Dystrophin Dp71 Expression Is Down-regulated during Myogenesis
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m411571200
ISSN1083-351X
AutoresMario Bermúdez de León, Cecilia Montáñez, Pablo Gómez, Sara L. Morales‐Lázaro, Victor Tapia‐Ramírez, Viviana Valadéz-Graham, Félix Recillas‐Targa, David Yaffe, Uri Nudel, Bulmaro Cisneros,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoDp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5′-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3. Dp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5′-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3. Duchenne muscular dystrophy (DMD) 1The abbreviations used are: DMD, Duchenne muscular dystrophy; DAP, dystrophin-associated proteins; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; FBS, fetal bovine serum; CAT, chloramphenicol acetyltransferase; RT, reverse transcription; r, ribosomal. is an inherited disorder characterized by progressive muscle degeneration due to the absence of dystrophin (1Ahn A.H. Kunkel L.M. Nat. Genet. 1993; 3: 283-291Crossref PubMed Scopus (577) Google Scholar). Dystrophin is a 427-kDa protein consisting of four major domains as follows: an N-terminal actin-binding domain, a central spectrin-like rod domain consisting of 24 triple helix structures, a cysteine-rich domain, and a unique C-terminal domain (2Koenig M. Monaco A.P. Kunkel L.M. Cell. 1988; 53: 219-226Abstract Full Text PDF PubMed Scopus (1279) Google Scholar). In skeletal muscle, dystrophin is associated with a group of sarcolemmal proteins and glycoproteins known collectively as the dystrophin-associated proteins (DAP) (3Ozawa E. Yoshida M. Suzuki A. Mizuno Y. Hagiwara Y. Noguchi S. Hum. Mol. Genet. 1995; 4: 1711-1716Crossref PubMed Scopus (218) Google Scholar). One of the proposed functions of dystrophin is to provide a structural link between the actin-based cytoskeleton and the extracellular matrix (4Mehler M.F. Brain Res. Brain Res. Rev. 2000; 32: 277-307Crossref PubMed Scopus (158) Google Scholar). The DMD gene exhibits a complex transcriptional regulation due to the presence of at least seven independent promoters that generate three full-length dystrophins (Dp427) and N-terminally truncated gene products (Dp260, Dp140, Dp116, and Dp71) (5Barnea E. Zuk D. Simantov R. Nudel U. Yaffe D. Neuron. 1990; 5: 881-888Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 6Boyce F.M. Beggs A.H. Feener C. Kunkel L.M. Proc. Natl. Acad. Sci. 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Differentiation. 1992; 49: 187-193Crossref PubMed Scopus (65) Google Scholar). Conversely, Dp427 is not expressed until after the cells begin myogenic differentiation and is the major isoform expressed in mature fibers (18Lev A.A. Feener C.C. Kunkel L.M. Brown Jr., R.H. J. Biol. Chem. 1987; 262: 15817-15820Abstract Full Text PDF PubMed Google Scholar, 19Nudel U. Robzyk K. Yaffe D. Nature. 1988; 331: 635-638Crossref PubMed Scopus (116) Google Scholar). Based on obvious structural differences between Dp71 and Dp427, it is unlikely that these proteins are functionally interchangeable. In fact, ectopic expression of Dp71 in skeletal muscle of mdx mice, which lack dystrophin, restored the normal levels of DAP but did not alleviate muscle damage (20Greenberg D.S. Sunada Y. Campbell K.P. Yaffe D. Nudel U. Nat. Genet. 1994; 8: 340-344Crossref PubMed Scopus (119) Google Scholar, 21Cox G.A. Sunada Y. Campbell K.P. Chamberlain J.S. Nat. Genet. 1994; 8: 333-339Crossref PubMed Scopus (162) Google Scholar), and more surprisingly, ectopic expression of Dp71 in skeletal muscle of transgenic wild-type mice results in muscle damage, similar to that observed in mdx mice (22Leibovitz S. Meshorer A. Fridman Y. Wieneke S. Jockusch H. Yaffe D. Nudel U. Neuromuscul. Disord. 2002; 12: 836-844Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The performance of stage-specific tasks by Dp427 and Dp71 in muscle cells indicates that their expression is tightly controlled (23Howard P.L. Dally G.Y. Ditta S.D. Austin R.C. Worton R.G. Klamut H.J. Ray P.N. Muscle Nerve. 