Title: Identification and Functional Characterization of Distinct Critically Important Bone Morphogenetic Protein-specific Response Elements in the Id1 Promoter
Abstract: Transforming growth factor-β (TGF-β) family members, which include bone morphogenetic proteins (BMPs) and TGF-βs, elicit their cellular effects by activating specific Smad proteins, which control the transcription of target genes. BMPs and TGF-βs have overlapping as well as specific effects on mesenchymal cell differentiation for which the mechanisms are incompletely understood. Here we report that Id1, a dominant negative inhibitor of basic helix-loop-helix proteins, is a direct target gene for BMP. BMP, but not TGF-β, strongly activates the Id1 promoter in an Smad-dependent manner. We identified two BMP-responsive regions in the mouse Id1 promoter, which contain three distinct sequence elements; one region contains two Smad binding elements (SBEs), and the other region contains a GGCGCC palindromic sequence flanked by two CAGC and two CGCC motifs. Whereas SBEs and GGCGCC sequence are critically important, the CAGC and CGCC motifs are needed for efficient BMP-induced Id1 promoter activation. Smads are part of nuclear transcription factor complexes that specifically bind to SBEs and GGCGCC sequence in response to BMP but not TGF-β. Multimerization of the all three distinct sequence motifs is needed to generate a highly sensitive and BMP/Smad-dependent specific enhancer. Our results provide important new insights into how the BMP/Smad pathway can specifically activate target genes. Transforming growth factor-β (TGF-β) family members, which include bone morphogenetic proteins (BMPs) and TGF-βs, elicit their cellular effects by activating specific Smad proteins, which control the transcription of target genes. BMPs and TGF-βs have overlapping as well as specific effects on mesenchymal cell differentiation for which the mechanisms are incompletely understood. Here we report that Id1, a dominant negative inhibitor of basic helix-loop-helix proteins, is a direct target gene for BMP. BMP, but not TGF-β, strongly activates the Id1 promoter in an Smad-dependent manner. We identified two BMP-responsive regions in the mouse Id1 promoter, which contain three distinct sequence elements; one region contains two Smad binding elements (SBEs), and the other region contains a GGCGCC palindromic sequence flanked by two CAGC and two CGCC motifs. Whereas SBEs and GGCGCC sequence are critically important, the CAGC and CGCC motifs are needed for efficient BMP-induced Id1 promoter activation. Smads are part of nuclear transcription factor complexes that specifically bind to SBEs and GGCGCC sequence in response to BMP but not TGF-β. Multimerization of the all three distinct sequence motifs is needed to generate a highly sensitive and BMP/Smad-dependent specific enhancer. Our results provide important new insights into how the BMP/Smad pathway can specifically activate target genes. Depending on their extracellular stimuli, mesenchymal precursor cells can differentiate into muscle, fat, bone, or cartilage. Members of the transforming growth factor-β superfamily, which includes TGF-βs, 1The abbreviations used are:TGF-βtransforming growth factor-βBMPbone morphogenetic proteinCHIcycloheximideSBESmad binding elementALKactivin receptor kinaseIdinhibitors of differentiationMLPmajor late promoterEMSAelectrophoretic mobility shift assayGSTglutathioneS-transferaseActDactinomycin Dcaconstitutively active 1The abbreviations used are:TGF-βtransforming growth factor-βBMPbone morphogenetic proteinCHIcycloheximideSBESmad binding elementALKactivin receptor kinaseIdinhibitors of differentiationMLPmajor late promoterEMSAelectrophoretic mobility shift assayGSTglutathioneS-transferaseActDactinomycin Dcaconstitutively active activins, and bone morphogenetic proteins (BMPs), have important roles in directing cell fate choices of mesenchymal cells (1Centrella M. Horowitz M.C. Wozney J.M. McCarthy T.L. Endocr. Rev. 1994; 15: 27-39PubMed Google Scholar, 2Alliston T. Choy L. Ducy P. Karsenty G. Derynck R. 