Vascular Smooth Muscle α-Actin Gene Transcription during Myofibroblast Differentiation Requires Sp1/3 Protein Binding Proximal to the MCAT Enhancer
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m203232200
ISSN1083-351X
AutoresJohn G. Cogan, Sukanya Subramanian, John A. Polikandriotis, Robert J. Kelm, Arthur R. Strauch,
Tópico(s)Cancer-related gene regulation
ResumoThe conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor β1 (TGF-β1) and accompanied by transient activation of the vascular smooth muscle α-actin (SmαA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmαA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-β1. However, TGF-β1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmαA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmαA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmαA enhancer required for TGF-β1 amplification. Additionally, the TGF-β1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmαA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmαA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmαA enhancer provides new insight into the poorly understood process of myofibroblast differentiation. The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor β1 (TGF-β1) and accompanied by transient activation of the vascular smooth muscle α-actin (SmαA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmαA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-β1. However, TGF-β1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmαA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmαA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmαA enhancer required for TGF-β1 amplification. Additionally, the TGF-β1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmαA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmαA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmαA enhancer provides new insight into the poorly understood process of myofibroblast differentiation. smooth muscle α-actin transforming growth factor β1 TGF-β1 control element TGF-β1 hypersensitivity region fetal bovine serum chloramphenicol acetyltransferase vascular smooth muscle electrophoretic mobility shift assay downstream element Myofibroblasts have phenotypic properties intermediate between fibroblasts and smooth muscle cells and are thought to provide mechanical force necessary for wound contraction and closure (1Darby I. Skalli O. Gabbiani G. Lab. Invest. 1990; 63: 21-29PubMed Google Scholar, 2Desmoulière A. Gabbiani G. Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 391-423Google Scholar, 3Skalli O. Gabbiani G. Clark R.A.F. Henson P.M. The Molecular and Cellular Biology of Wound Repair. Plenum Publishing Corp., New York1988: 373-401Crossref Google Scholar, 4Grinnell F. J. Cell Biol. 1994; 124: 401-404Crossref PubMed Scopus (973) Google Scholar). Believed to arise from stromal fibroblasts in response to growth factors and inflammatory cytokines released during tissue injury, myofibroblasts contain abundant contractile microfilaments composed of vascular smooth muscle α-actin (SmαA)1 (1Darby I. Skalli O. Gabbiani G. Lab. Invest. 1990; 63: 21-29PubMed Google Scholar, 5Desmoulière A. Geinoz A. Gabbiani F. Gabbiani G. J. Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1859) Google Scholar, 6Ronnov-Jessen L. Petersen O.W. J. Cell Biol. 1996; 134: 67-80Crossref PubMed Scopus (191) Google Scholar, 7Ronnov-Jessen L. Petersen O.W. Lab. Invest. 1993; 68: 696-707PubMed Google Scholar, 8Dugina V. Alexandrova A. Chaponnier C. Vasiliev J. Gabbiani G. Exp. Cell Res. 1998; 238: 481-490Crossref PubMed Scopus (68) Google Scholar, 9Serini G. Gabbiani G. Exp. Cell Res. 1999; 250: 273-283Crossref PubMed Scopus (499) Google Scholar), smooth muscle myosin (10Chiavegato A. Bochaton-Piallat M.-L. D'Amore E. Sartore S. Gabbiani G. Virchows Arch. Int. J. Pathol. 