FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2016228757> ?p ?o ?g. }

• W2016228757 endingPage "45772" @default.
• W2016228757 startingPage "45766" @default.
• W2016228757 abstract "Bone morphogenetic protein-2 (BMP-2) induces a switch in differentiation of mesenchymal cells from the myogenic to the osteogenic lineage. Here we describe that in C2C12 cells, BMP-2 decreases myogenin expression induced by des-(1,3) insulin-like growth factor-1 (des-(1,3)IGF-1) or ectopically expressed from a constitutive promoter, even in conditions where myogenin mRNA levels were unaffected. Addition of BMP-2 decreases myogenin protein half-life to 50%, whereas proteasome inhibitors abolish these effects. Forced expression of Id1, either by transient transfection or under the control of an inducible system, causes degradation of myogenin in the absence of BMP-2. In contrast, E47 overexpression blocks the inhibitory effect of BMP-2 on myogenin levels. Finally, expression of E47 in 293 cells stabilizes myogenin, an effect that is dependent on the heterodimerization mediated by their helix-loop-helix. Our findings indicate that induction of Id1 not only blocks transcriptional activity but also induces myogenin degradation by blocking formation of myogenin-E47 protein complexes. Bone morphogenetic protein-2 (BMP-2) induces a switch in differentiation of mesenchymal cells from the myogenic to the osteogenic lineage. Here we describe that in C2C12 cells, BMP-2 decreases myogenin expression induced by des-(1,3) insulin-like growth factor-1 (des-(1,3)IGF-1) or ectopically expressed from a constitutive promoter, even in conditions where myogenin mRNA levels were unaffected. Addition of BMP-2 decreases myogenin protein half-life to 50%, whereas proteasome inhibitors abolish these effects. Forced expression of Id1, either by transient transfection or under the control of an inducible system, causes degradation of myogenin in the absence of BMP-2. In contrast, E47 overexpression blocks the inhibitory effect of BMP-2 on myogenin levels. Finally, expression of E47 in 293 cells stabilizes myogenin, an effect that is dependent on the heterodimerization mediated by their helix-loop-helix. Our findings indicate that induction of Id1 not only blocks transcriptional activity but also induces myogenin degradation by blocking formation of myogenin-E47 protein complexes. Mesenchymal cells differentiate into distinct cell types, such as adipocytes, osteoblasts, or myoblasts. Differentiation has two stages; first is the commitment to a particular cell lineage, and secondly, cells start to express the proteins that characterize their final phenotype. Commitment to a specific lineage depends on mutually exclusive factors. Thus, signals that induce a particular phenotype repress others. For example, signals that induce the osteoblastic phenotype, such as BMPs (bone morphogenetic proteins), 1The abbreviations used are: BMP, bone morphogenetic protein; bHLH, basic helix-loop-helix; MRF, muscle regulatory factor; IGF, insulin-like growth factor; LLnL, N-acetyl-Leu-Leu-norleucinal; DMEM, Dulbecco's modified Eagle's medium; tTa, tetracycline-regulated transactivator; PBS, phosphate-buffered saline; HEK, human embryonic kidney; ERK, extracellular signal-regulated kinase; E3, ubiquitin-protein isopeptide ligase. repress myogenic differentiation in vitro (1Yamaguchi A. Katagiri T. Ikeda T. Wozney J.M. Rosen V. Wang E.A. Kahn A.J. Suda T. Yoshiki S. J. Cell Biol. 1991; 113: 681-687Crossref PubMed Scopus (656) Google Scholar, 2Viñals F. Lopez-Rovira T. Rosa J.L. Ventura F. FEBS Lett. 2002; 510: 99-104Crossref PubMed Scopus (112) Google Scholar) and induce bone after implantation in muscle in vivo (3Urist M.R. DeLange R.J. Finerman G.A. Science. 1983; 220: 680-686Crossref PubMed Scopus (621) Google Scholar). The capacity of mesenchymal cells to differentiate into distinct cell types is a result of the expression and function of determination genes, which include distinct families of transcription factors known as master genes. Thus, the fate of the mesenchymal precursors depends on the expression of a specific combination of master genes: the myogenic basic helix-loop-helix (bHLH) family for myoblasts, the peroxisome proliferator-activated receptor-γ (PPAR-γ) and CAAAT/enhancer-binding protein (C/EBP) families for adipocytes, and core binding factor α-1 (Cbfa-1) and Osterix for osteoblasts (4Molkentin J.D. Olson E.N. Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (388) Google Scholar, 5Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1053) Google Scholar, 6Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2797) Google Scholar). The myogenic bHLH family, also known as muscle regulatory factors (MRFs), is formed by four members, MyoD, myogenin, Myf5, and MRF4 (4Molkentin J.D. Olson E.N. Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (388) Google Scholar, 7Parker M.H. Seale P. Rudnicki M.A. Nat. Rev. Genet. 2003; 4: 497-507Crossref PubMed Scopus (282) Google Scholar). These were identified by their ability to induce the differentiation of non-muscle cells into a muscle phenotype (8Davis R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2479) Google Scholar, 9Edmondson D.G. Olson E.N. Genes Dev. 1989; 3: 628-640Crossref PubMed Scopus (600) Google Scholar). Whereas MyoD and Myf5 are involved in the initial determination state, myogenin and MRF4 participate in terminal differentiation (7Parker M.H. Seale P. Rudnicki M.A. Nat. Rev. Genet. 2003; 4: 497-507Crossref PubMed Scopus (282) Google Scholar). The MRFs, which belong to the superfamily of bHLH transcription factors, contain a conserved basic DNA-binding domain and a helix-loop-helix motif, which is essential for dimerization. MRFs heterodimerize with the E family of ubiquitously expressed bHLH factors (10Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1303) Google Scholar, 11Hu J.S. Olson E.N. Kingston R.E. Mol. Cell. Biol. 1992; 12: 1031-1042Crossref PubMed Scopus (254) Google Scholar). Assembly of the heterodimer allows correct juxtaposition of the two basic regions, which leads to binding and activation of E-boxes (CANNTG), DNA motifs in the promoters of skeletal muscle-specific genes (12Ephrussi A. Church G.M. Tonegawa S. Gilbert W. Science. 1985; 227: 134-140Crossref PubMed Scopus (491) Google Scholar, 13Blackwell T.K. Weintraub H. Science. 1990; 250: 1104-1110Crossref PubMed Scopus (759) Google Scholar). Mesenchymal cells use redundant mechanisms to ensure that the muscle differentiation program is activated only at the appropriate moment. These mechanisms probably also control the timing of other mesenchymal differentiation programs with the concomitant block of muscle differentiation. The first checkpoint is the expression of the master genes for the myogenic program. Thus, myogenin is absent in proliferating myoblasts, and its expression is induced by factors that stimulate the myogenic program, such as the decrease in growth factors or presence of IGF-1 (9Edmondson D.G. Olson E.N. Genes Dev. 1989; 3: 628-640Crossref PubMed Scopus (600) Google Scholar, 14Wright W.E. Sassoon D.A. Lin V.K. Cell. 1989; 56: 607-617Abstract Full Text PDF PubMed Scopus (936) Google Scholar, 15Florini J.R. Ewton D.Z. Magri K.A. Annu. Rev. Physiol. 1991; 53: 201-216Crossref PubMed Scopus (349) Google Scholar). Moreover, expression of MyoD and myogenin mRNA is suppressed by incubation with BMP-2 (16Katagiri T. Akiyama S. Namiki M. Komaki M. Yamaguchi A. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. Exp. Cell Res. 1997; 230: 342-351Crossref PubMed Scopus (120) Google Scholar). The second level of control is covalent modification, mainly phosphorylation of the transcription factors. Thus, myogenin is phosphorylated by protein kinase C (17Li L. Zhou J. James G. Heller-Harrison R. Czech M.P. Olson E.N. Cell. 1992; 71: 1181-1194Abstract Full Text PDF PubMed Scopus (281) Google Scholar) and calmodulin-dependent kinase II (CaMKII) (18Blagden C.S. Fromm L. Burden S.J. Mol. Cell. Biol. 2004; 24: 1983-1989Crossref PubMed Scopus (12) Google Scholar, 19Tang H. Macpherson P. Argetsinger L.S. Cieslak D. Suhr S.T. CarterSu C. Goldman D. Cell. Signalling. 