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The mechanisms that govern the ability of different TGF-β family members to induce similar but also specific differentiation effects are unclear. transforming growth factor-β bone morphogenetic protein cycloheximide Smad binding element activin receptor kinase inhibitors of differentiation major late promoter electrophoretic mobility shift assay glutathioneS-transferase actinomycin D constitutively active transforming growth factor-β bone morphogenetic protein cycloheximide Smad binding element activin receptor kinase inhibitors of differentiation major late promoter electrophoretic mobility shift assay glutathioneS-transferase actinomycin D constitutively active Like other members of the TGF-β superfamily, BMPs exert their effects through distinct combinations of two different types of serine/threonine kinase receptors, i.e. type I receptors (also termed activin receptor-like kinases, or ALKs) and type II receptors (10Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (505) Google Scholar, 11Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar). For BMPs, three distinct type II receptors for BMP,i.e. BMP type II receptor (BMPR-II) and activin type II receptors (ActR-II and ActR-IIB), and three distinct type I receptor,i.e. BMPR-IA, BMPR-IB, and ALK-2, have been identified. The mechanism of receptor activation has been best characterized for TGF-β but is very likely to occur in an analogous fashion for BMPs (10Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (505) Google Scholar, 11Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar). Receptor activation involves BMP-induced hetero-oligomerization of two sequentially acting kinases, with the type I receptor acting as a substrate for the type II receptor kinase. This is consistent with the notion that the type I receptor has been shown to confer signaling specificity to the type I-type II heteromeric complex (10Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (505) Google Scholar, 11Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar). Activated type I receptors initiate intracellular signaling through phosphorylating specific Smad proteins (12Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 13Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar, 14Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). Whereas Smad2 and Smad3 are phosphorylated by TGF-β and activin receptors, Smad1, Smad5, and Smad8 are phosphorylated by BMP type I receptors. Based upon their functional properties, Smads can be divided into three distinct subclasses: they are (i) receptor-regulated Smads (R-Smads), which transiently interact with activated type I receptors and become phosphorylated on two serine residues in their carboxyl-terminal SSXS motif, (ii) common partner Smads (Co-Smads), which assemble into heteromeric complexes with R-Smads that accumulate into the nucleus, where these complexes regulate the transcription of target genes, and (iii) inhibitory Smads (i.e. Smad6 and Smad7), which potently interfere with TGF-β/Smad signaling by competing with R-Smads for interaction with type I receptors or by inducing receptor degradation (12Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 13Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar, 14Massagué J. Wotton D. 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Smad3 and Smad4 were found to preferentially recognize, via their conserved amino-terminal regions known as MH1 domains, the 5′-GTCT-3′ sequence (also termed the Smad binding element (SBE)) (17Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1566) Google Scholar, 18Shi Y. Wang Y.F. Jayaraman L. Yang H. Massagué J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 19Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar). Multimers of SBE, when placed in front of a minimal promoter, can provide a strong enhancer function for TGF-β. SBE-like sequences are critically important for TGF-β-induced activation of multiple TGF-β-responsive genes (12Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 13Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar, 14Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). BMP R-Smads have been shown to weakly bind GCAT motifs or GCCG containing sequences found in promoters of the BMP target genesXVent2B and Smad6, respectively (20Henningfeld K.A. Rastegar S. Adler