1995; 426: 77-86Crossref PubMed Scopus (50) Google Scholar), the calponin-like, smooth muscle microfilament-binding protein SM22α (11Faggin E. Puato M. Zardo L. Franch R. Millino C. Sarinella F. Pauletto P. Sartore S. Chiavegato A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1393-1404Crossref PubMed Scopus (70) Google Scholar), and caldesmon, a smooth muscle-specific calcium-regulatory protein (12Ando Y. Moriyama T. Oka K. Takatsuji K. Miyazaki M. Akagi Y. Kawada N. Isaka Y. Izumi M. Yokoyama K. Yamauchi A. Horio M. Ando A. Ueda N. Sobue K. Imai E. Hori M. Nephrol. Dial. Transplant. 1999; 14: 1408-1417Crossref PubMed Scopus (22) Google Scholar). Normally transient cellular participants in wound-healing processes, chronic accumulation of α-actin-positive myofibroblasts has been associated with pathologic tissue remodeling processes such as hypertrophic scarring, stromal responses to certain neoplasias, and interstitial fibrosis associated with repair of myocardial infarction (13Frangogiannis N.G. Michael L.H. Entman M.L. Cardiovasc. Res. 2000; 48: 89-100Crossref PubMed Scopus (172) Google Scholar, 14Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Am. J. Physiol. Cell Physiol. 1999; 277: C1-C19Crossref PubMed Google Scholar). Although the molecular control of myofibroblast differentiation is largely unknown, several studies have shown that transforming growth factor β1 (TGF-β1) may be particularly important in their recruitment to sites of tissue inflammatory damage. TGF-β1 is a potent SmαA gene transcriptional activator in both granulation tissue and isolated fibroblasts (2Desmoulière A. Gabbiani G. Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 391-423Google Scholar, 5Desmoulière A. Geinoz A. Gabbiani F. Gabbiani G. J. Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1859) Google Scholar, 7Ronnov-Jessen L. Petersen O.W. Lab. Invest. 1993; 68: 696-707PubMed Google Scholar, 15Becker N.A. Kelm Jr., R.J. Vrana J.A. Getz M.J. Maher L.J.I. J. Biol. Chem. 2000; 275: 15384-15391Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). TGF-β1 also induces expression of SmαA in cultured rat aortic smooth muscle cells, bovine aortic endothelial cells, and rat fibroblasts, where specific transcriptional control elements contained within the rat SmαA gene promoter were shown to mediate TGF-β1-dependent activation (16Hautmann M.B. Madsen C.S. Owens G.K. J. Biol. Chem. 1997; 272: 10948-10956Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 17Hautmann M.B. Adam P.J. Owens G.K. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2049-2058Crossref PubMed Scopus (103) Google Scholar). However, the sequence context and position of DNA-regulatory elements identified in studies on the rat SmαA promoter and aortic smooth muscle cells differed from those in the mouse SmαA enhancer that undergoes TGF-β1-dependent chromatin conformational changes in AKR-2B stromal fibroblasts (15Becker N.A. Kelm Jr., R.J. Vrana J.A. Getz M.J. Maher L.J.I. J. Biol. Chem. 2000; 275: 15384-15391Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). TGF-β1 elicits both tissue-specific and cell culture context-specific responses (18Orlandi A. Ropraz P. Gabbiani G. Exp. Cell Res. 1994; 214: 528-536Crossref PubMed Scopus (84) Google Scholar, 19Majack R.A. J. Cell Biol. 1987; 105: 465-471Crossref PubMed Scopus (177) Google Scholar), yet studies performed to date have not resolved whether sequence elements required for activation from TGF-β1-dependent promoters are distinctly different or evolutionarily conserved. Previous studies from our laboratories have indicated that the mouse SmαA gene is regulated by an array of both positive and negativecis-acting elements that behaved differently in fibroblasts compared with muscle cells (20Cogan J.G. Sun S. Stoflet E.S. Schmidt L.J. Getz M.J. Strauch A.R. J. Biol. Chem. 1995; 270: 11310-11321Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Foster D.N. Min B. Foster L.K. Stoflet