2004; 16: 551-563Crossref PubMed Scopus (32) Google Scholar) in its DNA-binding domain, thereby inhibiting DNA binding. Similarly, overexpression of protein kinase A inhibits the transcriptional activity of MRF4 (20Hardy S. Kong Y. Konieczny S.F. Mol. Cell. Biol. 1993; 13: 5943-5956Crossref PubMed Scopus (54) Google Scholar) and myogenin (21Li L. Heller-Harrison R. Czech M. Olson E.N. Mol. Cell. Biol. 1992; 12: 4478-4485Crossref PubMed Scopus (85) Google Scholar). The third level of control is through interaction with other proteins that regulate the activity of transcription factors. For example, the hypophosphorylated form of the retinoblastoma protein (Rb) associates with MyoD and is required for the efficient transactivation of E-box-containing muscle-specific promoters (22Gu W. Schneider J.W. Condorelli G. Kaushal S. Mahdavi V. Nadal-Ginard B. Cell. 1993; 72: 309-324Abstract Full Text PDF PubMed Scopus (643) Google Scholar). Moreover, several proteins that participate in the control of the cell cycle, such as Fos, Jun, or the adenoviral protein E1A, directly interact with MRFs and inhibit their transcriptional activity (23Bengal E. Ransone L. Scharfmann R. Dwarki V.J. Tapscott S.J. Weintraub H. Verma I.M. Cell. 1992; 68: 507-519Abstract Full Text PDF PubMed Scopus (324) Google Scholar, 24Li L. Chambard J.C. Karin M. Olson E.N. Genes Dev. 1992; 6: 676-689Crossref PubMed Scopus (189) Google Scholar, 25Taylor D.A. Kraus V.B. Schwarz J.J. Olson E.N. Kraus W.E. Mol. Cell. Biol. 1993; 13: 4714-4727Crossref PubMed Google Scholar). Finally, the helix-loop-helix transcription factors can be negatively regulated by the Id family of proteins (26Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1804) Google Scholar, 27Norton J.D. Deed R.W. Craggs G. Sablitzky F. Trends Cell Biol. 1998; 8: 58-65Abstract Full Text PDF PubMed Google Scholar, 28Ruzinova M.B. Benezra R. Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Id members are helix-loop-helix factors that lack the basic region that allows DNA binding. Thus, Ids heterodimerize with the E-factors and inhibit their binding to DNA. Ids sequester ubiquitous E-factors (27Norton J.D. Deed R.W. Craggs G. Sablitzky F. Trends Cell Biol. 1998; 8: 58-65Abstract Full Text PDF PubMed Google Scholar, 29Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar) and act as dominant negative regulators with respect to the tissue-specific helix-loop-helix proteins (MyoD, myogenin, etc.). Here we describe a new posttranscriptional mechanism implicated in the inhibition of the myogenic program by BMP-2, the abrupt degradation of myogenin by a mechanism that involves the proteasome. This mechanism is dependent on the induction of Id proteins by BMP-2. Materials—Human recombinant BMP-2 was obtained from the Genetics Institute, and N-acetyl-Leu-Leu-norleucinal (LLnL) was from Sigma. Des-(1,3)IGF-1 was from Angelika F. Schutzdeller (Tubingen, Germany). Cell culture media, fetal bovine serum, glutamine, and antibiotics were obtained from Invitrogen. The other reagents were of analytical or molecular biology grade and were purchased from Sigma or Roche Applied Science. Cell Culture and Transfections—C2C12 mouse cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin sulfate. Confluent cells were differentiated in DMEM containing 2% horse serum in the presence or absence of 2 nm BMP-2 or 2 nm des-(1,3)IGF-1 for the times indicated. For short incubations, C2C12 cells were first depleted of growth factors for 24 h in DMEM containing 0.1% fetal bovine serum. C2C12 cells were transiently transfected after addition of differentiation medium with pIRESbleo-Id1 (the murine Id1 cDNA was obtained by PCR, sequenced, and subcloned into pIRESbleo from Clontech), pcDNA3-E47 (a generous gift from Dr. Pura Muñoz-Canoves), or pcDNA3 using polyethylenimine (PEI) (Aldrich) (30Abdallah B. Hassan A. Benoist C. Goula D. Behr J.P. Demeneix B.A. Hum. Gene Ther. 1996; 7: 1947-1954Crossref PubMed Scopus (529) Google Scholar). Stable clones of C2C12 cells were generated by transfecting 10 μg of pcDNA3-myogenin or pcDNA3 and selection using G418 (400 μg/ml) for 2–3 weeks. We used three clones that expressed distinct levels of myogenin (Fig. 2, Clones 5, 7, and 8) or the C2C12-pcDNA3 control cells. We generated an inducible clone of Id1 in C2C12 cells (C2C12-pTISN-Id1) following the Tet-Off protocol described by Chambard and Pognonec (31Chambard J.C. Pognonec P. Nucleic Acids Res. 1998; 26: 3443-3444Crossref PubMed Scopus (19) Google Scholar). First, we