G. Knochel W. J. Biol. Chem. 2000; 275: 21827-21835Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Ishida W. Hamamoto T. Kusanagi K. Yagi K. Kawabata M. Takehara K. Sampath T.K. Kato M. Miyazono K. J. Biol. Chem. 2000; 275: 6075-6079Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Mutation of these sequences decreased BMP-induced promoter activation. However, BMP inducibility of reporter constructs containing multimerized GC-rich sequences is very low and requires R-Smad overexpression (22Kusanagi K. Inoue H. Ishidou Y. Mishima H.K. Kawabata M. Miyazono K. Mol. Biol. Cell. 2000; 11: 555-565Crossref PubMed Scopus (154) Google Scholar, 23Yoshida Y. Tanaka S. Umemori H. Minowa O. Usui M. Ikematsu N. Hosoda E. Imamura T. Kuno J. Yamashita T. Miyazono K. Noda M. Noda T. Yamamoto T. Cell. 2000; 103: 1085-1097Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Therefore, the mechanism on how the BMP/Smad pathway specifically activates target genes is still incompletely understood. The affinity of Smads for DNA is weak (18Shi Y. Wang Y.F. Jayaraman L. Yang H. Massagué J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Smads need thus to co-operate with other DNA binding factors to bind efficiently to promoters of target genes. The 30-zinc finger nuclear protein OAZ associates with BMP R-Smads in response to BMP (24Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massagué J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Expression of OAZ is cell type-specific and cannot be detected in mesenchymal cells. A member of the core binding factor (CBF) family of transcription factors, Cbfa1, also termed Runt-related gene 2 (RUNX2) was shown to interact directly with Smad1 and Smad5 (25Hanai J. Chen L.F. Kanno T. Ohtani-Fujita N. Kim W.Y. Guo W.H. Imamura T. Ishidou Y. Fukuchi M. Shi M.J. Stavnezer J. Kawabata M. Miyazono K. Ito Y. J. Biol. Chem. 1999; 274: 31577-31582Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 26Pardali E. Xie X.Q. Tsapogas P. Itoh S. Arvanitidis K. Heldin C.H. ten Dijke P. Grundstrom T. Sideras P. J. Biol. Chem. 2000; 275: 3552-3560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). RUNX2 precedes the appearance of osteoblasts, and mice deficient in RUNX2 lack osteoblasts, and bone ossification is completely blocked (27Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (697) Google Scholar, 28Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3569) Google Scholar). RUNX2 and Smads have been shown to co-operate in transcriptional responses and BMP-induced osteoblast differentiation (29Lee K.S. Kim H.J., Li, Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell. Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (748) Google Scholar, 30Zhang Y.W. Yasui N. Ito K. Huang G. Fujii M. Hanai J. Nogami H. Ochi T. Miyazono K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10549-10554Crossref PubMed Scopus (307) Google Scholar). Ectopic expression of RUNX2 in C2C12 cells was found to induce many of the extracellular matrix proteins induced by BMPs (29Lee K.S. Kim H.J., Li, Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell. Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (748) Google Scholar). However, RUNX2 alone is not sufficient to induce the whole onset of osteoblast differentiation without co-operation with Smad5 (29Lee K.S. Kim H.J., Li, Q.L. Chi X.Z. Ueta C. Komori T. Wozney J.M. Kim E.G. Choi J.Y. Ryoo H.M. Bae S.C. Mol. Cell. Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (748) Google Scholar). Thus, other BMP targets genes with critical roles in BMP-induced osteogenesis remain to be identified. Inhibitors of differentiation (Id) proteins act as dominant negative inhibitors of basic helix-loop-helix transcription factors, including members of the MyoD family of myogenic transcription factors, and also pocket proteins, including the retinoblastoma protein (31Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar). Id and basic helix-loop-helix proteins dictate in an opposite manner cellular programs of differentiation and proliferation in various cell types. Because BMPs can induce Id expression (32Hollnagel A. Oehlmann V. Heymer J. Ruther U. Nordheim A. J. Biol. Chem. 1999; 274: 19838-19845Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 33Ogata T. Wozney J.M. Benezra R. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9219-9222Crossref PubMed Scopus (134) Google Scholar) and ectopic expression of Id proteins can mimic certain BMP-induced responses, e.g.inhibiting myoblast differentiation (34Jen Y. Weintraub H. Benezra R. Genes Dev. 1992; 6: 1466-1479Crossref PubMed Scopus (393) Google Scholar,35Melnikova I.N. Christy B.A. Cell Growth Differ. 1996; 7: 1067-1079PubMed Google Scholar), 2O. Korchynskyi, unpublished results. 2O. Korchynskyi, unpublished results. Id proteins may serve an important effector function for BMPs. In the present paper we show that BMP specifically transcriptionally activates Id1 expression in C2C12 myoblasts and map two distinct BMP-specific responsive regions containing three pivotal distinct sequence motifs in the Id1 promoter. Expression plasmids for Smads and type I receptors have been previously described (36Nakao 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 (1534) Google Scholar). Mouse Id1 promoter fragment (−1575/+88) (37Tournay O. Benezra R. Mol. Cell. Biol. 1996; 16: 2418-2430Crossref PubMed Scopus (110) Google Scholar) and human Id1 promoter have been described before (38Nehlin J.O. Hara E. Kuo W.L. Collins C. Campisi J. Biochem. Biophys. Res. Commun. 1997; 231: 628-634Crossref PubMed Scopus (35) Google Scholar). Id1, Id2, and Id3 cDNAs were provided by the Dr. H. Weintraub laboratory. Mouse Id1 promoter 5′ deletion constructs were made by PCR approach using mouse Id1-(−1575/+88) fragment as a template. For precise mapping of the BMP-responsive element, Id1 promoter fragments were PCR-amplified using Id1-(−1231/+88) fragment as a template and subcloned between KpnI and XmaI sites upstream of minimal adenoviral major late promoter (MLP) reporter construct (17Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1566) Google Scholar). Site directed mutagenesis was performed using QuikChange site-directed mutagenesis kit (Stratagene) protocol. The wild-type GTCT sites were replaced by the mutated GagT, the wild-type CAGC sites were changed into the mutated Ctca sequence, the wild-type GCCG elements were replaced by GaCG-mutated sequence, and the wild type GGCGCC palindrome, GC′-1, and GC′-2 sequence elements were replaced with GaattC, tGCa, tcaC, respectively. All constructs were DNA sequence-verified. C2C12, HepG2, and MDA-MB468 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal calf serum (Sigma). Cells were grown in a 5% CO2-containing atmosphere at 37 °C. Recombinant BMPs were a gift from Dr. K. Sampath (Curis, Inc.), activin A was from Dr. Y. Eto (Ajinomoto Co.), and recombinant TGF-β3 was obtained from Dr. K. Iwata (OSI Pharmaceuticals). Cells were seeded at a density of 1.5 × 104 cells/cm2 in 12-well (C2C12 cells) or 6-well (MDA-MB468 cells) plates. The next day, cells were transiently transfected with the reporters in the absence or presence of expression plasmids using pcDNA4 plasmid to keep total amount of transfected DNA constant. Transfection was carried out using the calcium phosphate co-precipitation method (4 μg of total plasmid DNA/well) in the case of MDA-MB468 and HepG2 cells or FuGENE 6 (Roche Molecular Biochemicals) transfection reagent (500 ng of total plasmid DNA/well) in the case of C2C12 cells, following the manufacturer's protocol. β-Galactosidase co-transfection was used as an internal control for normalizing transfection efficiency. Luciferase and β-galactosidase activity were quantified using the luciferase assay (Promega) with Victor luminometer (Wallac) as described previously (39Jonk L.J. Itoh S. Heldin C.H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). RNA was isolated with using TriZol reagent (Invitrogen) in accordance with manufacturer protocol. 