E.S. Sun S. Getz M.J. Strauch A.R. J. Biol. Chem. 1992; 267: 11995-12003Abstract Full Text PDF PubMed Google Scholar, 22Kelm Jr., R.J. Cogan J.G. Elder P.K. Strauch A.R. Getz M.J. J. Biol. Chem. 1999; 274: 14238-14245Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Kelm Jr., R.J. Elder P.K. Strauch A.R. Getz M.J. J. Biol. Chem. 1997; 272: 26727-26733Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24Kelm Jr., R.J. Sun S. Strauch A.R. Getz M.J. J. Biol. Chem. 1996; 271: 24278-24285Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 25Min B. Foster D.N. Strauch A.R. J. Biol. Chem. 1990; 265: 16667-16675Abstract Full Text PDF PubMed Google Scholar, 26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar, 27Sun S. Stoflet E.S. Cogan J.G. Strauch A.R. Getz M.J. Mol. Cell. Biol. 1995; 15: 2429-2436Crossref PubMed Google Scholar). These studies resulted in the identification of a minimal enhancer element that was constitutively active in fibroblasts, immature myoblasts, and cultured aortic smooth muscle cells, but not in quiescent, differentiated skeletal muscle myocytes. The aim of the present work was to determine whether the mouse SmαA enhancer also conveyed the TGF-β1 response in fibroblasts and whether sequence elements and transcription factors previously identified as responsible for TGF-β1 inducibility in rat aortic smooth muscle cells also directed this effect in mouse fibroblasts. Our results indicate that TGF-β1 amplifies basal expression of the mouse SmαA enhancer by concerted action at five positive regulatory sites. Interestingly, stoichiometric changes in nuclear protein binding activity at these specific sites were not observed in TGF-β1-activated mouse fibroblasts. Whereas three of these sites bind activating proteins of known identity, proteins interacting with the previously identified TGF-β1 control element (TCE) plus a newly identified TGF-β1 hypersensitivity region (THR) in fibroblasts were determined to be the Sp1 and Sp3 transcriptional regulatory proteins. Our data indicate that both Sp1 and Sp3 can mediate basal expression from both the TCE and THR but convey TGF-β1-inducible expression only at the THR in the mouse SmαA enhancer. A rate-limiting role in the activation of the mouse SmαA enhancer is indicated for the TGF-β1 receptor-regulated Smad proteins previously shown to govern transcription of TGF-β1-dependent genes associated with wound healing and extracellular matrix remodeling in fibroblasts. AKR-2B embryonic fibroblasts were maintained in McCoy's 5A medium (Biowhittaker, Walkersville, MD) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen) and penicillin-streptomycin (20Cogan J.G. Sun S. Stoflet E.S. Schmidt L.J. Getz M.J. Strauch A.R. J. Biol. Chem. 1995; 270: 11310-11321Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar, 27Sun S. Stoflet E.S. Cogan J.G. Strauch A.R. Getz M.J. Mol. Cell. Biol. 1995; 15: 2429-2436Crossref PubMed Google Scholar). Nonhuman primate COS7 kidney fibroblasts were maintained in Dulbecco's modified Eagle's medium (4.5 g/liter d-glucose) supplemented with penicillin-streptomycin and 10% FBS. Primary cultures of mouse embryonic fibroblasts derived from wild-type and Smad3 knockout mice were kindly provided by Dr. X.-F. Wang (Duke University, Durham, NC) and maintained in Dulbecco's modified Eagle's medium containing 10% FBS and penicillin-streptomycin. All fibroblast preparations were rendered quiescent by a 48-h exposure to HEPES-buffered Dulbecco's modified Eagle's medium (1.0 g/liter d-glucose), 0.5% FBS, and penicillin-streptomycin. Recombinant human TGF-β1 (2–5 ng/ml, final concentration; R&D Systems, Minneapolis, MN) or an equivalent volume of vehicle (1 mg/ml bovine serum albumin, 4 mm HCl) was added to fibroblast cultures for varying periods before preparation of cell extracts for either reporter gene determinations or electrophoretic mobility shift