1999; 22: 16-27Crossref PubMed Scopus (30) Google Scholar). The transcriptional regulation of Dp427 in muscle cells has been well established. It is known that high levels of YY1 protein down-regulate the Dp427 promoter in undifferentiated muscle cells, but upon the induction of muscular differentiation, YY1 protein levels are negatively controlled by the action of the protease m-calpain, allowing the dystrophin promoter bending factor to regulate positively the promoter activity (24Galvagni F. Cartocci E. Oliviero S. J. Biol. Chem. 1998; 273: 33708-33713Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In contrast, regulation of Dp71 during myogenesis remains to be approached, and the Dp71 promoter region, which exhibits the structure of a typical housekeeping promoter, has been only partially characterized (11Lederfein D. Yaffe D. Nudel U. Hum. Mol. Genet. 1993; 2: 1883-1888Crossref PubMed Scopus (69) Google Scholar). In this study, we analyzed the expression of Dp71 and characterized the activity of its promoter region during muscle cell differentiation of the C2C12 cells, a sub-culture derived from the C2 cell line. Deletion analysis showed that the major Dp71 promoter activity in proliferating myoblasts depends on the proximal 224-bp promoter region, which contains several Sp-binding sites. Altogether, gel shift, chromatin immunoprecipitation, and site-directed mutagenesis assays, as well as transient transfection experiments in Drosophila SL2 cells, established that Sp1 and Sp3 transcription factors interact with the Sp-binding sites and transactivate the Dp71 promoter. In differentiating muscle cells, the Dp71 promoter activity is down-regulated; the 224-bp proximal promoter region seems to be sufficient to exert such control, and a concomitant reduction in the endogenous Sp1 and Sp3 protein levels was observed. Restoration of significant promoter activity in differentiating cells was obtained after exogenous expression of Sp1 or Sp3 proteins. Our results indicate that the developmentally regulated expression of Dp71 in muscle cells during differentiation is based on the differential expression of Sp1 and Sp3 transcription factors. Cell Cultures—C2C12 cells (ATCC CRL-1772), a sub-culture derived from C2 cell line (25Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1574) Google Scholar), were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 4.5 g of glucose/liter, supplemented with 10% fetal bovine serum (FBS) and 2 mm l-glutamine (proliferation medium), and maintained at 37 °C in a humidified incubator with a 5% CO2 atmosphere. In some experiments, subconfluent C2C12 cells were induced to differentiate by lowering FBS to a final concentration of 1% (differentiation medium). Schneider's Drosophila Line 2 (SL2, ATCC CRL-1963) cells were cultured in Schneider's Drosophila Medium (Invitrogen) containing l-glutamine and were maintained at 25 °C without CO2. All culture media contained 100 units of penicillin and 100 μg/ml streptomycin. Real Time RT-PCR—Dp71 transcript levels during C2C12 muscle cell differentiation were measured by quantitative real time RT-PCR using the comparative CT method described by Applied Biosystems. Total RNA was extracted using the TRIzol reagent (Invitrogen) from C2C12 cells induced to differentiate for 0, 3, 6, or 9 days. 5 μg of the extracted total RNA was primed with random hexanucleotides and reversed-transcribed by the Moloney murine leukemia virus-reverse transcriptase (Invitrogen), according to the manufacturer's instructions. Real time PCRs were set up in a reaction volume of 25 μl using the TaqMan Universal PCR Master Mix (Applied Biosystems). The probe and primers specific for Dp71 detection were designed using the Primer express software from PerkinElmer Life Sciences. The Dp71 fluorogenic probe was 5′-(FAM™)-CCCCAAAGGACTCAAAGAACCT-(TAMRA™)-3′, and the Dp71 PCR primers were Dp71F 5′-TGTATTGCATTTAGAGCCCCAA-3′ and Dp71R 5′-CTTCCTCTGCGCTTAATTGC-3′ (Synthetic Genetics, San Diego, CA). As endogenous control, real time RT-PCR analysis of the eukaryotic 18 S ribosomal (r)RNA gene was performed in parallel. The r18 S fluorogenic probe, labeled with VIC™ dye-TAMRA™ dye, and PCR primers were purchased from Applied Biosystems. DNA amplifications were carried out in a 96-well reaction plate format in a PE Applied Biosystems 7700 Sequence Detector (PerkinElmer Life Sciences). Both Dp71 and r18 S PCRs were carried out in triplicate. Multiple negative control water blanks were included in each analysis. Plasmid Constructs—Plasmids p1800CAT, p1500CAT, p900CAT, p700CAT, p1.8StuI-delCAT and pSV0CAT were described previously (11Lederfein D. Yaffe D. Nudel U. Hum. Mol. Genet. 