used two distinct vectors that code for tetracycline-regulated transactivator (tTA) and puromycin resistance under the control of a tTA-responsive promoter (tetO-CMV) to generate C2C12 cells that stably expressed the tTA. After selection of clones resistant to puromycin, we transfected Id1 cloned in the pTISN vector (which expressed neomycin resistance), where Id1 was under the control of the tTA-responsive promoter. After selection, clones expressed Id1 in inverse proportion to the tetracycline concentration in the culture medium. The concentration of tetracycline was 100 ng/ml in all experiments. To reverse the effect of tetracycline, cells were incubated for 12 h in the presence of tetracycline, rinsed five times with phosphate-buffered saline (PBS), and incubated in normal medium in the absence of tetracycline for the times indicated. HEK-293 cells were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics. Cells were transiently transfected using polyethylenimine. The expression vectors encoding full-length E47, E47 lacking the helix-loop-helix (E47ΔHLH, which codes for amino acids 1–386), or E47 containing the helix-loop-helix (E47+HLH, which codes for amino acids 387–649) were kindly provided by Dr. P. Muñoz-Canoves and tagged with Myc. Western Blot Analysis—Cells were washed twice in cold PBS and lysed in Triton X-100 lysis buffer (50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 50 mm NaF, 5 mm EDTA, 40 mm β-glycerophosphate, 200 μm sodium orthovanadate, 100 μm phenylmethylsulfonyl fluoride, 1 μm pepstatin A, 1 μg/ml leupeptin, 4 μg/ml aprotinin, 1% Triton X-100) for 15 min at 4 °C. Western blots were performed as described previously (2Viñals F. Lopez-Rovira T. Rosa J.L. Ventura F. FEBS Lett. 2002; 510: 99-104Crossref PubMed Scopus (112) Google Scholar). The blots were incubated with polyclonal Id1 antibody (Santa Cruz Biotechnology), monoclonal antimyogenin 1F8 antibody (a generous gift from Dr. Pura Muñoz-Canoves, Centre de Regulació Genòmica, Barcelona, Spain), polyclonal E47 antibody (Santa Cruz Biotechnology), monoclonal anti-β-actin (Sigma), polyclonal anti-ERK2 (32McKenzie F.R. Pouyssegur J. J. Biol. Chem. 1996; 271: 13476-13483Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), or monoclonal anti-Myc (Sigma) in blocking solution overnight at 4 °C. Northern Blot—Total RNA from cells was extracted using the phenol/chloroform method (33Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar), and Northern blot with 20 μg of RNA was performed as described previously (2Viñals F. Lopez-Rovira T. Rosa J.L. Ventura F. FEBS Lett. 2002; 510: 99-104Crossref PubMed Scopus (112) Google Scholar). Blots were hybridized to the mouse myogenin cDNA or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA labeled with [α-32P]dCTP (Amersham Biosciences). Immunofluorescence Studies—Cells were cultured on glass coverslips for 24 h and transfected with the plasmids indicated. 48 h after transfection, cells were rinsed three times with PBS and fixed in 3% paraformaldehyde for 20 min. After four washes with PBS, they were permeabilized with PBS, 0.2% Triton X-100 for 5 min, rinsed four times with PBS, and blocked for 30 min at room temperature in PBS containing 2% bovine serum albumin. Coverslips were incubated with mouse monoclonal 1F8 antimyogenin antibody, rabbit polyclonal anti-Id1 antibody (Santa Cruz Biotechnology), or rabbit polyclonal anti-E47 antibody (Santa Cruz Biotechnology) in blocking solution for 1 h at room temperature, followed by Texas Red- or fluorescein isothiocyanate-conjugated anti-mouse or anti-rabbit antibody (Molecular Probes) for 1 h at room temperature. Finally, cells were incubated with Hoechst 333258 (Sigma) for 5 min at room temperature. Coverslips were mounted using Mowiol (Calbiochem), and immunofluorescence was visualized with a Nikon Eclipse E800 microscope. BMP-2 Induces the Degradation of Myogenin in C2C12 Cells—C2C12 cells incubated for 3 days with a limited supply of growth factors (2% horse serum) or in the presence of 2 nm des-(1,3)IGF-1 (a potent myogenic inducer) (15Florini J.R. Ewton D.Z. Magri K.A. Annu. Rev. Physiol. 1991; 53: 201-216Crossref PubMed Scopus (349) Google Scholar, 34Kaliman P. Canicio J. Shepherd P.R. Beeton C.A. Testar X. Palacin M. Zorzano A. Mol. Endocrinol. 