OAZ Regulates Bone Morphogenetic Protein Signaling through Smad6 Activation
2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês
10.1074/jbc.m510004200
ISSN1083-351X
AutoresManching Ku, Shavonne Howard, Weihua Ni, Giorgio Lagna, Akiko Hata,
Tópico(s)Kruppel-like factors research
ResumoThe intensity and duration of activation of a signal transduction system are important determinants of the specificity of the cellular response to the stimulus. It is unclear how different cells can generate a signal of varying intensity and duration in response to the same cytokine. We investigated the role of the transcriptional activator and Smad1/4 cofactor OAZ in regulating bone morphogenetic protein (BMP) signaling. We demonstrate that upon BMP4 stimulation, an OAZ-Smad1/4 complex binds to and activates the gene encoding Smad6, a specific inhibitor of the BMP pathway. Removal of endogenous OAZ from pluripotent embryonal carcinoma cells prevents the induction of Smad6 by BMP4 and extends the period of detection of phosphorylated Smad1 after BMP stimulation. Conversely, in cells that do not normally express OAZ, such as myoblasts and smooth muscle cells, forced OAZ expression leads to faster and higher Smad6 induction in response to BMP4, decrease of Smad1 phosphorylation, and attenuation of BMP-mediated responses. Our results demonstrate that OAZ can alter the intensity and duration of the BMP stimulus through Smad6 and indicate that the tissue-specific expression of OAZ is a critical determinant of the cellular response to the BMP signal. The intensity and duration of activation of a signal transduction system are important determinants of the specificity of the cellular response to the stimulus. It is unclear how different cells can generate a signal of varying intensity and duration in response to the same cytokine. We investigated the role of the transcriptional activator and Smad1/4 cofactor OAZ in regulating bone morphogenetic protein (BMP) signaling. We demonstrate that upon BMP4 stimulation, an OAZ-Smad1/4 complex binds to and activates the gene encoding Smad6, a specific inhibitor of the BMP pathway. Removal of endogenous OAZ from pluripotent embryonal carcinoma cells prevents the induction of Smad6 by BMP4 and extends the period of detection of phosphorylated Smad1 after BMP stimulation. Conversely, in cells that do not normally express OAZ, such as myoblasts and smooth muscle cells, forced OAZ expression leads to faster and higher Smad6 induction in response to BMP4, decrease of Smad1 phosphorylation, and attenuation of BMP-mediated responses. Our results demonstrate that OAZ can alter the intensity and duration of the BMP stimulus through Smad6 and indicate that the tissue-specific expression of OAZ is a critical determinant of the cellular response to the BMP signal. Growth factors of the transforming growth factor β (TGFβ) 2The abbreviations used are: TGFβ, transforming growth factor β; ALP, alkaline phosphatase; bHLH, basic helix-loop-helix; BMP, bone morphogenetic protein; BRE, BMP response element; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; Luc, luciferase; OBS, OAZ binding sequence; PASMCs, primary pulmonary smooth muscle cells; PPH, primary pulmonary hypertension; RFP, red fluorescence protein; R-Smad, receptor-regulated Smad protein; RT, reverse transcription; SBE, Smad-binding element; siRNA, small interference RNA; Smurf, Smad ubiquitination regulatory factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. family are secreted homodimeric proteins that regulate cell growth and differentiation in all metazoan organisms. Perturbations of signals evoked by TGFβs and transduced by Smad proteins have been implicated in several developmental disorders and in various human diseases, including cancer, fibrosis, autoimmunity, and pulmonary hypertension. Such diversity of effects and responses is caused in large part by the cell type specificity of the