assays. Mithramycin A (0.1–0.8 μm, final concentration; Sigma) was used in some experiments as specified in the figure legends to specifically block Sp1 binding at GC-rich motifs within the SmαA enhancer (30Greenwel P. Imagaki Y., Hu, W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Plasmids used for transfections were purified using QiagenTM preparative resin and a protocol provided by the manufacturer (Qiagen, Chatsworth, CA). In studies using AKR-2B and mouse embryonic fibroblasts, SmαA enhancer:CAT reporter gene fusion plasmids were combined with 5 μg of either pSV-β-galactosidase (Promega, Madison, WI) or pXGH5 (28Seldin R.F. Burke-Howie K. Rowe M.E. Goodman H.M. Moore D.D. Mol. Cell. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (470) Google Scholar) reporter gene constructs to normalize CAT expression for variation in transfection efficiency. pXGH5 encodes secreted human growth hormone under control of the constitutive metallothionein promoter. CAT, β-galactosidase, and human growth hormone assays were performed in triplicate using commercial enzyme-linked immunosorbent assay kits (Roche Molecular Biochemicals) in accordance with the manufacturer's instructions. For Sp and Smad protein overexpression studies using high transfection efficiency SL2 and COS7 cells (see below), SmαA:CAT fusion gene output was normalized to total protein rather than output from a secondary β-galactosidase or human growth hormone reporter gene. This was done because direct trans-regulation of the pSV-β-galactosidase and pXGH5 promoters by the overexpressed proteins could compromise their role in monitoring transfection efficiency. For transfection, cells at 50% confluence in 6-well plates first were washed with serum- and antibiotic-free medium. Optimized mixtures of SmαA promoter:reporter fusion plasmids (pC3VSMP4 and pC3VSMP3) and plasmids encoding various transcriptional regulatory proteins (see below) were combined (total plasmid payloads were between 0.5 and 2.5 μg) along with 15 μl of LipofectAMINETM reagent (Invitrogen) and incubated for 30 min at room temperature before applying them to cell monolayer for a 5-h period. After washing out unincorporated DNA, the cells were maintained in complete medium for an additional 36–48 h before harvesting for reporter gene assays as described above. Transfections were routinely performed in triplicate, and each experiment was repeated three to five times. Mean values for normalized CAT activity (±S.E.) were evaluated by analysis of variance with statistical significance set at p ≤ 0.05. Schneider Drosophila 2 (SL2; kindly provided by Dr. M. Seeger; Ohio State University) cells were grown in Schneider's medium (Invitrogen) supplemented with penicillin, streptomycin, and 10% FBS. For transfection, replicate preparations of SL2 cells were plated at 50% confluence 1 day before DNA delivery and washed briefly with 2 ml of serum-free Schneider's medium just before transfection. For each transfection, 2 μg of SmαA promoter:reporter fusion plasmid (pC3VSMP3 or pC3VSMP4) plus 4 μg of Sp insect expression plasmid (empty pPAC vector, pPACSp1, and pPACSp3 driven by theDrosophila actin promoter and kindly provided by Dr. G. Suske; University of Marburg) were combined with 9 μl of CellfectinTM reagent (Invitrogen), incubated for 15 min at room temperature, diluted with serum-free medium, and then distributed to SL2 cells. After 20 h, the DNA-containing medium was removed, and the cells were washed briefly and incubated with complete growth medium for 48 h before harvesting extracts for CAT assays. Recombinant forms of human Sp1 and Sp3 (kindly provided by Dr. J. Horowitz; North Carolina State University) and the human Smad proteins (kindly provided by Drs. L. Choy and R. Derynck; UCSF) were subcloned into mammalian expression plasmids under control of the cytomegalovirus promoter. Transversion mutants of