1998; 12: 66-77Crossref PubMed Scopus (100) Google Scholar) differentiated to multinucleated myotubes. These myotubes expressed typical skeletal muscle markers, such as myogenin (Fig. 1A). Treatment with BMP-2 not only blocked myotube formation (2Viñals F. Lopez-Rovira T. Rosa J.L. Ventura F. FEBS Lett. 2002; 510: 99-104Crossref PubMed Scopus (112) Google Scholar, 35Chalaux E. Lopez-Rovira T. Rosa J.L. Bartrons R. Ventura F. J. Biol. Chem. 1998; 273: 537-543Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and myogenin expression (Fig. 1A) but also stimulated typical osteoblastic markers, such as osteocalcin or alkaline phosphatase (2Viñals F. Lopez-Rovira T. Rosa J.L. Ventura F. FEBS Lett. 2002; 510: 99-104Crossref PubMed Scopus (112) Google Scholar, 36Katagiri 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 (1296) Google Scholar). Simultaneous treatment of cells with both factors abolished myogenin expression (Fig. 1A), whereas the osteoblastic markers were maintained (data not shown). Furthermore, the addition of des-(1,3)IGF-1 12 or 24 h after BMP-2 did not reverse its inhibitory effects, and levels of myogenin remained low. In contrast, addition of BMP-2 12 or 24 h after IGF-1 caused a progressive decrease in myogenin levels (Fig. 1A). Thus, the effects of BMP-2 on myogenin expression were dominant over those of des-(1,3)IGF-1. BMP-2 inhibits transcription of the myogenin gene (16Katagiri T. Akiyama S. Namiki M. Komaki M. Yamaguchi A. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. Exp. Cell Res. 1997; 230: 342-351Crossref PubMed Scopus (120) Google Scholar). To evaluate the time course of the inhibitory effect of BMP-2 on myogenin expression, we incubated C2C12 cells in the presence of BMP-2, des-(1,3)IGF-1, or both factors for 4 or 8 h. After a 4-h incubation, BMP-2 induced Id1, an inhibitor of the family of helix-loop-helix transcription factors (28Ruzinova M.B. Benezra R. Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 29Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 37Ogata T. Wozney J.M. Benezra R. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9219-9222Crossref PubMed Scopus (135) Google Scholar), and its expression was maintained until 8 h (Fig. 1B). Des-(1,3)IGF-1 induced myogenin mRNA and protein at 4 and 8 h (Fig. 1C) but did not inhibit Id1 induction by BMP-2. Incubation in the presence of BMP-2 blocked the myogenin protein induction not only at 8 h but also at 4 h, in conditions where the myogenin mRNA induction was unaffected (Fig. 1, B and C). To identify the mechanisms involved in the effect of BMP-2 on myogenin protein levels over short times, we first evaluated a possible effect of BMP-2 on the IGF-1 signaling pathways. C2C12 cells were preincubated in the presence of BMP-2 followed by the addition of des-(1,3)IGF-1 for 30 min or 1 h. We did not detect any difference in ERK1/2 or phosphatidylinositol 3-kinase stimulation by des-(1,3)IGF-1 in the presence or absence of BMP-2 (data not shown). The observation that BMP-2 blocked myogenin protein induction at 4 h, independently of transcriptional regulation, led us to hypothesize that BMP-2 affects myogenin protein stability. To test this hypothesis, we generated stable clones of C2C12 that express myogenin under the control of an independent promoter (cytomegalovirus promoter of the vector pcDNA3). We generated three independent clones (Fig. 2A, Clones 5, 7, and 8) with distinct expression levels of the myogenin mRNA and protein. Myogenin transcription from these clones did not respond to BMP-2 (Fig. 2B). In all the cases BMP-2 caused a time-dependent decrease in the levels of myogenin protein, which was already observed 4 h after the addition of BMP-2 (Fig. 2A). The rate of myogenin decrease depended on the initial levels of myogenin expressed in each clone. Thus, in those that expressed low levels, the rate of decrease was faster than in those that expressed high myogenin levels (Fig. 2A), indicating the dependence of a limiting factor that controlled myogenin degradation by BMP-2. To confirm the data obtained by Western blot experiments, we performed immunofluorescence studies with antibodies