1993; 2: 1883-1888Crossref PubMed Scopus (69) Google Scholar). New truncated promoter versions were prepared on p1800CAT by restriction endonuclease digestion in the appropriate sites, followed by blunt end generation and ligation, to generate p1159CAT (NdeI and AccI enzymes), p939CAT (NdeI and StuI), p1.8Xba-delCAT (XbaI), p711CAT (NdeI and XbaI), p588CAT (NdeI and EcoRV), and p224CAT (NdeI and BstXI). The ligated products were transformed into competent DH5-α cells, and selected individual colonies were cultured overnight in 5 ml of LB + ampicillin (100 μg/ml). Plasmid DNA was isolated from these cultures to verify the joined sequences of the new constructs. Finally, plasmids were purified using the plasmid midi kit columns (Qiagen). Transient Cell Transfection—Each Dp71 promoter construct was introduced into C2C12 cells by means of the Lipofectamine Plus reagent (Invitrogen), together with the pRSV-β-gal vector or with both the pRSV-β-gal and pPac, AP2γ/pcDNA3.1(+), pPacSp1, and/or PacSp3 expression plasmids according to the manufacturer's instructions. Defined amounts of each expression vector were incubated with 8 μl of plus reagent in 250 μl of Opti-MEM medium, mixed well, and added to an equal volume of Opti-MEM medium containing 12 μl of Lipofectamine reagent. The lipid/DNA mixture was mixed well, incubated for 15 min at room temperature, and added to cell cultures (previously washed with Opti-MEM medium) covered by 2 ml of Opti-MEM medium. After 48 h of incubation at 37 °C in a 5% CO2-humidified incubator, Opti-MEM medium was replaced with either proliferation or differentiation medium. For transfection of the SL2 cell line, freshly grown cells from 3- to 4-day-old cultures were plated at density of 2.5 × 106 cells/60-mm dish and the day after were transfected using Cellfectin reagent (Invitrogen), according to the manufacturer's instructions. Each plate was transfected previously with defined amounts of pRSV-β-gal, each Dp71 promoter-CAT reporter plasmids, and pPacSp1 and/or pPacUSp3 expression plasmids. After transfection, C2C12 and SL2 cells were maintained in their respective growth medium for 48 h, and the CAT and β-galactosidase assays were performed. CAT and β-Galactosidase Assays—To prepare cell extracts for the CAT and β-galactosidase expression assays, cells were scraped into 1× phosphate-buffered saline and centrifuged at 2600 × g at 4 °C for 5 min. The cell pellet was resuspended in 150 μl of 0.25 m Tris and 1 mm EDTA, pH 8.0, and then subjected to six freeze-thaw cycles with dry ice. Cell debris was removed by centrifugation at 4 °C for 5 min at 3000 × g, and the resulting supernatant was removed and its protein content determined by the Bradford assay (Bio-Rad). These clarified extracts were used directly in enzymatic assays. β-Galactosidase activity was measured using 2.5 mmo-nitrophenyl β-d-galactopyranoside (Sigma) as substrate and 30 μl of cell extract in a reaction mixture consisting of 60 mm Na2HPO4, 40 mm NaH2PO4, 2 mm MgCl2, and 50 mm β-mercaptoethanol. After incubation for 30 min at 37 °C, the reaction was stopped by adding 1 m Na2CO3, and the absorbance of the reaction product was read at 405 nm. CAT activity was determined by using 20 μg of cell extract, 80 mm acetyl-CoA (Sigma), and 0.15 μCi of [14C]chloramphenicol (Amersham Biosciences) in a total volume of 180 μl. The reaction mixture was incubated at 37 °C for 60 min, extracted with 800 μl ethyl acetate, and dried in a vacuum centrifugal evaporator. The dry reaction products were resuspended in 20 ml of ethyl acetate, and the acetylated and nonacetylated forms of [14C]chloramphenicol