TGFβ signal, the local concentration of ligand, and the activity of other signal transduction pathways. Recent studies have provided insights into several of the determinants of specificity in the Smad signaling pathways, including combinatorial ligand-receptor associations, selective interactions between Smads and other pathway components that are mediated through defined binding motifs, and the regulation of duration and intensity of signaling. Typically, signaling initiates with TGFβs binding and activating specific dual cell surface receptors (type I and type II receptors) that have intrinsic serine/threonine kinase activity. Activated receptors phosphorylate receptor-regulated Smad proteins (R-Smads). Among the nine mammalian R-Smads, Smad1, Smad5, and Smad8 are phosphorylated by type I receptors specific for BMPs, whereas Smad2 and Smad3 are activated by TGFβs, activins, or Nodals. Upon phosphorylation, R-Smads form a complex with Smad4 (known as common partner of R-Smads or Co-Smad) and accumulate in the nucleus. Here the R-Smads·Co-Smad complex can bind DNA or be recruited by interactions with transcription factors to specific promoter elements of target genes. Low affinity direct binding can occur to the palindromic sequence GTCTAGAC, known as the Smad-binding element (SBE) (1Zawel 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 (892) Google Scholar). Several TGFβ-regulated promoters (e.g. plasminogen activator inhibitor-1, collagenase I, c-jun, IgA, and junB) contain one or multiple copies of the SBE half-site (5′-AGAC-3′), which has been shown to bind the Smad3-Smad4 complex. Activation of other promoters requires the cooperation of sequence-specific cofactors including FoxH1 family member (FAST1 and FAST2), Mixer/Milk, c-Jun/c-Fos, PEBP2/Runx, and TFII-I family members (TFII-I and BEN) (2Ku M.C. Sokol S.Y. Wu J. Tussie-Luna M.I. Roy A.L. Hata A. Mol. Cell. Biol. 2005; 25: 7144-7157Crossref PubMed Scopus (38) Google Scholar, 3Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1759Crossref PubMed Google Scholar). The interaction between Smads and cofactors provides Smads with increased sequence specificity and higher affinity to DNA. Because these cofactors are often expressed in a tissue-specific manner, specific Smads-cofactor complex formation is recognized as one of the determinants of specificity of the TGFβ signaling pathway (3Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1759Crossref PubMed Google Scholar, 4Attisano L. Tuen Lee-Hoeflich S. Genome Biol. 2001; 2Crossref PubMed Google Scholar). The first identified Smad-interacting transcription cofactor of the BMP signaling pathway was Xenopus OAZ (also known as EBFAZ, ROAZ, or ZNF423). OAZ is composed of 30 Krüppel-like zinc fingers; it can interact with the Smad1-Smad4 complex after BMP stimulation, bind to the promoter region of the Xenopus homeobox gene Vent-2, and activate its transcription. Two different sets of OAZ zinc fingers are involved in the interaction with Smads and in binding to DNA, and no intrinsic transcriptional stimulatory activity has been found (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Poly-(ADP-ribose) polymerase 1 (Parp1) was identified as a protein partner of OAZ by a yeast two-hybrid screen. The enzymatic activity of Parp1 is essential for BMP-mediated activation of the Xvent-2 promoter (6Ku M.C. Stewart S. Hata A. Biochem. Biophys. Res. Commun. 2003; 311: 702-707Crossref PubMed Scopus (25) Google Scholar), presumably because the poly(ADP-ribosylation) of OAZ, Smads, or other proteins facilitates the recruitment of cofactors or of the basal transcription machinery. XVent-2 is a transcriptional repressor that carries out several of the embryological effects of BMPs in Xenopus, such as ventralization of mesoderm and suppression of neuralization (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 7Onichtchouk D. Gawantka V. Dosch R. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. Development. 1996; 122: 3045-3053Crossref PubMed Google Scholar, 8Onichtchouk D. Glinka A. Niehrs C. Development. 1998; 125: 1447-1456Crossref PubMed Google Scholar). The BMP response element (BRE) in the Xvent-2 promoter includes one SBE and one OAZ binding sequence (OBS; GCTCCA) (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). The essential role of OAZ and OBS in the regulation of Xvent-2 expression has been established by different approaches in Xenopus: 1) expression of a dominant-negative mutant form of OAZ into the ventral side of embryos leads to inhibition of Xvent-1 and Xvent-2 transcription and dorsalization of mesoderm (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar); and 2) in transgenic Xenopus embryos expressing the green fluorescence protein reporter gene under the regulation of the Xvent-2 promoter, mutation of the OBS in the Xvent-2 promoter leads to misexpression of green fluorescence protein (9Karaulanov E. Knochel W. Niehrs C. EMBO J. 2004; 23: 844-856Crossref PubMed Scopus (116) Google Scholar). The human and mouse orthologs of OAZ are expressed in embryonic tissues responsive to BMP, suggesting that OAZ might be involved in BMP responses during early embryogenesis in mammals. However, a mammalian gene regulated by BMP through OAZ has not been identified. Recently, a protein structurally similar to OAZ, named Early Hematopoietic Zinc Finger protein (EHZF) (also known as Evi3 or ZNF521), has been identified (10Bond H.M. Mesuraca M. Carbone E. Bonelli P. Agosti V. Amodio N. De Rosa G. Di Nicola M. Gianni A.M. Moore M.A. Hata A. Grieco M. Morrone G. Venuta S. Blood. 2004; 103: 2062-2070Crossref PubMed Scopus (79) Google Scholar, 11Hentges K.E. Weiser K.C. Schountz T. Woodward L.S. Morse H.C. Justice M.J. Oncogene. 2005; 24: 1220-1230Crossref PubMed Scopus (28) Google Scholar, 12Warming S. Suzuki T. Yamaguchi T.P. Jenkins N.A. Copeland N.G. Oncogene. 2004; 23: 2727-2731Crossref PubMed Scopus (23) Google Scholar). Both OAZ and EHZF contain 30 Krüppel-like zinc fingers and have an overall homology of 63.5% (10Bond H.M. Mesuraca M. Carbone E. Bonelli P. Agosti V. Amodio N. De Rosa G. Di Nicola M. Gianni A.M. Moore M.A. Hata A. Grieco M. Morrone G. Venuta S. Blood. 2004; 103: 2062-2070Crossref PubMed Scopus (79) Google Scholar). The homology is lower (50% on average) in the region known to interact with the BRE of the Xvent-2 promoter (zinc fingers 9-13), as well as in some of the zinc fingers responsible for the interaction with Smads (zinc fingers 14-19) (10Bond H.M. Mesuraca M. Carbone E. Bonelli P. Agosti V. Amodio N. De Rosa G. Di Nicola M. Gianni A.M. Moore M.A. Hata A. Grieco M. Morrone G. Venuta S. Blood. 2004; 103: 2062-2070Crossref PubMed Scopus (79) Google Scholar). Furthermore, unlike OAZ, EHZF is expressed highly in hematopoietic cells (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 10Bond H.M. Mesuraca M. Carbone E. Bonelli P. Agosti V. Amodio N. De Rosa G. Di Nicola M. Gianni A.M. Moore M.A. Hata A. Grieco M. Morrone G. Venuta S. Blood. 2004; 103: 2062-2070Crossref PubMed Scopus (79) Google Scholar, 11Hentges K.E. Weiser K.C. Schountz T. Woodward L.S. Morse H.C. Justice M.J. Oncogene. 2005; 24: 1220-1230Crossref PubMed Scopus (28) Google Scholar, 12Warming S. Suzuki T. Yamaguchi T.P. Jenkins N.A. Copeland N.G. Oncogene. 2004; 23: 2727-2731Crossref PubMed Scopus (23) Google Scholar). Therefore, it has been speculated that OAZ and EHZF might recognize distinct arrays of molecular partners and regulate distinct sets of target genes. The mouse Smad6 gene was characterized as a target gene of the BMP signaling pathway (13Ishida 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 (232) Google Scholar, 14Takase M. Imamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Kawabata M. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (135) Google Scholar). With Smad7, Smad6 belongs to a distinct subclass of Smads (Inhibitory Smads or I-Smads) that antagonize R-Smads and inhibit signaling. Smad7 acts as a general inhibitor of TGFβ signaling by interacting with activated type I receptors and preventing their phosphorylation of R-Smad (4Attisano L. Tuen Lee-Hoeflich S. Genome Biol. 2001; 2Crossref PubMed Google Scholar, 15Attisano L. Wrana J.L. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1138) Google Scholar, 16Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar, 17Miyazono K. ten Dijke P. Heldin C.H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar, 18Zhang Y. Derynck R. Trends Cell Biol. 1999; 9: 274-279Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Smad7 can also recruit to the activated type I receptor a family of E3 ubiquitin ligases known as Smad ubiquitination regulatory factor (Smurf)-1 and Smurf-2, resulting in receptor ubiquitination and degradation and subsequent termination of signaling. Unlike Smad7, Smad6 acts as a specific inhibitor of BMP signaling by competing with Smad4 for complex formation with Smad1 (and presumably Smad5 and Smad8) (19Hata A. Lagna G. Massagué J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (587) Google Scholar, 20Horiki M. Imamura T. Okamoto M. Hayashi M. Murai J. Myoui A. Ochi T. Miyazono K. Yoshikawa H. Tsumaki N. J. Cell Biol. 2004; 165: 433-445Crossref PubMed Scopus (95) Google Scholar, 21Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (873) Google Scholar). When overexpressed, Smad6 can also associate with type I receptors and inhibit Smad phosphorylation. In this report, we demonstrate that the BMP-mediated transcriptional activation of Smad6 requires OAZ as a cofactor. Down-regulation of OAZ in OAZ-positive embryonic carcinoma P19 cells leads to prolonged duration of the BMP signal because of a lack of Smad6 induction. Furthermore, misexpression of OAZ in OAZ-negative C2C12 myoblasts and primary pulmonary smooth muscle cells leads to early attenuation of the BMP signal and inhibition of BMP-dependent responses, such as osteoblastic differentiation and apoptosis, respectively. Cell Culture and DNA Transfection—Both P19 and C2C12 cell lines (American Type Culture Correction) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FCS, Hyclone). C2C12 and P19 cells were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Human primary pulmonary smooth muscle cells (PASMCs) were purchased from Cambrex and were maintained in Sm-GM2 medium (Cambrex) containing 10% FCS. PASMCs were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Antibodies—BMP receptor-phosphorylated Smad1 was detected with anti-phospho-Smad1/5/8 rabbit monoclonal antibody (41D10, Cell Signaling Technology). OAZ was detected with goat anti-OAZ polyclonal antibodies (E-20, Santa Cruz Biotechnology). Smad1 and Smad6 were detected with rabbit polyclonal antisera raised against synthetic peptides (Upstate Biotechnology and Zymed Laboratories Inc.). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected with mouse anti-GAPDH monoclonal antibody (clone 1D4, COVANCE). Plasmid Constructs—A reporter containing three copies of the BRE of the mouse Smad6 promoter was generated by inserting the oligonucleotides containing the wild type or mutated BRE sequence into the pE1b-Luc vector (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). The wild type and mutant BRE sequences are: 5′-CGGGCCGCGCCGGCTCCAGGGCAGGAGCGGCCTTAA-3′ (WT), 5′-CGGTCCGCTCCGATCCAGGGCAGGAGCGGCCTTAA-3′ (MUT1), and 5′-CGGGCCGCGCCGAATCCAGGGCAGGAGCGGCCTTAA-3′(MUT2). Reverse Transcription (RT)-PCR Assay—Cells were treated with 200 ng/ml BMP4 (R&D Systems) in 0.2% FCS and DMEM. Total RNA was extracted by TRIzol (Invitrogen), and 5 μg of total RNA was subjected to RT using a first-strand cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. One-tenth of the reaction mixture was used as template for each PCR. The products of semiquantitative PCR were separated