20 μg of total RNA/lane was loaded on denaturating (6% formaldehyde) 1% agarose gels. Blotting and hybridization using 32P-labeled (Amersham Biosciences, Inc.) probes were performed as described previously (36Nakao 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 (1534) Google Scholar). Synthetic oligonucleotides 5′CGCGG¯¯¯CGC¯C¯¯AGC¯CTGACAGC¯CCG3′(sense) and 5′AGGACGGGCTG¯TCAGGCTG¯GCGC3′(antisense), containing two CAGC sites (underlined), GGCGCC palindrome (overlined) and MluI 5′-overhang (double-underlined), on the sense strand and, correspondingly, 5′TCCTG¯GC¯G¯¯TCT¯AACGGTCT¯GAG3′(sense) and 5′CTAG¯¯CTCAGAC¯CGTTAGAC¯GCC3′(antisense) containing two SBE sites (underlined), one GGCG site (overlined), and NheI 5′-overhang (double-underlined), on the antisense strand were ligated and inserted into NheI sites in pGL3-MLP-luc minimal promoter vector. Thereby a construct was created with a 5′ site for NheI site followed by 2 SBEs, 1 GGCG site, 2 CAGC′ sites, 2 GGCGCC palindromes, 2 CAGC′ sites, 1 GGCG site, 2 SBEs, and an NheI 3′ site. The annealed oligonucleotides containing palindrome and two CAGC sites or two SBE sites were also used as specific competitors in electrophoretic mobility shift assays (EMSAs). To determine the functional importance of different elements that are present in BRE-Luc, the following four derivative constructs were made. (i) The CAGC-1,2- and GC′3,4-containing oligonucleotides were multimerized twice and subcloned into the SmaI site of MLP-Luc, (ii) SBE-2,3- and GC′-5-containing oligonucleotides were multimerized twice and inserted into the NheI site of MLP-Luc, and (iii) GC′-3,4-containing oligonucleotides and (iv) CAGC-1,2-containing oligonucleotides were multimerized twice, and both were subcloned into BglII site of 2× (SBE-2,3 and GC′-5) reporter construct. Western blotting was performed as described previously (36Nakao 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 (1534) Google Scholar). Polyclonal rabbit PS1 antibody that specifically recognizes phosphorylated Smad1 and/or Smad5 (used in a dilution 1:1000) has been previously described (40Persson U. Izumi H. Souchelnytskyi S. Itoh S. Grimsby S. Engstrom U. Heldin C.H. Funa K. ten Dijke P. FEBS Lett. 1998; 434: 83-87Crossref PubMed Scopus (335) Google Scholar). Antibody against Id1 (diluted in assay 1:1000) was from Santa Cruz Biotechnology. Secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Amersham Biosciences, Inc.) was used in a 104-fold dilution. Detection was performed by enhanced chemiluminescence (ECL, Amersham Biosciences, Inc.). Nuclear extracts from untreated C2C12 cells and C2C12 cells stimulated with BMP-6 (100 ng/ml) or TGF-β3 (5 ng/ml) were prepared according to Schreiber et al. (41Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). GST-Smad fusion proteins were prepared as described before (17Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1566) Google Scholar). The binding reaction containing 20 pm32P-end-labeled oligonucleotides and appropriate nuclear extracts (15 μg of protein/sample) or GST-Smad proteins (500 μg of purified protein/sample) were performed as described in Dennleret al. (17Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1566) Google Scholar). A ×100, ×250, or ×500 molar excess of unlabeled oligonucleotides was used as a competitor where indicated. One μg of purified anti-Smad5 (42Tamaki K. Souchelnytskyi S. Itoh S. Nakao A. Sampath K. Heldin C.H. ten Dijke P. J. Cell. Physiol. 1998; 177: 355-363Crossref PubMed Scopus (72) Google Scholar) and anti-Smad2 and three μg of anti-Smad4 (43Nakao 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 (891) Google Scholar) rabbit polyclonal antibodies were used for supershift experiments. Previous studies show that BMPs, but not TGF-β, potently induce the expression of Id proteins (32Hollnagel A. Oehlmann V. Heymer J. Ruther U. Nordheim A. J. Biol. Chem. 1999; 274: 19838-19845Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 33Ogata T. Wozney J.M. Benezra R. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9219-9222Crossref PubMed Scopus (134) Google Scholar, 44Brennan T.J. Edmondson D.G., Li, L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3822-3826Crossref PubMed Scopus (111) Google Scholar), which are inhibitors of basic helix-loop-helix proteins (31Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar). Because we wanted to obtain new insights into the molecular mechanism of how certain genes can be selectively induced by BMP, but not by TGF-β, the Id genes were selected for further study. We found that BMP4 and BMP7 potently induce mRNA levels of Id1, Id2, and Id3 in C2C12 cells (Fig. 1 A). Induction of Id expression by BMPs peaks around 1–2 