against myogenin. As described previously (38Brennan T.J. Olson E.N. Genes Dev. 1990; 4: 582-595Crossref PubMed Scopus (174) Google Scholar), myogenin was immunolocalized in the nucleus of C2C12 cells (Fig. 2C). Incubation for 4 h with BMP-2 caused a significant decrease in the myogenin signal, without causing a clear effect of protein relocalization at the subcellular level. Myogenin Is Degraded by a Proteasome-dependent Mechanism—To confirm an induction of the degradation of the myogenin protein by BMP-2, we incubated cells with BMP-2 for 1 h, and after this preincubation, we added cycloheximide, a protein synthesis inhibitor, and then harvested cells after a range of times. In control conditions myogenin had a short half-life of about 60 min (Fig. 3A), which is consistent with the findings of other studies (39Edmondson D.G. Brennan T.J. Olson E.N. J. Biol. Chem. 1991; 266: 21343-21346Abstract Full Text PDF PubMed Google Scholar). Addition of BMP-2 increased the rate of myogenin degradation, causing the half-life to fall to 30 min, confirming that the short term effect of BMP-2 was because of an increase in myogenin degradation. The BMP-2-dependent decrease in myogenin protein levels may be explained by its degradation by a ubiquitin-proteasome system. Proteasome is one of the systems most often used by the cellular machinery to control the levels of subtly regulated proteins (40Baumeister W. Walz J. Zuhl F. Seemuller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar, 41Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6902) Google Scholar). To assess this possibility, we preincubated cells in the presence of LLnL, a potent inhibitor of the 26 S proteasome (42Seemuller E. Lupas A. Stock D. Lowe J. Huber R. Baumeister W. Science. 1995; 268: 579-582Crossref PubMed Scopus (584) Google Scholar). Addition of the inhibitor in the absence of BMP-2 caused a clear increase in the levels of myogenin (Fig. 3B) both in its major 36-kDa form and in a higher Mr form because of its phosphorylation (data not shown) (39Edmondson D.G. Brennan T.J. Olson E.N. J. Biol. Chem. 1991; 266: 21343-21346Abstract Full Text PDF PubMed Google Scholar, 43Zhou J. Olson E.N. Mol. Cell. Biol. 1994; 14: 6232-6243Crossref PubMed Scopus (30) Google Scholar). These results indicate that myogenin was degraded in basal conditions by a proteasome-dependent mechanism. More importantly, addition of LLnL before incubation with BMP-2 abolished the effect of the cytokine, and the accumulation of myogenin was at levels comparable with those obtained in the LLnL condition (Fig. 3B). Id1 Is Sufficient to Mediate the BMP-2-induced Degradation of Myogenin—One of the mediators of the antimyogenic response of BMP-2 is the induction of the Id proteins that act as dominant negative inhibitors of the tissue-specific helix-loophelix transcription factors (36Katagiri 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 (1296) Google Scholar, 37Ogata T. Wozney J.M. Benezra R. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9219-9222Crossref PubMed Scopus (135) Google Scholar, 44Lopez-Rovira T. Chalaux E. Massague J. Rosa J.L. Ventura F. J. Biol. Chem. 2002; 277: 3176-3185Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Given these effects, one possibility was that the induction of Id by BMP-2, by sequestering E-factors, not only blocked the transcriptional activity of myogenin but also served as an intermediate for the increase in myogenin degradation. To test this, we transiently transfected clones of C2C12 that expressed ectopic myogenin with Id1, and we immunolocalized both proteins. Cells that did not express Id1 clearly expressed myogenin in the nucleus (Fig. 4A). Id1 was localized in the nucleus and cytoplasm as described before (45Jen Y. Weintraub H. Benezra R. Genes Dev. 1992; 6: 1466-1479Crossref PubMed Scopus (397) Google Scholar, 46Iavarone A. Garg P. Lasorella A. Hsu J. Israel M.A. Genes Dev. 1994; 8: 1270-1284Crossref PubMed Scopus (340) Google Scholar). The overexpression of Id1 in the absence of BMP-2 always caused a decrease in the levels of myogenin. To further confirm these effects in a more controlled system, we used the Tet-Off system to generate an inducible Id1 expression system in C2C12 