were separated by thin layer silica gel chromatography for 60 min at room temperature with chloroform/methanol (19:1, v/v) as mobile phase. Percentage conversion of chloramphenicol to its acetylated forms was determined using a radioactive image analyzer (AMBIS 4000). Isolation of Nuclear Extracts—Nuclear extracts were prepared from undifferentiated and differentiated C2C12 cells, as described previously (26Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar). Briefly, cells were washed with ice-cold phosphate-buffered saline and resuspended in 400 μl of lysis buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). Cell lysates were incubated on ice for 15 min and then centrifuged for 3 min at 3,000 × g at 4 °C. The pellet was resuspended in 50 μl of extraction buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride). After that, cells were vigorously shaken at 4 °C for 15 min and pelleted by centrifuging for 5 min at 12,000 × g at 4 °C. The supernatant was recovered as the nuclear extract, and its protein concentration was determined by the Bradford assay. Nuclear extracts were stored at –70 °C until use. Electrophoretic Mobility Shift Assay—Synthetic complementary deoxyoligonucleotides corresponding to the AP2 and Sp DNA elements of the Dp71 promoter were annealed and radioactively labeled by incubating 4 pmol of dephosphorylated double-stranded oligonucleotides, 10 units of T4 polynucleotide kinase (Invitrogen), 5 μl of T4 polynucleotide kinase buffer, and 20 μCi of [γ-32P]dATP (Amersham Biosciences) for 30 min at 37 °C and inactivated at 65 °C for 10 min. For the electrophoretic mobility shift assay (EMSA), 10 μg of nuclear extract proteins from either undifferentiated or differentiated cells were mixed in the binding buffer (12 mm HEPES, pH 7.8, 4 mm MgCl2, 1 mm dithiothreitol, 0.5 mm EDTA, 60 mm KCl, 10% glycerol), with 1 μg of poly(dI-dC) (Amersham Biosciences) as nonspecific competitor and the appropriate double-stranded 32P-labeled oligonucleotides as follows: Sp-A, 5′-TCCCAGCCCCGCCCCGTCTGC-3′; Sp-BC, 5′-CTGTCCCGCCCGCCCGCCAG-3′; Sp-D, 5′-GAGCCTGGGCGGCGGGCGCTTGACT-3′; and AP2, 5′-GCCCGCCAGCCCGCCAGCCAG-3′. In competition experiments, a 100-fold molar excess of the appropriate unlabeled double-stranded oligonucleotides were incubated with nuclear extracts before the addition of 32P-labeled oligonucleotides. Competitor double-stranded oligonucleotides were as follows: consensus Sp1, 5′-ATTCGATCGGGGCGGGGCGAGC-3′; mutated Sp1, 5′-ATTCGATCGGTTCGTGGCGAGC-3′; consensus AP2, 5′-GATCGAACTGACCGCCCGCGGCCCGT-3′; mutated AP2, 5′-GATCGAACTGACCGCTTGCGGCCCGT-3′; consensus OCT1 5′-TGTCGAATGCAAATCACTAGAA-3′; mutated bases are underlined. For supershift analysis, 10 μg of anti-Sp1- or anti-Sp3-specific antibodies (Santa Cruz Biotechnology) were incubated with nuclear extract proteins for 12 h at 4 °C after addition of the DNA probe. Protein-DNA complexes were fractionated by electrophoresis in nondenaturing 6% polyacrylamide gels and visualized by autoradiography. Antibodies and Western Blot Analysis—Antibodies directed to Sp1, Sp3, and myogenin were purchased from Santa Cruz Biotechnology. The anti-Dp71 polyclonal antibody 2166 was a donation of Dr. D. J. Blake (27Loh N.Y. Newey S.E. Davies K.E. Blake D.J. J. Cell Sci. 2000; 113: 2715-2724PubMed Google Scholar). The anti-actin monoclonal antibody was generously provided by Dr. J. M. Hernandez. C2C12 cells cultured in 35-mm culture dishes were scraped, lysed as described previously (28Acosta R. Montanez C. Fuentes-Mera L. Gonzalez E. Gomez P. Quintero-Mora L. Mornet D. Alvarez-Salas L.M. Cisneros B. Exp. Cell Res. 2004; 296: 265-275Crossref PubMed Scopus (39) Google Scholar), and centrifuged at 12,000 × g for 10 min at 4 °C. Protein samples were quantified by the Bradford assay and denatured at 100 °C for 5 min in a protein sample buffer containing 1% SDS and 1% dithiothreitol. One hundred μg of total protein extracts were loaded in each lane and subjected to 10% PAGE under denaturing conditions (SDS-PAGE) and transferred to a nitrocellulose membrane for immunoblotting. Immunoblots were probed with the