitrary units of β-gal activities for yeast bearing different plasmids were plotted as shown. Open table in a new tab The initial Smad6 cDNA clone (Smad6C in Table 1) encodes amino acids 281 to 496 of a 496-amino acid protein. The interaction between Hoxc-8 and Smad6 was further confirmed with a β-gal filter lift assay (data not shown) and quantified by a liquid β-gal assay (Table 1). When the full length of Smad6 fused with the GAL4 transcriptional activation domain was tested in the two-hybrid system, it showed a weaker interaction with Hoxc-8 in comparison with the carboxyl-terminal domain (Smad6C). Deletion of the Smad6 amino-terminal domain may change the protein conformation in a way that the carboxyl-terminal region becomes easier to interact with Hoxc-8. The assays of both empty bait vector (pGBT9) with Smad6C or Smad6 full-length cDNAs in prey plasmids and empty prey vector (pACT2) with full-length Hoxc-8 in bait vector showed very little activity. Compared with the interaction between Smad1 and Hoxc-8, the interaction of Smad6 with Hoxc-8 is about 5 times stronger (Table 1).To investigate the interaction of Smad6 with Hoxc-8 in mammalian cells and the effect of BMP stimulation on this interaction, COS-1 cells were transiently co-transfected with expression plasmids for FLAG-Smad6, HA-Hoxc-8, and/or constitutively active BMP type IA receptor ALK3 (Q233D). The cell lysates were immunoprecipitated with anti-FLAG M2 antibody and immunoblotted with anti-HA antibody. The results represented in Fig. 1 demonstrate that Smad6 (see Fig. 1, lanes 7 and 8) was co-immunoprecipitated with HA-Hoxc-8. BMP induces Smad6 mRNA expression (25.Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.H. Heldin N.E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar, 26.Takase M. Imamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Kawabata M. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar), and overexpression of ALK3 (Q233D) did not significantly change the interaction of Smad6 with Hoxc-8 (lane 8), indicating that BMP stimulation is not required for the interaction between Smad6 and Hoxc-8. Our initial Smad6 clone only encodes the carboxyl-terminal domain, indicating that this region of the protein may be involved in the interaction with Hoxc-8. To further investigate this observation, two FLAG-tagged Smad6 truncation expression plasmids were constructed. As shown in Fig.1, Smad6C exhibits a strong interaction with Hoxc-8 (Fig. 1, lanes 4 and 5). In contrast, the Smad6 amino-terminal with the linker region (Smad6NL) failed to bind to Hoxc-8 in immunoprecipitation assay (Fig. 1, lane 6). Smad proteins contain highly conserved amino- and carboxyl-terminal domains (referred to as MH1 and MH2 domains, respectively). The MH1 domain inhibits biological activities of the MH2 domain because of interactions between these two distal sites (27.Shi Y. Wang Y.F. Jayaraman L. Yang H. Massague J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Like other regulatory Smads, Smad6 also contains a conserved MH2 domain and short segments of MH1 domain homology (28.Newfeld S.J. Wisotzkey R.G. Kumar S. Genetics. 1999; 152: 783-795Crossref PubMed Google Scholar). Therefore, our results suggest that the carboxyl-terminal domain of Smad6 interacts with Hoxc-8 and that the amino terminus negatively regulates interaction between the two proteins.We examined the effect of the interaction between Hoxc-8 and Smad6 on Hoxc-8 DNA binding activity. Gel shift assays were performed with purified GST-Smad6 and GST-Hoxc-8 fusion proteins using osteopontin Hoxc-8 DNA binding element as a probe. As expected, Hoxc-8 protein binds to the DNA probe, which is inhibited by Smad1 (Fig.2 a, lanes 5 and6). Smad6 alone did not bind to the DNA element (lane 4). Interestingly, Incubation of both Hoxc-8 and Smad6 proteins yields a distinct shifted band with a molecular weight higher than Hoxc-8 binding alone, indicating that Hoxc-8 and Smad6 bind to the DNA element as a complex (lane 7). More importantly, the formation of the Smad6-Hoxc-8 complex eliminated the inhibitory effects of Smad1 on Hoxc-8 DNA binding (lane 8). Yeast two-hybrid assays already demonstrated that the interaction between Hoxc-8 and Smad6 is much stronger than that between Hoxc-8 and Smad1 (Table 1).Hox proteins have been demonstrated to interact with Smad1, but not Smad2 and -3, in response to BMP stimulation (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To examine whether the interaction between Smad6 and Hoxc-8 is also specifically involved in the BMP signaling pathway, Smad7 