h. Among the three genes analyzed, Id3 was found to be most stably induced within the 1–24-h time period, and Id1 was found to be most rapidly induced upon BMP challenge (Fig. 1 A); Id1, likely to be an immediate early BMP response gene, was therefore chosen for further analysis. Induction of Id1 by BMP6 was indeed observed in the presence of cycloheximide (CHI), indicating that de novo protein synthesis is not required for this response (Fig. 1 B). Id1 is thus a direct BMP target gene. Id1 mRNA was actually more induced in the presence of BMP6 and CHI (Fig. 1 B), probably as a result of an increase in mRNA stability or loss of transcriptional repressors by CHI. The addition of actinomycin D (ActD), an inhibitor of transcription, inhibited the BMP-induced effect on Id1 expression (Fig. 1 B), indicating that the up-regulation of the Id1 protein is transcriptionally dependent. BMP6 stimulation also resulted in an up-regulation of the Id1 protein in C2C12 cells. This effect was blocked by ActD (and CHI) (Fig.1 C), indicating that in these cells BMP does not increase the Id1 expression by increasing the stability of the Id1 protein. When we transfected the mouse or human Id1-Luc reporter constructs in C2C12 cells and stimulated the cells with BMP or TGF-β, we found that only BMPs activated these Id1 reporters (Fig.2, A and B). On another transcriptional reporter, TGF-β activated a transcriptional response, indicating that C2C12 cells are TGF-β-responsive, as previously reported (5Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). Consistent with these findings, BMP-induced activation of reporter is potently inhibited by dominant negative BMP type I receptors and stimulated by constitutively active (ca) BMP type I receptors, i.e. caALK2, caALK3, and caALK6 but not caALK4 and caALK5 (which are type I receptors for TGF-β and activin, respectively) (Fig. 2 C and data not shown). To examine whether Smad proteins are involved in the BMP6-induced transcriptional activation of Id1-Luc reporter, we co-transfected C2C12 cells with the mouse Id1-Luc reporter and expression constructs for R-Smads,i.e. Smad1 or Smad5 or inhibitory Smads, i.e.Smad6 or Smad7. We found that Smad1 or Smad5 strongly promoted both basal and BMP-induced activation of this reporter (Fig. 2 D) and that both I-Smads strongly inhibited both basal and BMP-induced luciferase levels (Fig. 2 E). These findings suggest an involvement of Smad proteins in BMP-induced activation of Id1-Luc reporter. The observation that basal levels are reduced by I-Smads suggests that there is autocrine BMP signaling in C2C12 cells. To show that Smad4 is required for the BMP6-induced activation of Id1-Luc reporter, we used MDA-MB468 cells. MDA-MB468 cells are human epithelial cells deficient in Smad4. In these cells, BMP6 has no effect on Id1-Luc reporter activity (Fig. 2 F). However, co-transfection of an expression construct encoding Smad4 restored the BMP6 activation of Id1-Luc reporter, demonstrating that Smad4 is necessary for the BMP6 transcriptional effect. This BMP6-induced activation of Id1-Luc reporter can be further enhanced by co-expressing Smad4 with Smad1 or Smad5. These results suggest that the Co-Smad4 and R-Smads (i.e. Smad1 and Smad5) co-operate in BMP6-induced activation of Id1-Luc reporter. By performing sequential deletion analysis of mouse Id1 promoter (Fig. 3 A), we found that Id1 promoter fragment −1231 to +88 (Id-(−1231/+88)) had similar activity as Id-(−1575/+88). The activity was completely lost in Id1-(−1070/+88) reporter construct. Subsequently, we sub-cloned Id1-(−1231/−996) fragment into a minimal promoter reporter construct and showed that it was highly sensitive to BMP when transfected into C2C12 cells (Fig. 3 B). Upon subdividing this fragment into two smaller fragments Id-(−1231/−1071) and Id(−1070/−996), we (nearly) lost completely the BMP responsiveness. In addition, Id1-(−1231 to −1055) reporter construct shows only little BMP responsiveness, which was regained when we extended this fragment to −1025. The minimal region, which remained BMP responsive, was Id1-(−1133/−1025) fragment (Fig. 3 B). Interestingly, when we compared the mouse Id1 with the human Id1 promoter sequences, which are both strongly acti