cells. Id1 expression in these cells was an inverse function of the tetracycline in the medium. Thus, in the presence of 100 ng/ml tetracycline in the medium for 12 h Id1 expression was abolished (Fig. 4B). After removal of tetracycline, expression levels increased with time but did not exceed the physiological levels obtained after 4 h of stimulation with BMP-2 (Fig. 4B). In these conditions of Id1 expression and in the absence of other effects caused by the presence of BMP-2, the myogenin protein decreased in an inverse function of Id1. These results confirmed that Id1 was sufficient to cause the drop in the levels of myogenin working as a mediator for the effects of BMP-2. E47 Protects Myogenin from Degradation Induced by BMP-2—The E family of transcription factors is essential for the transcriptional activity of the MRFs on the specific E-boxes of the muscle-specific gene promoters (12Ephrussi A. Church G.M. Tonegawa S. Gilbert W. Science. 1985; 227: 134-140Crossref PubMed Scopus (491) Google Scholar, 13Blackwell T.K. Weintraub H. Science. 1990; 250: 1104-1110Crossref PubMed Scopus (759) Google Scholar). If Id1 promotes degradation of myogenin by releasing this factor from the heterodimer with the E-members, overexpression of an E-member should prevent the effect of BMP-2. To evaluate this, we transfected E47 expression constructs in C2C12 cells expressing ectopic myogenin. Incubation for 8 h with BMP-2 caused a large decrease in the myogenin expression in these cells (Fig. 5A). In contrast, most cells that overexpressed E47 (Fig. 5A, green nuclei) maintained the expression of myogenin and were refractory to the BMP-2 effect. 5% of the control cells that expressed green fluorescent protein still maintained the myogenin expression after 8 h of incubation in the presence of BMP-2. In contrast, 90 ± 4% of cells that overexpressed E47 maintained the myogenin expression. To biochemically confirm these data, mock or E47 expression plasmids were cotransfected with a plasmid that encoded puromycin resistance. After 24 h of transfection, puromycin was added to the culture medium to select cells that had incorporated the expression plasmids. The resistant cells were incubated for 0, 4, or 8 h in the presence of 2 nm BMP-2. Overexpression of E47 blocked myogenin degradation even 8 h after the addition of BMP-2 (Fig. 5B). In contrast, myogenin was degraded by BMP-2 in the control cells that expressed pcDNA3. These results confirmed the hypothesis that Id was a mediator for the effects of BMP-2 observed through a mechanism that involved the sequestering of endogenous E-proteins. To confirm these data obtained analyzing the endogenous myogenin protein in C2C12 cells, we expressed myogenin in HEK-293 cells. Basal levels of myogenin expression in these cells were low (Fig. 6A). As it was observed in C2C12 cells, incubation with LLnL caused a strong increase in the levels of myogenin. Co-expression of E47 with myogenin caused the stabilization of the protein in the absence of LLnL, confirming the stabilizing role of heterodimerization with E-proteins. In contrast, addition of a Myc6 tag to the N terminus of myogenin blocked its high degradation rate as it was highly expressed in the absence of LLnL (Fig. 6A). Finally, to analyze the importance of myogenin-E47 heterodimer formation in myogenin stability, we made similar assays using two E47 deletion mutants (E47ΔHLH, defective in heterodimer formation (47Voronova A. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4722-4726Crossref PubMed Scopus (233) Google Scholar), and E47+HLH, which contains the bHLH). The E47 mutant unable to heterodimerize with myogenin was also defective" @default.
• W2016228757 created "2016-06-24" @default.
• W2016228757 creator A5032606260 @default.
• W2016228757 creator A5041712131 @default.
• W2016228757 date "2004-10-01" @default.
• W2016228757 modified "2023-09-26" @default.
• W2016228757 title "Myogenin Protein Stability Is Decreased by BMP-2 through a Mechanism Implicating Id1" @default.
• W2016228757 cites W1549203021 @default.
• W2016228757 cites W1569019154 @default.
• W2016228757 cites W1748935412 @default.
• W2016228757 cites W1768681792 @default.
• W2016228757 cites W1800167367 @default.