on a SDS-PAGE. The quantitative analysis of the change in expression levels was calculated by real time PCR (Bio-Rad) following the manufacturer's protocol. Primers used are: 5′-CCACTTGGAGACTTCTTCTTCTTCG-3′ and 5′-ATGTAGGGCAGCATCTGGTGCG-3′ for mouse Smad6, 5′-GTCGACCGAGGAGCCTCTTA-3′ and 5′-AAGCTCCTCTTGTCCTTGGAG-3′ for mouse Id3, and 5′-CACCCGCAGCTGTCCGAGAAGGC-3′ and 5′-CTCCACAGAGGCGCTGGAGTCGGG-3′ for mouse OAZ. Human GAPDH primers and mouse hypoxanthine-guanines phosphoribosyltransferase (HPRT) primers were described before (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 22Zhang S. Fantozzi I. Tigno D.D. Yi E.S. Platoshyn O. Thistlethwaite P.A. Kriett J.M. Yung G. Rubin L.J. Yuan J.X. Am. J. Physiol. 2003; 285: L740-L754Crossref PubMed Scopus (212) Google Scholar). RNA Interference—Synthetic small interference RNA (siRNA) was purified and annealed according to the manufacturer's instructions (Dharmacon). The siRNA sequence targeting OAZ corresponds to nucleotides 17-37 after the start codon. siRNA with a nontargeting sequence (Dharmacon) was used as a negative control. The siRNAs were transfected by OligofectAMINE (Invitrogen) according to the manufacturer's instructions. 48 h after transfection, cells were treated with BMP4 and harvested. Transfection efficiencies as determined by fluorescent-labeled siRNA (Dharmacon) were in the range of 80-90%. Specific silencing of OAZ was confirmed by Western blotting using anti-Smad1 (Zymed Laboratories Inc.), anti-lamin A, and anti-vimentin antibodies as described previously (23Elbashir S.M. Harborth J. Weber K. Tuschi T. Methods. 2002; 2: 199-213Crossref Scopus (1031) Google Scholar). Luciferase Assay—After transfection, the cells were reseeded onto 12-well plates and treated with 200 ng/ml BMP4 for 20 h in 0.2% FCS and DMEM. Luciferase assays were carried out essentially as described previously (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Immunoblot Assay—Cells were treated with 200 ng/ml BMP4 and lysed in TNE buffer (1% Nonidet P-40, 10 mm Tris-HCl (pH 7.5), 1 mm EDTA, 150 mm NaCl). Proteins were separated on a SDS-PAGE, transferred to polyvinylidene difluoride membranes, immunoblotted with antibodies, and visualized using an enhanced chemiluminescence detection system (Amersham Biosciences). Constructions of Recombinant Adenoviruses—Recombinant adenoviruses were constructed as described previously (24He T.-C. Zhou S. Da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). Briefly, FLAG-epitope tagged OAZ and Smad6 cDNAs were subcloned into the pShuttle-CMV vector. The recombinant adenoviruses generated by homologous recombination were isolated, and high titer stocks of recombinant viruses were grown in 293 cells and purified. Infection of recombinant adenoviruses was performed at a multiplicity of infection of <8 × 102 plaque-forming units/cell. As a control, adenovirus driving β-galactosidase (LacZ) expression was used (Vector Biolabs, Inc.) Chromatin Immunoprecipitation Assay—The chromatin immunoprecipitation assay was performed as described previously (25Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar, 26Seki K. Hata A. J. Biol. Chem. 2004; 279: 18544-18549Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Chromatin was sheared to an average length of 400 bp. We used a set of PCR primers for the detection of the mSmad6 BRE (-901/-832) and a control set corresponding to the 5′-upstream region of mSmad6 (-2031/-1853). Alkaline Phosphatase Assay—Histochemical analysis of alkaline phosphatase (ALP) activity was performed in 12-well plates as described previously (27Katagiri 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 (1302) Google Scholar, 28Fujii M. Takeda K. Imamura T. Aoki H. Smapath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (370) Google Scholar). After histochemical ALP analysis by phase contrast microscopy, the ALP activity was quantified by measuring the absorbance