the VSM α-actin promoter:CAT reporter fusion constructs pC3VSMP3 and pC3VSMP4 (abbreviated henceforth in this work as VSMP3 or SMP3 and VSMP4 or SMP4) were constructed using PCR amplification or site-directed mutagenesis using a commercial mutagenesis kit (Altered SitesTM; Promega). Custom oligonucleotide primers harboring transversion mutations of nucleotides comprising regulatory elements in VSMP3 and VSMP4 were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Primers were annealed to 0.1–1 μg of template DNA (VSMP4) and amplified for 30–35 cycles in a PerkinElmer Life Sciences DNA ThermacyclerTM using PCR kit (PerkinElmer Life Sciences) reagents and reaction times (94 °C × 1 min; 50 °C × 2 min; 72 °C × 3 min). PCR products were purified from 1–2% agarose gel slices using Spin-XTM columns (Costar, Cambridge, MA). The promoter construct harboring the MCAT mutation (MCAT-mut) was created by amplifying the VSMP4 insert with a forward primer containing a 5′SalI restriction site and substituting CTTCCGT for the native MCAT site between −182 and −176. The promoter construct harboring the THR mutation (THR-mut) was created by amplifying the VSMP4 insert with a forward primer containing a 5′ SalI restriction site that substitutes CTTA for AGGC between −160 and −157. Mutation of the putative TGF-β1 control element (TCE-mut) was made using a primer that substituted TT for GG at positions −50 and −49 in VSMP4. Promoters harboring the downstream element mutation (DE-mut) were created by amplification with a reverse primer containing a 3′ BamH1 restriction site and substituting AATTTCACAACAA for CCGGGACACCACC between −2 and +11 in both VSMP3 and VSMP4 context. Promoter constructs harboring StuI recognition site substitution mutations in place of CARG-like elements A, B, or D (A-mut, B-mut, and D-mut, respectively) were prepared as described previously (26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar). All sequence modifications were confirmed by double-stranded dideoxy sequencing. EMSA reactions typically contained 5–10 μg of nuclear protein extract, 1 μg of poly(dI-dC), 10 mm Tris, pH 7.5, 50 mm NaCl, 0.5 mmdithiothreitol, 0.5 mm EDTA, 0.12 mmphenylmethylsulfonyl fluoride, 4% glycerol, and 20,000 cpm (5–50 fmol) of 32P-labeled probe in a 10-μl reaction volume. EMSA probes were constructed in native copy number context with the exception of the CARG D oligonucleotide, which contained a dimerized version of the native motif to improve binding affinity. Mutated versions of each EMSA probe contained nucleotide substitutions identical to those contained in the reporter gene counterparts described in the previous section. Oligonucleotide probes were labeled with Klenow enzyme (Invitrogen) and [γ-32P]ATP (ICN Biomedical, Costa Mesa, CA), purified by electrophoresis on 8% polyacrylamide gels, eluted, and ethanol-precipitated before use. EMSA reaction mixtures were incubated for 30 min at room temperature before electrophoretic analysis on 5% nondenaturing polyacrylamide gels in 0.5× TBE buffer (0.045 m Tris borate and 1 mmEDTA). For competition EMSAs, an excess of unlabeled oligonucleotide competitor was added to the reaction before adding protein extract or labeled probe. Antibody supershift EMSAs using commercial Sp1 and Sp3 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were performed as described above but included a 20 min preincubation with 2 μl of anti-Sp antibody before the addition of probe. Purified mouse IgG was used as a negative control in antibody supershift experiments. Reaction mixtures containing nuclear extract (100 μg of protein) and biotinylated oligonucleotides (100 pmol; Integrated DNA Technologies) corresponding to the Sp1 consensus site, native TCE site, or mutated TCE site (TCE-mut) were incubated under conditions that mimicked an EMSA reaction. Sp1/Sp3-biotin-DNA complexes were captured on streptavidin-immobilized