was tested for its interaction with Hox proteins (Fig. 2 b). Like Smad2 and -3, Smad7 did not interact with either Hoxc-8 or Hoxa-9 (Fig. 2 b, lanes 4, 7, and 10). Considering Smad6 as only interacting with phosphorylated Smad1 in the cytoplasm (14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar), our results also suggest that Smad6 be preferentially involved in BMP signaling. Smad4, the common partner for all receptor-regulated Smads and interaction with Hoxc-8, was examined for the same purpose (Fig.2 c). In comparison with Smad1, the complex of Smad6 and Hoxc-8 did not block the interaction of Smad4 with Hoxc-8 completely (Fig. 2 c, lanes 7 and 9). In fact, Smad4 can only be passively translocated into the nucleus by forming hetero-oligomers with any of the receptor-regulated Smads (29.Liu F. Pouponnot C. Massague J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (397) Google Scholar). Again, these results support that Smad6 is an important antagonist preferentially for the BMP signaling pathway.To investigate whether the Smad6-Hoxc-8 complex inhibits the interaction of Smad1 with Hoxc-8 in activating gene transcription, we utilized the model described in our earlier studies (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Overexpression of the Smad1-Hoxc-8 interaction domain linked to a nuclear localization signal (Smad1B) stimulates BMP downstream gene expression and induces osteoblast differentiation from osteogenic cells (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 30.Yang X. Ji X. Shi X. Cao X. J. Biol. Chem. 2000; 275: 1065-1072Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). When the BMP-inducible construct (Hox-pGL3) was co-transfected in Mv1Lu cells with the Smad1B expression plasmid, the luciferase activity was stimulated in a dose-dependent manner (Fig.3 a). This model provides an ideal assay to directly examine the Smad6 antagonistic function in the nucleus. Because Smad1B mimics BMP-induced gene transcription without BMP receptor phosphorylation involving and interaction with Smad6 of Smad1 (13.Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar, 30.Yang X. Ji X. Shi X. Cao X. J. Biol. Chem. 2000; 275: 1065-1072Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), this assay avoids Smad6 inhibitory function in the cytoplasm. Hox-pGL3 construct was co-transfected in Mv1Lu cells with Hoxc-8 and/or Smad6 expression plasmid. As shown in Fig. 3 b, overexpression of Hoxc-8 or Smad6 alone moderately inhibited Smad1B-induced transcription activity. Most importantly, co-transfection of both Hoxc-8 and Smad6 plasmids completely abolished the Smad1B-induced luciferase activity. To validate this observation, we transfected Mv1Lu cells with a mutated construct, mHox-pGL3, in which the core nucleotides of the Hoxc-8 binding site were mutated from TAAT to GCCG. Transfection of the mutant construct dramatically reduced Smad1B-induced reporter activity. As expected, the inhibition mediated by co-transfection of Smad6 and Hoxc-8 was also reduced (Fig.3 c).Figure 3The Hoxc-8 DNA binding site is required for Smad6-mediated transcription inhibition. a, Smad1-Hoxc-8 interaction domain (Smad1B) induces transcription in a dose-dependent manner. Hox-pGL3 construct (500 ng), containing the osteopontin Hox binding site linked to the SV40 promoter, was co-transfected in Mv1lu cells with different amounts of Smad1B expression plasmid. b, Smad6 inhibits Smad1B-induced transcription in the presence of Hoxc-8. Hox-pGL3 construct was co-transfected with Smad1B (300 ng), Hoxc-8 (25 ng), and/or Smad6 (100 ng) expression plasmids. c, mutation of the Hox binding site abolishes Smad1B stimulation. mHox-pGL3 (500 ng), containing the mutated osteopontin Hox binding site in Hox-pGL3 construct, was co-transfected with Smad1B (300 ng), Hoxc-8 (25 ng), and/or Smad6 (100 ng) plasmids. Cell lysates in a,b, and c were assayed for luciferase activity normalized to Renilla luciferase levels 48 h after transfection. Experiments were repeated 3 times in triplicate.d, both Smad6 and Hoxc-8 are localized in the nucleus. Mv1Lu cells were co-transfected with HA-tagged Hoxc-8 and GFP-Smad6 in the presence or absence of BMP-4. 48 h after transfection, cells were stained with monoclonal antibody against HA tag. Hoxc-8 was detected with secondary antibody conjugated to Cy3. Cells transfected with GFP expression plasmid alone serve as the control. All images are magnified by 1000.