with the image documentation system Imagestore 7500 (Packard). Apoptosis Assay—Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed following manufacturer's instructions. Briefly, PASMCs were cultured in serum-free DMEM for 48 h, followed by stimulation with 200 ng/ml BMP4 for 48 h. Cells were then subjected to TUNEL assay using a Cell Death Detection kit (Roche Applied Science). Statistical Analysis—Statistical significance was determined by analysis of variance and Fisher's least significant difference, or by Student's t test analysis (p < 0.05) as appropriate. All data are plotted as the mean ± S.E. A Putative OAZ Binding Sequence Is Conserved in the Mouse and Human Smad6 Promoters—Smad6 is a known direct BMP target gene in vertebrates. The BMP receptor-specific Smads, Smad1 and Smad5, as a complex with Smad4 (13Ishida 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 (232) Google Scholar, 14Takase M. Imamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Kawabata M. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (135) Google Scholar), induce Smad6 expression. To identify a conserved region of the Smad6 promoter potentially involved in BMP-mediated transcriptional regulation, we aligned the mouse Smad6 promoter with the promoter of its human ortholog (Fig. 1A). A 57-bp region (-891/-834) of the mouse Smad6 promoter is highly conserved in the human Smad6 promoter (-1062/-1003) (Fig. 1A, sequence highlighted). A 28-bp sequence (-874/-847) within this region (Fig. 1A, underlined) has been identified previously as a region critical for BMP-mediated activation of the mouse Smad6 promoter and has been named BMP response element (13Ishida 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 (232) Google Scholar). We found two head-to-head repeats of a putative OBS, which was identified previously in the Xenopus BMP target genes Vent-2 and Vent-1B, in both the mouse and human Smad6 promoter (Fig. 1A, shown with arrows, and Fig. 1B, black boxes) (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). This observation suggests that OAZ might be involved in the transcriptional activation of Smad6 by the BMP signaling pathway. Furthermore, two sequences resembling the BREs found in the Xvent genes, comprising an OBS-like sequence adjacent to an SBE, were recently found in the Xenopus Id3 promoter by genome-wide in silico sequence analysis (Fig. 1B) (9Karaulanov E. Knochel W. Niehrs C. EMBO J. 2004; 23: 844-856Crossref PubMed Scopus (116) Google Scholar, 29von Bubnoff A. Peiffer D.A. Blitz I.L. Hayata T. Ogata S. Zeng Q. Trunnell M. Cho K.W.Y. Dev. Biol. 2005; 281: 210-226Crossref PubMed Scopus (51) Google Scholar). Id3 is transcriptionally regulated by the BMP signaling pathway both in Xenopus embryos and P19 cells (29von Bubnoff A. Peiffer D.A. Blitz I.L. Hayata T. Ogata S. Zeng Q. Trunnell M. Cho K.W.Y. Dev. Biol. 2005; 281: 210-226Crossref PubMed Scopus (51) Google Scholar). 3M. Ku and A. Hata, unpublished observation. Therefore, it is possible that Id3 might be regulated by BMP through the BRE in an OAZ-dependent manner. The Putative OAZ Binding Sequence Is Essential for Activation of Smad6 by BMP4—To test the functional significance of a putative OBS in the Smad6 promoter, we transfected a luciferase reporter construct containing three copies of the wild type Smad6 BRE sequence (-874/-847) from the mSmad6 promoter together with increasing amounts of an OAZ expression plasmid into the mouse embryonic carcinoma cell line P19 (Fig. 2A). P19 cells express all known BMP signaling molecules including OAZ (5Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massague J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Overexpression of OAZ weakly augmented the basal activity of the reporter, but the induction by BMP4 stimulation was strongly increased by OAZ in a dose-dependent manner (Fig. 2A, WT). In agreement with previous observations (13Ishida 