corresponding primary antibodies and developed by using the ECL Western blotting analysis system (Amersham Biosciences). Chromatin Immunoprecipitation Assay—C2C12 cells (2 × 107) were treated with 1% formaldehyde to cross-link protein-DNA complexes. Immunoprecipitates of cross-linked complexes were prepared with anti-Sp1 and anti-Sp3 antibodies, treated with proteinase K for 2 h, and then incubated at 65 °C to eliminate cross-linking. DNA was purified by phenol/chloroform extraction and ethanol precipitation. DNA samples were quantified by the picogreen assay (Molecular Probes) and then analyzed by PCR amplification of the Dp71 promoter sequence located between –127 and +78 with 30 cycles of PCR using the following of 32P-labeled oligonucleotides: Dp71 (upper), 5′-CTATCCAGGTTTCCCCAGGTC-3′, and Dp71 (lower), 5′-CGGAGGAGTAAGCTTGCCCAA-3′. Different cycle numbers were employed to ensure linearity of the amplification reaction. As negative control, a DMD gene region localized on the junction of the DMD gene intron 63 and exon 64 (from +41,508 to +41,646, relative to the Dp71 transcription start site, GenBank™ accession number AL645848) was amplified with the following 32P-labeled oligonucleotides: Dys 64/64 (upper), 5′-ATAATGTCAGGTTCTCCGCG-3′, and Dys 64/64 (lower), 5′-TCAAAAATCCCCAAGCCCCA-3′. As positive control, a DNA region of the human telomerase promoter, which contains several Sp-binding sites, was amplified as positive control with the following 32P-labeled oligonucleotides Tert (upper), 5′-AACACATCCAGCAACCACTGA-3′, and Tert (lower), 5′-AAGATGAGGAGGGAACGGAGG-3′. PCR products were analyzed by 6% PAGE and visualized by autoradiography. Construction of Mutant Plasmids—A series of mutations was performed in the Dp71 proximal promoter region (p224CAT) by using the QuickChange™ site-directed mutagenesis kit (Stratagene) and each of the following double-stranded oligonucleotides: mutSp-A, 5′-CCCTTCCCAGCCCTGCTCCGTCTGCACGC-3′; mutSp-B, 5′-CCCCTCCCTGTCCTGTCCGCCCGCCAGC-3′; mutSp-C, 5′-CCCCTCCCTGTCCCGCCTGTCCGCCAGC-3′; mutSp-D, 5′-GGCTGCGAGCCTGGGCTTCGTGCGCTTGAC-3′; and mutAP2, 5′-CCCGCCCGCCAGCCAACCAGGCAGCGGCGG-3′. The underlined letters indicate the mutated bases. Briefly, 50 ng of the p224CAT promoter plasmid was used in a PCR containing 5 μl of Pfu polymerase buffer, 2.5 units of Pfu turbo DNA polymerase (Stratagene), 10 mm dNTPs, 125 ng of each oligonucleotide, and water was added to 50 μl. Following temperature cycling (PerkinElmer Life Sciences), DpnI treatment was performed to cleave parental DNA and to improve the efficiency of the mutant plasmid screening. The reaction was transferred into XL-1 Blue competent cells, and the transformation mixture was plated on LB ampicillin plates. The authenticity of the mutants was established by DNA sequencing. Data Analysis—All data are depicted as mean ± S.E. Differences between two groups were validated by Student's t test. Dp71 Expression Is Down-regulated during C2C12 Myogenesis—To evaluate the expression of Dp71 during C2C12 muscle cell differentiation, its mRNA and protein levels were determined. Dp71 mRNA expression was measured by real time RT-PCR using Dp71-specific primers, whereas Dp71 protein was assessed by Western blotting using the polyclonal antibody 2166, directed against its C-terminal domain. Myoblast cultures were induced to differentiate for 3, 6, and 9 days by lowering serum concentration, and cell-differentiated morphology was monitored by light microscopy analysis. As shown in Fig. 1A, Dp71 mRNA expression was detected in myoblasts but decreased to minimum levels in myotube cultures since day 3 of induced differentiation, at which point myotube formation became apparent under microscopy visualization (data not shown). Dp71 protein expression pattern during C2C12 myogenesis was similar to that of Dp71 transcript; it was immunodetected in undifferentiated cells but disappeared totally from differentiated cultures of 3, 6, and 9 days (Fig. 1B, upper panel). In contrast, C2C12 maintained in differentiation medium resulted in up-regulation of myogenin, a myogenic gene marker; this protein was undetectable in myoblast cells but appeared by day 3 and remained steady throughout the rest of the differentiation treatment (Fig. 1B, lower panel), in agreement with previous reports (29Lopez-Casillas F. Riquelme C. Perez-Kato Y. Ponce-Castaneda M.V. Osses N. Esparza-Lopez J. Gonzalez-Nunez G. Cabello-Verrugio C. Mendoza V. Troncoso V. Brandan E. J. Biol. Chem. 2003; 278: 382-390Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 30Molkentin J.D. Olson E.N. Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (390) Google Scholar, 31Sabourin L.A. Rudnicki M.A. Clin. Genet. 2000; 57: 16-25Crossref PubMed Scopus (565) Google Scholar, 32Shimokawa T. Kato M. Ezaki O. Hashimoto S. Biochem. Biophys. Res. Commun. 1998; 246: 287-292Crossref PubMed Scopus (85) Google Scholar). Thus, the concomitant decreases of Dp71 mRNA and protein levels were negatively correlated with the differentiation process of C2C12 cells and evaluated by the acquisition of a differentiated morphology and induction of myogenin expression. Furthermore, these results indicated that the reduction of Dp71 expression during skeletal muscle differentiation was regulated, at least in part, at a transcriptional level. Dp71 Promoter Activity during Muscle Cell Differentiation—To characterize the mechanisms controlling the negative transcriptional regulation of Dp71 that occurred during myogenic differentiation, C2C12 myoblasts were transiently cotransfected with pRSV-β-gal and p1.8CAT vectors; the latter contains 1.8 kb from the mouse Dp71 promoter region fused to the reporter gene CAT (11Lederfein D. Yaffe D. Nudel U. Hum. Mol. Genet. 1993; 2: 1883-1888Crossref PubMed Scopus (69) Google Scholar). Myoblast cultures were induced to differentiate for 2 days, and the Dp71 promoter function was evaluated by CAT assays using β-galactosidase activity to normalize transfection efficiency. The 1.8-kb Dp71 promoter fragment (p1.8CAT) drove the efficient expression of CAT in C2C12 myoblast compared with promoterless pSV0CAT (Fig. 3). As expected from the mRNA results described above, reporter activity of the Dp71 promoter decreased by 70% in differentiated cells (Fig. 2). Because the induction of muscle cell differentiation was also modulated by the addition of cAMP (33Usuki F. Ishiura S. Sasagawa N. Sorimachi H. Suzuki K. Shimizu T. Terao T. Biochem. Biophys. Res. Commun. 1995; 210: 654-659Crossref PubMed Scopus (4) Google Scholar, 34Drab M. Haller H. Bychkov R. Erdmann B. Lindschau C. Haase H. Morano I. Luft F.C. Wobus A.M. FASEB J. 1997; 11: 905-915Crossref PubMed Scopus (196) Google Scholar), we decided to evaluate the effect of this alternative inductor of differentiation on the Dp71 promoter activity. For that purpose, myoblasts, transiently transfected with the p1.8CAT vector, were cultured in growth medium for 48 h with or without cAMP. Fig. 2 shows that cAMP treatment resulted in a 40% inhibition of CAT activity, whereas a negative control experiment, in which the nerve growth factor was added to the proliferation medium, provoked no changes in CAT activity. Altogether, these results indicate that Dp71 promoter responds negatively in a specific way to inducers of skeletal muscle differentiation.Fig. 2Effect of myogenesis induction on the Dp71 promoter activity. C2C12 cell cultures were cotransfected with vector p1.8CAT that contains the 1.8-kb Dp71 promoter region and pRSV-β-gal. Transfected cells were either maintained under proliferating conditions or induced to differentiate for 2 days by culturing in differentiation medium (1% FBS) or by adding 1 mm dibutyryl-cAMP (db-cAMP) to the proliferation medium. The resulting CAT activities were normalized against β-galactosidase activities, and the reporter activity of undifferentiated control cells (cells cultured in medium supplements with 10% FBS) was set at 100%. In negative control experiments, nerve growth factor (NGF) at 2 nm was added to cells cultured in proliferation medium. Data are expressed as the mean ± S.D. of at least three independent experiments, each performed in duplicate. Asterisks denote significant diffe
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