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, protein localization analysis demonstrated that both Smad6 and Hoxc-8 are highly expressed in the nucleus (Fig. 3 d). Differing from Smad7, Smad6 nuclear exportation was not induced with BMP-4. Taken together with immunocoprecipitation of Smad6 with Hoxc-8 and formation of the Smad6-Hoxc-8 complex in gel shift assays, these data suggest co-localization of Smad6 and Hoxc-8 in the nucleus. Thus, our results are the first to demonstrate that Smad6 has an antagonistic function toward BMP signaling in the nucleus in addition to its interaction with BMP type I receptor and Smad1 in the cytoplasm. Members of TGF-β1superfamily transduce their signals into the cell through a family of mediator proteins called Smads. Receptor-regulated Smad1, Smad5, and Smad8 mediate BMP signaling, whereas Smad2 and Smad3 respond to TGF-β (1.Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 2.Nishimura R. Kato Y. Chen D. Harris S.E. Mundy G.R. Yoneda T. J. Biol. Chem. 1998; 273: 1872-1879Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3.Nakayama T. Snyder M.A. Grewal S.S. Tsuneizumi K. Tabata T. Christian J.L. Development. 1998; 125: 857-867Crossref PubMed Google Scholar, 4.Nakao 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 (901) Google Scholar). Upon phosphorylation by their type I receptors, The receptor-regulated Smads interact with the common partner, Smad4, and translocate to the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription (5.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 6.Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar, 7.Heldin C-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar, 8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 9.Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Nature. 1997; 389: 85-89Crossref PubMed Scopus (490) Google Scholar). Smad6 and Smad7 are struturally divergent Smads as antagonists of TGF-β family signaling (5.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 6.Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar). They can associate with activated TGF-β and BMP type I receptors, thereby preventing phosphorylation of receptor-regulated Smads (11.Hayashi H. Abdollah S. Qiu Y. Cai J. Xu 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, 12.Nakao 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: 631-635Crossref PubMed Scopus (1546) Google Scholar, 13.Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar). In addition, Smad6 has also been demonstrated to interact with phosphorylated Smad1 to prevent the formation of an active signaling complex of Smad1 and Smad4, preferentially inhibiting the signaling pathways activated by BMPs (14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar, 15.Ishisaki A. Yamato K. Hashimoto S. Nakao A. Tamaki K. Nonaka K. ten Dijke P. Sugino H. Nishihara T. J. Biol. Chem. 1999; 274: 13637-13642Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Studies on the mechanism by which Smads mediate TGF-β-/activin-regulated gene transcription have led to the discovery of several Smad-interacting nuclear transcription factors and their cis-acting DNA elements. In particular, the Xenopus forkhead activin signal transducer-1 (FAST-1) binds to an activin response element upstream of the homeobox gene mix2. The transcription activation requires the presence of activin and assembly of a FAST-1-Smad2-Smad4 complex (9.Chen X. Weisberg E. Fridmacher V. Watanabe M. Naco G. Whitman M. Nature. 1997; 389: 85-89Crossref PubMed Scopus (490) Google Scholar). The mammalian homolog FAST-2 activates the hox gene goosecoid where formation of a higher order complex of FAST-2-Smad2-Smad4 is also essential for transactivation (17.Labbe E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Transcription factor μF3 (TFE3) binds to the E-box of the plasminogen activator inhibitor-1 (PAI-1) promoter, whereas Smad3 and Smad4 bind to a sequence adjacent to the TFE3 binding site to cooperatively activate PAI-1 gene transcription (10.Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (257) Google Scholar). We have reported that Smad1 interacts with homeodomain transcription factor Hoxc-8 in response to BMP signaling (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Hoxc-8 belongs to a highly conserved hox gene family and is expressed in limbs, backbone rudiments, the neural tube of mouse mid-gestation embryos, and in the cartilage and skeleton of newborns (19.Simeone A. Mavilio F. Acampora D. Giampaolo A. Faiella A. Zappavigna V. D'Esposito M. Pannese M. Russo G. Boncinelli E. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4914-4918Crossref PubMed Scopus (94) Google Scholar, 20.Yueh Y.G. Gardner D.P. Kappen C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9956-9961Crossref PubMed Scopus (91) Google Scholar, 21.Le Mouellic H. Condamine H. Brulet P. Genes Dev. 1988; 2: 125-135Crossref PubMed Scopus (46) Google Scholar). The interaction domains between the two proteins were characterized. Two regions within the amino-terminal 87 amino acid residues of Smad1 were mapped to interact with the homeodomain of Hoxc-8. Stable expression of recombinant cDNAs encoding the Hoxc-8 interaction domains of Smad1 in 2T3 osteoblast precursor cells stimulated osteoblast differentiation-related gene expression and lead to mineralized bone matrix formation. In this communication we show that Smad6 interacts Hoxc-8 as a complex when binding to DNA, thereby inhibiting Smad1-mediated transcriptional activity as negative feedback loop in the nucleus. RESULTS AND DISCUSSIONWe have previously demonstrated that Smad1 interacts with Hoxc-8 in response to BMP stimulation (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Hoxc-8 functions as a transcription repressor in BMP signaling. The interaction of Smad1 with Hoxc-8 dislodges Hoxc-8 binding from its element resulting in initiation of gene transcription (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To characterize the Hoxc-8-mediated transcription mechanism in BMP-induced gene activation, we used a yeast two-hybrid system to identify transcription factors that interact with Hoxc-8. An intact Hoxc-8 cDNA fused with the Gal4 DNA binding domain was used as a bait plasmid to screen a human U2 OS osteoblast-like cell cDNA library constructed in pACT2 plasmid. After two rounds of screening, we obtained 26 positive clones. DNA sequence analysis identified one clone as Smad6 (Table 1) . Smad6 and Smad7 are immunolocalized in the nucleus of rat epiphyseal plate (22.Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar), Xenopus embryo (23.Nakayama T. Gardner H. Berg L.K. Christian J.L. Genes Cells. 1998; 3: 387-394Crossref PubMed Scopus (66) Google Scholar), and Mink lung epithelial (Mv1Lu) cells (24.Itoh S. Landstrom M. Hermansson A. Itoh F. Heldin C.H. Heldin N.E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The interaction of Smad6 with Hoxc-8 suggests that Smad6 may have a novel antagonistic function in the nucleus.Table ISpecific interaction of Smad6 with Hoxc-8 in a yeast two-hybrid systemGroupBaitPreyβ-Gal activity1pGBT9pACT212pGBT9pACT2-Smad633pGBT9pACT2-Smad6c84pGBT9-Hoxc-8pACT225pGBT9-Smad1pACT2-Hoxc-8596pGBT9-Hoxc-8pACT2-Smad62867pGBT9-Hoxc-8pACT2-Smad6c1957The interaction was assayed in liquid culture of mutant yeast strain Y190, which requires His, Leu, and Trp to grow. pGBT9-Hox c-8 and pACT2-Smad6 plasmids carry Trp and Leu as their selective markers, respectively. The interaction between Smad6 and Hoxc-8 enables the yeast to synthesize His and induces β-gal expression. The arbitrary units of β-gal activities for yeast bearing different plasmids were plotted as shown. Open table in a new tab The initial Smad6 cDNA clone (Smad6C in Table 1) encodes amino acids 281 to 496 of a 496-amino acid protein. The interaction between Hoxc-8 and Smad6 was further confirmed with a β-gal filter lift assay (data not shown) and quantified by a liquid β-gal assay (Table 1). When the full length of Smad6 fused with the GAL4 transcriptional activation domain was tested in the two-hybrid system, it showed a weaker interaction with Hoxc-8 in comparison with the carboxyl-terminal domain (Smad6C). Deletion of the Smad6 amino-terminal domain may change the protein conformation in a way that the carboxyl-terminal region becomes easier to interact with Hoxc-8. The assays of both empty bait vector (pGBT9) with Smad6C or Smad6 full-length cDNAs in prey plasmids and empty prey vector (pACT2) with full-length Hoxc-8 in bait vector showed very little activity. Compared with the interaction between Smad1 and Hoxc-8, the interaction of Smad6 with Hoxc-8 is about 5 times stronger (Table 1).To investigate the interaction of Smad6 with Hoxc-8 in mammalian cells and the effect of BMP stimulation on this interaction, COS-1 cells were transiently co-transfected with expression plasmids for FLAG-Smad6, HA-Hoxc-8, and/or constitutively active BMP type IA receptor ALK3 (Q233D). The cell lysates were immunoprecipitated with anti-FLAG M2 antibody and immunoblotted with anti-HA antibody. The results represented in Fig. 1 demonstrate that Smad6 (see Fig. 1, lanes 7 and 8) was co-immunoprecipitated with HA-Hoxc-8. BMP induces Smad6 mRNA expression (25.Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.H. Heldin N.E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar, 26.Takase M. Imamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Kawabata M. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar), and overexpression of ALK3 (Q233D) did not significantly change the interaction of Smad6 with Hoxc-8 (lane 8), indicating that BMP stimulation is not required for the interaction between Smad6 and Hoxc-8. Our initial Smad6 clone only encodes the carboxyl-terminal domain, indicating that this region of the protein may be involved in the interaction with Hoxc-8. To further investigate this observation, two FLAG-tagged Smad6 truncation expression plasmids were constructed. As shown in Fig.1, Smad6C exhibits a strong interaction with Hoxc-8 (Fig. 1, lanes 4 and 5). In contrast, the Smad6 amino-terminal with the linker region (Smad6NL) failed to bind to Hoxc-8 in immunoprecipitation assay (Fig. 1, lane 6). Smad proteins contain highly conserved amino- and carboxyl-terminal domains (referred to as MH1 and MH2 domains, respectively). The MH1 domain inhibits biological activities of the MH2 domain because of interactions between these two distal sites (27.Shi Y. Wang Y.F. Jayaraman L. Yang H. Massague J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Like other regulatory Smads, Smad6 also contains a conserved MH2 domain and short segments of MH1 domain homology (28.Newfeld S.J. Wisotzkey R.G. Kumar S. Genetics. 1999; 152: 783-795Crossref PubMed Google Scholar). Therefore, our results suggest that the carboxyl-terminal domain of Smad6 interacts with Hoxc-8 and that the amino terminus negatively regulates interaction between the two proteins.We examined the effect of the interaction between Hoxc-8 and Smad6 on Hoxc-8 DNA binding activity. Gel shift assays were performed with purified GST-Smad6 and GST-Hoxc-8 fusion proteins using osteopontin Hoxc-8 DNA binding element as a probe. As expected, Hoxc-8 protein binds to the DNA probe, which is inhibited by Smad1 (Fig.2 a, lanes 5 and6). Smad6 alone did not bind to the DNA element (lane 4). Interestingly, Incubation of both Hoxc-8 and Smad6 proteins yields a distinct shifted band with a molecular weight higher than Hoxc-8 binding alone, indicating that Hoxc-8 and Smad6 bind to the DNA element as a complex (lane 7). More importantly, the formation of the Smad6-Hoxc-8 complex eliminated the inhibitory effects of Smad1 on Hoxc-8 DNA binding (lane 8). Yeast two-hybrid assays already demonstrated that the interaction between Hoxc-8 and Smad6 is much stronger than that between Hoxc-8 and Smad1 (Table 1).Hox proteins have been demonstrated to interact with Smad1, but not Smad2 and -3, in response to BMP stimulation (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To examine whether the interaction between Smad6 and Hoxc-8 is also specifically involved in the BMP signaling pathway, Smad7 was tested for its interaction with Hox proteins (Fig. 2 b). Like Smad2 and -3, Smad7 did not interact with either Hoxc-8 or Hoxa-9 (Fig. 2 b, lanes 4, 7, and 10). Considering Smad6 as only interacting with phosphorylated Smad1 in the cytoplasm (14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar), our results also suggest that Smad6 be preferentially involved in BMP signaling. Smad4, the common partner for all receptor-regulated Smads and interaction with Hoxc-8, was examined for the same purpose (Fig.2 c). In comparison with Smad1, the complex of Smad6 and Hoxc-8 did not block the interaction of Smad4 with Hoxc-8 completely (Fig. 2 c, lanes 7 and 9). In fact, Smad4 can only be passively translocated into the nucleus by forming hetero-oligomers with any of the receptor-regulated Smads (29.Liu F. Pouponnot C. Massague J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (397) Google Scholar). Again, these results support that Smad6 is an important antagonist preferentially for the BMP signaling pathway.To investigate whether the Smad6-Hoxc-8 complex inhibits the interaction of Smad1 with Hoxc-8 in activating gene transcription, we utilized the model described in our earlier studies (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Overexpression of the Smad1-Hoxc-8 interaction domain linked to a nuclear localization signal (Smad1B) stimulates BMP downstream gene expression and induces osteoblast differentiation from osteogenic cells (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 30.Yang X. Ji X. Shi X. Cao X. J. Biol. Chem. 2000; 275: 1065-1072Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). When the BMP-inducible construct (Hox-pGL3) was co-transfected in Mv1Lu cells with the Smad1B expression plasmid, the luciferase activity was stimulated in a dose-dependent manner (Fig.3 a). This model provides an ideal assay to directly examine the Smad6 antagonistic function in the nucleus. Because Smad1B mimics BMP-induced gene transcription without BMP receptor phosphorylation involving and interaction with Smad6 of Smad1 (13.Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar, 30.Yang X. Ji X. Shi X. Cao X. J. Biol. Chem. 2000; 275: 1065-1072Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), this assay avoids Smad6 inhibitory function in the cytoplasm. Hox-pGL3 construct was co-transfected in Mv1Lu cells with Hoxc-8 and/or Smad6 expression plasmid. As shown in Fig. 3 b, overexpression of Hoxc-8 or Smad6 alone moderately inhibited Smad1B-induced transcription activity. Most importantly, co-transfection of both Hoxc-8 and Smad6 plasmids completely abolished the Smad1B-induced luciferase activity. To validate this observation, we transfected Mv1Lu cells with a mutated construct, mHox-pGL3, in which the core nucleotides of the Hoxc-8 binding site were mutated from TAAT to GCCG. Transfection of the mutant construct dramatically reduced Smad1B-induced reporter activity. As expected, the inhibition mediated by co-transfection of Smad6 and Hoxc-8 was also reduced (Fig.3 c).Finally, protein localization analysis demonstrated that both Smad6 and Hoxc-8 are highly expressed in the nucleus (Fig. 3 d). Differing from Smad7, Smad6 nuclear exportation was not induced with BMP-4. Taken together with immunocoprecipitation of Smad6 with Hoxc-8 and formation of the Smad6-Hoxc-8 complex in gel shift assays, these data suggest co-localization of Smad6 and Hoxc-8 in the nucleus. Thus, our results are the first to demonstrate that Smad6 has an antagonistic function toward BMP signaling in the nucleus in addition to its interaction with BMP type I receptor and Smad1 in the cytoplasm. We have previously demonstrated that Smad1 interacts with Hoxc-8 in response to BMP stimulation (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Hoxc-8 functions as a transcription repressor in BMP signaling. The interaction of Smad1 with Hoxc-8 dislodges Hoxc-8 binding from its element resulting in initiation of gene transcription (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To characterize the Hoxc-8-mediated transcription mechanism in BMP-induced gene activation, we used a yeast two-hybrid system to identify transcription factors that interact with Hoxc-8. An intact Hoxc-8 cDNA fused with the Gal4 DNA binding domain was used as a bait plasmid to screen a human U2 OS osteoblast-like cell cDNA library constructed in pACT2 plasmid. After two rounds of screening, we obtained 26 positive clones. DNA sequence analysis identified one clone as Smad6 (Table 1) . Smad6 and Smad7 are immunolocalized in the nucleus of rat epiphyseal plate (22.Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar), Xenopus embryo (23.Nakayama T. Gardner H. Berg L.K. Christian J.L. Genes Cells. 1998; 3: 387-394Crossref PubMed Scopus (66) Google Scholar), and Mink lung epithelial (Mv1Lu) cells (24.Itoh S. Landstrom M. Hermansson A. Itoh F. Heldin C.H. Heldin N.E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The interaction of Smad6 with Hoxc-8 suggests that Smad6 may have a novel antagonistic function in the nucleus. The interaction was assayed in liquid culture of mutant yeast strain Y190, which requires His, Leu, and Trp to grow. pGBT9-Hox c-8 and pACT2-Smad6 plasmids carry Trp and Leu as their selective markers, respectively. The interaction between Smad6 and Hoxc-8 enables the yeast to synthesize His and induces β-gal expression. The arbitrary units of β-gal activities for yeast bearing different plasmids were plotted as shown. The initial Smad6 cDNA clone (Smad6C in Table 1) encodes amino acids 281 to 496 of a 496-amino acid protein. The interaction between Hoxc-8 and Smad6 was further confirmed with a β-gal filter lift assay (data not shown) and quantified by a liquid β-gal assay (Table 1). When the full length of Smad6 fused with the GAL4 transcriptional activation domain was tested in the two-hybrid system, it showed a weaker interaction with Hoxc-8 in comparison with the carboxyl-terminal domain (Smad6C). Deletion of the Smad6 amino-terminal domain may change the protein conformation in a way that the carboxyl-terminal region becomes easier to interact with Hoxc-8. The assays of both empty bait vector (pGBT9) with Smad6C or Smad6 full-length cDNAs in prey plasmids and empty prey