paramagnetic particles (Promega; 0.6 ml/reaction, 30-min incubation). After washing four times with buffer containing 25 mm Tris-HCl, pH 7.5, 1 mmEDTA, and 100 mm NaCl, bound protein was eluted using 2× protein denaturation buffer and analyzed by Western blot (22Kelm Jr., R.J. Cogan J.G. Elder P.K. Strauch A.R. Getz M.J. J. Biol. Chem. 1999; 274: 14238-14245Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Eluted protein was processed by SDS-PAGE using 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schüll). After overnight blocking at 4 °C in TBS (25 mm Tris-HCl, pH 7.5, 150 mm NaCl) containing 3% (w/v) nonfat dry milk, blots were incubated with anti-Sp1 or anti-Sp3 rabbit polyclonal antibodies for a 60–90-min incubation at ambient temperature with gentle mixing. Goat anti-rabbit IgG-HRP (horseradish peroxidase) (Vector Laboratories, Burlingame, CA) diluted 1:2000 in TBST (TBS containing 0.05% Tween 20) was then applied for 30–45 min. After washing in TBST, the blot was incubated with VectastainTM reagent for 30 min. Blots were washed four times over a 30-min period and processed for chemiluminescence development for 1 min (ECLTM, reagents; PerkinElmer Life Sciences), and the immune complexes were visualized on x-ray film (X-Omat; Eastman Kodak) after a 5–10-s exposure. Both antibodies recognized multiple Sp1 and Sp3 isoforms. Northern blot analysis was performed using a SmαA-specific 3′(untranslated region) cDNA probe as described previously (15Becker N.A. Kelm Jr., R.J. Vrana J.A. Getz M.J. Maher L.J.I. J. Biol. Chem. 2000; 275: 15384-15391Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 53Lee S. Reeser J. Dillman J. Strauch A.R. J. Cell. Physiol. 1995; 164: 172-186Crossref PubMed Scopus (4) Google Scholar). Basal transcription of the native SmαA gene in mouse AKR-2B stromal fibroblasts was minimally enhanced 6-fold up to as much as 20-fold in higher cell density preparations within 6 h of treatment with TGF-β1 (Fig.1 a). To localize sequence elements responsible for TGF-β1 activation, we performed deletion analysis of a 3.6-kb segment of the mouse SmαA promoter (VSMP8) that exhibits correct developmental regulation in transgenic mice. At the low cell density required for efficient transfection, VSMP8 transcription was increased ∼4-fold by TGF-β1, indicating that elements contained within the 5′-flanking/first intronic region mediated growth factor responsiveness (Fig. 1, b andc). A subfragment of the 5′-flanking region, located between −191 and +46 relative to the start of transcription (Fig.1 b), contains a potent core enhancer element previously shown to contain a MCAT motif required for high-level constitutive expression and serum inducibility in stromal fibroblasts (21Foster D.N. Min B. Foster L.K. Stoflet E.S. Sun S. Getz M.J. Strauch A.R. J. Biol. Chem. 1992; 267: 11995-12003Abstract Full Text PDF PubMed Google Scholar, 26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar), aortic smooth muscle cells (25Min B. Foster D.N. Strauch A.R. J. Biol. Chem. 1990; 265: 16667-16675Abstract Full Text PDF PubMed Google Scholar, 29Carlini L.E. Getz M.J. Strauch A.R. Kelm Jr., R.J. J. Biol. Chem. 2002; 277: 8682-8692Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and undifferentiated striated muscle myoblasts (21Foster D.N. Min B. Foster L.K. Stoflet E.S. Sun S. Getz M.J. Strauch A.R. J. Biol. Chem. 1992; 267: 11995-12003Abstract Full Text PDF PubMed Google Scholar). The VSMP4 construct containing this enhancer was induced about 6-fold by TGF-β1. Further 5′ truncation by 41 bp (VSMP5) or 101 bp (VSMP6) completely eliminated basal and TGF-β1-inducible transcriptional activity, indicating that the region of the mouse SmαA promoter between −191 and −150 was minimally required to mediate the TGF-β1 response in fibroblasts (Fig. 1,b and c). Site-specific mutations were created within VSMP4 context to evaluate