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 (232) Google Scholar), the reporter completely lost its responsiveness to BMP4 when both the upstream OBS and the 5′-GC-rich sequence of the BRE were mutated (Fig. 2A, MUT1) (13Ishida 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 (232) Google Scholar). Overexpression of OAZ did not alter its activity (Fig. 2A, MUT1). Similar results were obtained when mutations were introduced only in the upstream OBS (Fig. 2A, MUT2). Mutations at the 3′-end of the BRE did not alter its response to BMP4 and exogenous OAZ (Fig. 2A, MUT3) (13Ishida 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 (232) Google Scholar). These results demonstrate that the upstream OBS in the SBE sequence is necessary for the BMP-OAZ-mediated activation of Smad6. We did not investigate the potential function of the downstream, inverted OBS; however, our results indicate that it is not sufficient to mediate OAZ or BMP activation of the reporter construct (Fig. 2A, MUT2). Recruitment of OAZ to OBS in Response to BMP4—Recruitment of OAZ to the BRE of the Smad6 promoter was confirmed in vivo by chromatin immunoprecipitation assay. Soluble chromatin was prepared after formaldehyde treatment of cells treated with BMP4 for 2 h. Antibodies against Smad1, Smad4, and OAZ were used to immunoprecipitate Smad1-, Smad4-, or OAZ-bound genomic DNA fragments. These genomic regions were analyzed by PCR using specific pairs of primers spanning the BRE of Smad6 (Fig. 2B, BRE). The negative control was provided by PCR primers corresponding to a 5′-upstream region of the BRE (-2031/-1853) which is not involved in the BMP-dependent regulation of mSmad6 and has no sequence homology with human Smad6 (Fig. 2B, control). No PCR signal was detected in BRE or control primers samples in the absence of BMP stimulation. However, in BMP4-treated cells, Smad1, Smad4, and OAZ were recruited to the BRE, but not to the 5′-control region (Fig. 2B). DNA fragments immunoprecipitated by nonspecific IgGs and amplified with the BRE primers yielded no signal (Fig. 2B), suggesting that recruitment of Smad1, Smad4, and OAZ to the BRE is specific. These results demonstrate that activation of Smad6 by BMP4 correlates with the recruitment of Smads and OAZ to the BRE. Thus, Smad6 is the first mammalian gene regulated directly by OAZ. OAZ Is Essential for the Activation of the Smad6 Gene in P19 Cells—To test whether OAZ is necessary for Smad6 induction by BMP4 in vivo, we decreased the expression of endogenous OAZ in P19 cells by RNA interference. A synthetic double-stranded siRNA complementary to the OAZ mRNA specifically down-regulated the expression of OAZ, but not of the control proteins Smad1, lamin A, and vimentin (Fig. 2C, left panel). We then measured by RT-PCR the mRNA level of Smad6 before and after BMP stimulation (Fig. 2C, right panel). In P19 cells treated with a control nontargeting siRNA (Fig. 2C, right panel, control), OAZ expression was unchanged, and Smad6 mRNA was strongly induced by BMP4 treatment. In OAZ siRNA-treated cells, OAZ expression was blocked, and Smad6 induction by BMP4 was abolished (Fig. 2C, right panel, OAZ siRNA). This result suggests that OAZ is essential for the activation of Smad6 by BMP4. Extended Duration of BMP Signal in P19 Cells by Down-regulation of OAZ—To assess the effect of OAZ-mediated regulation of Smad6 on the BMP signaling pathway, P19 cells were transiently transfected with control siRNA or OAZ-specific siRNA to down-regulate endogenous expression of OAZ, stimulated with BMP4, and subjected to anti-phospho-Smad1/5/8 Western blot. Cells treated with control siRNA showed a robust increase of phosphorylation of Smad1/5/8 between 30 min and 2 h after BMP treatment. However, the level of phospho-Smad1/5/8 was dramatically reduced after 4 h of treatment (Fig. 2D). The same membrane blotted with anti-Sma
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