vector (pACT2) with full-length Hoxc-8 in bait vector showed very little activity. Compared with the interaction between Smad1 and Hoxc-8, the interaction of Smad6 with Hoxc-8 is about 5 times stronger (Table 1). To investigate the interaction of Smad6 with Hoxc-8 in mammalian cells and the effect of BMP stimulation on this interaction, COS-1 cells were transiently co-transfected with expression plasmids for FLAG-Smad6, HA-Hoxc-8, and/or constitutively active BMP type IA receptor ALK3 (Q233D). The cell lysates were immunoprecipitated with anti-FLAG M2 antibody and immunoblotted with anti-HA antibody. The results represented in Fig. 1 demonstrate that Smad6 (see Fig. 1, lanes 7 and 8) was co-immunoprecipitated with HA-Hoxc-8. BMP induces Smad6 mRNA expression (25.Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.H. Heldin N.E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar, 26.Takase M. Imamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Kawabata M. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar), and overexpression of ALK3 (Q233D) did not significantly change the interaction of Smad6 with Hoxc-8 (lane 8), indicating that BMP stimulation is not required for the interaction between Smad6 and Hoxc-8. Our initial Smad6 clone only encodes the carboxyl-terminal domain, indicating that this region of the protein may be involved in the interaction with Hoxc-8. To further investigate this observation, two FLAG-tagged Smad6 truncation expression plasmids were constructed. As shown in Fig.1, Smad6C exhibits a strong interaction with Hoxc-8 (Fig. 1, lanes 4 and 5). In contrast, the Smad6 amino-terminal with the linker region (Smad6NL) failed to bind to Hoxc-8 in immunoprecipitation assay (Fig. 1, lane 6). Smad proteins contain highly conserved amino- and carboxyl-terminal domains (referred to as MH1 and MH2 domains, respectively). The MH1 domain inhibits biological activities of the MH2 domain because of interactions between these two distal sites (27.Shi Y. Wang Y.F. Jayaraman L. Yang H. Massague J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Like other regulatory Smads, Smad6 also contains a conserved MH2 domain and short segments of MH1 domain homology (28.Newfeld S.J. Wisotzkey R.G. Kumar S. Genetics. 1999; 152: 783-795Crossref PubMed Google Scholar). Therefore, our results suggest that the carboxyl-terminal domain of Smad6 interacts with Hoxc-8 and that the amino terminus negatively regulates interaction between the two proteins. We examined the effect of the interaction between Hoxc-8 and Smad6 on Hoxc-8 DNA binding activity. Gel shift assays were performed with purified GST-Smad6 and GST-Hoxc-8 fusion proteins using osteopontin Hoxc-8 DNA binding element as a probe. As expected, Hoxc-8 protein binds to the DNA probe, which is inhibited by Smad1 (Fig.2 a, lanes 5 and6). Smad6 alone did not bind to the DNA element (lane 4). Interestingly, Incubation of both Hoxc-8 and Smad6 proteins yields a distinct shifted band with a molecular weight higher than Hoxc-8 binding alone, indicating that Hoxc-8 and Smad6 bind to the DNA element as a complex (lane 7). More importantly, the formation of the Smad6-Hoxc-8 complex eliminated the inhibitory effects of Smad1 on Hoxc-8 DNA binding (lane 8). Yeast two-hybrid assays already demonstrated that the interaction between Hoxc-8 and Smad6 is much stronger than that between Hoxc-8 and Smad1 (Table 1). Hox proteins have been demonstrated to interact with Smad1, but not Smad2 and -3, in response to BMP stimulation (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To examine whether the interaction between Smad6 and Hoxc-8 is also specifically involved in the BMP signaling pathway, Smad7 was tested for its interaction with Hox proteins (Fig. 2 b). Like Smad2 and -3, Smad7 did not interact with either Hoxc-8 or Hoxa-9 (Fig. 2 b, lanes 4, 7, and 10). Considering Smad6 as only interacting with phosphorylated Smad1 in the cytoplasm (14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar), our results also suggest that Smad6 be preferentially involved in BMP signaling. Smad4, the common partner for all receptor-regulated Smads and interaction with Hoxc-8, was examined for the same purpose (Fig.2 c). In comparison with Smad1, the complex of Smad6 and Hoxc-8 did not block the interaction of Smad4 with Hoxc-8 completely (Fig. 2 c, lanes 7 and 9). In fact, Smad4 can only be passively translocated into the nucleus by forming hetero-oligomers with any of the receptor-regulated Smads (29.Liu F. Pouponnot C. Massague J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (397) Google Scholar). Again, these results support that Smad6 is an important antagonist preferentially for the BMP signaling pathway. To investigate whether the Smad6-Hoxc-8 complex inhibits the interaction of Smad1 with Hoxc-8 in activating gene transcription, we utilized the model described in our earlier studies (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Overexpression of the Smad1-Hoxc-8 interaction domain linked to a nuclear localization signal (Smad1B) stimulates BMP downstream gene expression and induces osteoblast differentiation from osteogenic cells (8.Shi X.M. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 30.Yang X. Ji X. Shi X. Cao X. J. Biol. Chem. 2000; 275: 1065-1072Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). When the BMP-inducible construct (Hox-pGL3) was co-transfected in Mv1Lu cells with the Smad1B expression plasmid, the luciferase activity was stimulated in a dose-dependent manner (Fig.3 a). This model provides an ideal assay to directly examine the Smad6 antagonistic function in the nucleus. Because Smad1B mimics BMP-induced gene transcription without BMP receptor phosphorylation involving and interaction with Smad6 of Smad1 (13.Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 14.Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar, 30.Yang X. Ji X. Shi X. Cao X. J. Biol. Chem. 2000; 275: 1065-1072Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), this assay avoids Smad6 inhibitory function in the cytoplasm. Hox-pGL3 construct was co-transfected in Mv1Lu cells with Hoxc-8 and/or Smad6 expression plasmid. As shown in Fig. 3 b, overexpression of Hoxc-8 or Smad6 alone moderately inhibited Smad1B-induced transcription activity. Most importantly, co-transfection of both Hoxc-8 and Smad6 plasmids completely abolished the Smad1B-induced luciferase activity. To validate this observation, we transfected Mv1Lu cells with a mutated construct, mHox-pGL3, in which the core nucleotides of the Hoxc-8 binding site were mutated from TAAT to GCCG. Transfection of the mutant construct dramatically reduced Smad1B-induced reporter activity. As expected, the inhibition mediated by co-transfection of Smad6 and Hoxc-8 was also reduced (Fig.3 c). Finally, protein localization analysis demonstrated that both Smad6 and Hoxc-8 are highly expressed in the nucleus (Fig. 3 d). Differing from Smad7, Smad6 nuclear exportation was not induced with BMP-4. Taken together with immunocoprecipitation of Smad6 with Hoxc-8 and formation of the Smad6-Hoxc-8 complex in gel shift assays, these data suggest co-localization of Smad6 and Hoxc-8 in the nucleus. Thus, our results are the first to demonstrate that Smad6 has an antagonistic function toward BMP signaling in the nucleus in addition to its interaction with BMP type I receptor and Smad1 in the cytoplasm. We are grateful to Albert Tousson for technical assistance with the fluorescence microscopy. We thank J. Warna for kindly providing the constitutively active BMP type IA (ALK3) receptor expression vector, R. Derynck for human Smad1 and -4 cDNA clones, H. Le Mouellic for Hoxc-8 cDNA, P. ten Dijke for Smad7 cDNA, and A. Hemmati-Brivanlou for Smad6 cDNA expression vectors." @default.
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• W2022751249 cites W1515719112 @default.
• W2022751249 cites W1553444759 @default.
• W2022751249 cites W1589859980 @default.
• W2022751249 cites W1663371441 @default.
• W2022751249 cites W1967781543 @default.
• W2022751249 cites W1969034753 @default.
• W2022751249 cites W1979208896 @default.
• W2022751249 cites W1980955079 @default.
• W2022751249 cites W2003573512 @default.
• W2022751249 cites W2012200818 @default.
• W2022751249 cites W2016311922 @default.
• W2022751249 cites W2023976317 @default.
• W2022751249 cites W2041750289 @default.
• W2022751249 cites W2047625373 @default.
• W2022751249 cites W2059437327 @default.
• W2022751249 cites W2082684861 @default.
• W2022751249 cites W2083879285 @default.
• W2022751249 cites W2085747258 @default.
• W2022751249 cites W2091746877 @default.
• W2022751249 cites W2093417867 @default.
• W2022751249 cites W2094579915 @default.
• W2022751249 cites W2098097317 @default.
• W2022751249 cites W2102868284 @default.
• W2022751249 cites W2103031848 @default.
• W2022751249 cites W2103802400 @default.
• W2022751249 cites W2120157258 @default.
• W2022751249 cites W2132008353 @default.
• W2022751249 cites W2145589640 @default.
• W2022751249 cites W2150073419 @default.
• W2022751249 cites W2189003807 @default.
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