• W2016228757 cites W1935346479 @default.
• W2016228757 cites W1964733862 @default.
• W2016228757 cites W1966387132 @default.
• W2016228757 cites W1976099082 @default.
• W2016228757 cites W1976154397 @default.
• W2016228757 cites W1990285237 @default.
• W2016228757 cites W1991222632 @default.
• W2016228757 cites W1991930087 @default.
• W2016228757 cites W1994377715 @default.
• W2016228757 cites W1994695193 @default.
• W2016228757 cites W2005097199 @default.
• W2016228757 cites W2008657934 @default.
• W2016228757 cites W2013046038 @default.
• W2016228757 cites W2013545053 @default.
• W2016228757 cites W2019672253 @default.
• W2016228757 cites W2019673480 @default.
• W2016228757 cites W2035452413 @default.
• W2016228757 cites W2037592320 @default.
• W2016228757 cites W2047449018 @default.
• W2016228757 cites W2047669760 @default.
• W2016228757 cites W2048928172 @default.
• W2016228757 cites W2050572437 @default.
• W2016228757 cites W2051541258 @default.
• W2016228757 cites W2051798857 @default.
• W2016228757 cites W2054483928 @default.
• W2016228757 cites W2056984915 @default.
• W2016228757 cites W2059509871 @default.
• W2016228757 cites W2060437500 @default.
• W2016228757 cites W2061937899 @default.
• W2016228757 cites W2076282408 @default.
• W2016228757 cites W2076497575 @default.
• W2016228757 cites W2076875187 @default.
• W2016228757 cites W2081544530 @default.
• W2016228757 cites W2084659477 @default.
• W2016228757 cites W2084948252 @default.
• W2016228757 cites W2090289340 @default.
• W2016228757 cites W2091301979 @default.
• W2016228757 cites W2095503159 @default.
• W2016228757 cites W2095723928 @default.
• W2016228757 cites W2097899015 @default.
• W2016228757 cites W2101927529 @default.
• W2016228757 cites W2102476333 @default.
• W2016228757 cites W2108490725 @default.
• W2016228757 cites W2121551386 @default.
• W2016228757 cites W2122043314 @default.
• W2016228757 cites W2122367477 @default.
• W2016228757 cites W2124398251 @default.
• W2016228757 cites W2125164793 @default.
• W2016228757 cites W2128940002 @default.
• W2016228757 cites W2132088839 @default.
• W2016228757 cites W2159511823 @default.
• W2016228757 cites W2166987747 @default.
• W2016228757 cites W2172326429 @default.
• W2016228757 cites W2185288066 @default.
• W2016228757 cites W4294216491 @default.
• W2016228757 doi "https://doi.org/10.1074/jbc.m408059200" @default.
• W2016228757 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15322112" @default.
• W2016228757 hasPublicationYear "2004" @default.
• W2016228757 type Work @default.
• W2016228757 sameAs 2016228757 @default.
• W2016228757 citedByCount "42" @default.
• W2016228757 countsByYear W20162287572012 @default.
• W2016228757 countsByYear W20162287572013 @default.
• W2016228757 countsByYear W20162287572014 @default.
• W2016228757 countsByYear W20162287572016 @default.
• W2016228757 countsByYear W20162287572017 @default.
• W2016228757 countsByYear W20162287572018 @default.
• W2016228757 countsByYear W20162287572019 @default.
• W2016228757 countsByYear W20162287572021 @default.
• W2016228757 countsByYear W20162287572023 @default.
• W2016228757 crossrefType "journal-article" @default.
• W2016228757 hasAuthorship W2016228757A5032606260 @default.
• W2016228757 hasAuthorship W2016228757A5041712131 @default.
• W2016228757 hasBestOaLocation W20162287571 @default.
• W2016228757 hasConcept C104317684 @default.
• W2016228757 hasConcept C111472728 @default.
• W2016228757 hasConcept C12554922 @default.
• W2016228757 hasConcept C138885662 @default.
• W2016228757 hasConcept C185592680 @default.
• W2016228757 hasConcept C2988375501 @default.
• W2016228757 hasConcept C55493867 @default.
• W2016228757 hasConcept C86339819 @default.
• W2016228757 hasConcept C86803240 @default.
• W2016228757 hasConcept C89611455 @default.
• W2016228757 hasConcept C95444343 @default.
• W2016228757 hasConcept C97816823 @default.

• next