the relative importance of the known positive elements in mediating TGF-β1 inducibility in fibroblasts. Altered sites included an inverted MCAT motif (AGGAATG, between −182 and −176) and CARG-like elements B and A (CCCTATATGG, between −120 and −111, and CCTTGTTTGG, between −70 and −61) that bind the TEF1 and serum response factor transcriptional activating proteins, respectively. Both sites were required for basal expression of VSMP4 in mouse fibroblasts (24Kelm Jr., R.J. Sun S. Strauch A.R. Getz M.J. J. Biol. Chem. 1996; 271: 24278-24285Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar), neonatal rat aortic A7r5 smooth muscle cells (29Carlini L.E. Getz M.J. Strauch A.R. Kelm Jr., R.J. J. Biol. Chem. 2002; 277: 8682-8692Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and undifferentiated mouse BC3H1 myoblasts (21Foster D.N. Min B. Foster L.K. Stoflet E.S. Sun S. Getz M.J. Strauch A.R. J. Biol. Chem. 1992; 267: 11995-12003Abstract Full Text PDF PubMed Google Scholar). Also included in the analysis was a variant containing a 4-bp transversion (AGGC to CTTA) within a recently described 20-bp subdomain of VSMP4 that exhibited differential reactivity to DNA-modifying agents in the presence and absence of TGF-β1 (15Becker N.A. Kelm Jr., R.J. Vrana J.A. Getz M.J. Maher L.J.I. J. Biol. Chem. 2000; 275: 15384-15391Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). This region, located between −170 and −150, is referred to as the THR. A construct containing a mutation within a putative TCE located between −53 and −43, previously identified in the rat SmαA promoter (16Hautmann M.B. Madsen C.S. Owens G.K. J. Biol. Chem. 1997; 272: 10948-10956Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), was also included in the analysis. Fig.2 shows that all mutations exhibited significantly reduced basal expression relative to native VSMP4. However, all VSMP4 mutants retained TGF-β1 inducibility, except for the construct harboring a THR mutation. Whereas fold induction in the presence of TGF-β1 (relative to that exhibited by native VSMP4) was highest for the construct lacking a functional MCAT element, mutations in the TCE, CARG A, or CARG B motifs all showed approximately the same fold increase. The slight induction exhibited by the THR mutation was not statistically significant (Fig. 2). Thus, with the exception of the THR, mutation of all other activating elements (MCAT, CARG B, CARG A, and TCE) did not fully eliminate the mouse SmαA enhancer responsiveness to TGF-β1 in transfected fibroblasts. EMSAs were performed using extracts prepared from TGF-β1-treated fibroblasts to examine nuclear protein binding to the SmαA enhancer. No net increase in binding activity to the MCAT, CARG B, CARG A, and TCE probes was observed after treatment with TGF-β1 (data not shown). Protein binding specificity was confirmed by competition with unlabeled excess DNA (see below). In the mouse SmαA enhancer, CARG B was previously shown to bind serum response factor (26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar, 29Carlini L.E. Getz M.J. Strauch A.R. Kelm Jr., R.J. J. Biol. Chem. 2002; 277: 8682-8692Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), whereas the MCAT element binds TEF1 (20Cogan J.G. Sun S. Stoflet E.S. Schmidt L.J. Getz M.J. Strauch A.R. J. Biol. Chem. 1995; 270: 11310-11321Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 26Stoflet E.S. Schmidt L.J. Elder P.K. Korf G.M. Foster D.N. Strauch A.R. Getz M.J. Mol. Biol. Cell. 1992; 3: 1073-1083Crossref PubMed Scopus (37) Google Scholar, 27Sun S. Stoflet E.S. Cogan J.G. Strauch A.R. Getz M.J. Mol. Cell. Biol. 1995; 15: 2429-2436Crossref PubMed Google Scholar). Whereas the identity of the mouse fibroblast TCE-binding protein was not known, an identical binding activity was noted when extracts were assayed using a probe cont
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