Osteopontin Transcription in Aortic Vascular Smooth Muscle Cells Is Controlled by Glucose-regulated Upstream Stimulatory Factor and Activator Protein-1 Activities
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m206235200
ISSN1083-351X
AutoresMiri Bidder, J.-S. Shao, Nichole Charlton-Kachigian, Arleen P. Loewy, Clay F. Semenkovich, Dwight A. Towler,
Tópico(s)dental development and anomalies
ResumoThe expression of the matrix cytokine osteopontin (OPN) is up-regulated in aortic vascular smooth muscle cells (VSMCs) by diabetes. OPN expression in cultured VSMCs is reciprocally regulated by glucose and 2-deoxyglucose (2-DG; inhibitor of cellular glucose metabolism). Systematic analyses of OPNpromoter-luciferase reporter constructs identify a CCTCATGAC motif at nucleotides −80 to −72 relative to the initiation site that supports OPN transcription in VSMCs. The region −83 to −45 encompassing this motif confers basal and glucose- and 2-DG-dependent transcription on an unresponsive promoter. Competition and gel mobility supershift assays identify upstream stimulatory factor (USF; USF1:USF2) and activator protein-1 (AP1; c-Fos:c-Jun) in complexes binding the composite CCTCATGAC element. Glucose up-regulates both AP1 and USF binding activities 2-fold in A7r5 cells and selectively up-regulates USF1 protein levels. By contrast, USF (but not AP1) binding activity is suppressed by 2-DG and restored by glucose treatment. Expression of either USF or AP1 activates the proximal OPN promoter in A7r5 VSMCs in part via the CCTCATGAC element. Moreover, glucose stimulates the transactivation functions of c-Fos and USF1, but not c-Jun, in one-hybrid assays. Mannitol does not regulate binding, transactivation functions, USF1 protein accumulation, or OPN transcription. Thus, OPN gene transcription is regulated by USF and AP1 in aortic VSMCs, entrained to changes in cellular glucose metabolism. The expression of the matrix cytokine osteopontin (OPN) is up-regulated in aortic vascular smooth muscle cells (VSMCs) by diabetes. OPN expression in cultured VSMCs is reciprocally regulated by glucose and 2-deoxyglucose (2-DG; inhibitor of cellular glucose metabolism). Systematic analyses of OPNpromoter-luciferase reporter constructs identify a CCTCATGAC motif at nucleotides −80 to −72 relative to the initiation site that supports OPN transcription in VSMCs. The region −83 to −45 encompassing this motif confers basal and glucose- and 2-DG-dependent transcription on an unresponsive promoter. Competition and gel mobility supershift assays identify upstream stimulatory factor (USF; USF1:USF2) and activator protein-1 (AP1; c-Fos:c-Jun) in complexes binding the composite CCTCATGAC element. Glucose up-regulates both AP1 and USF binding activities 2-fold in A7r5 cells and selectively up-regulates USF1 protein levels. By contrast, USF (but not AP1) binding activity is suppressed by 2-DG and restored by glucose treatment. Expression of either USF or AP1 activates the proximal OPN promoter in A7r5 VSMCs in part via the CCTCATGAC element. Moreover, glucose stimulates the transactivation functions of c-Fos and USF1, but not c-Jun, in one-hybrid assays. Mannitol does not regulate binding, transactivation functions, USF1 protein accumulation, or OPN transcription. Thus, OPN gene transcription is regulated by USF and AP1 in aortic VSMCs, entrained to changes in cellular glucose metabolism. Individuals with diabetes are characteristically afflicted with medial artery calcification (1Niskanen L.K. Suhonen M. Siitonen O. Lehtinen J.M. Uusitupa M.I. Atherosclerosis. 1990; 84: 61-71Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 2Niskanen L. Siitonen O. Suhonen M. Uusitupa M.I. Diabetes Care. 1994; 17: 1252-1256Crossref PubMed Scopus (138) Google Scholar, 3Lehto S. Niskanen L. Suhonen M. Ronnemaa T. Laakso M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 978-983Crossref PubMed Scopus (486) Google Scholar, 4Nicolosi A.C. Pohl L.L. Parsons P. Cambria R.A. Olinger G.N. J. Surg. Res. 2002; 102: 1-5Abstract Full Text PDF PubMed Scopus (40) Google Scholar). The pathobiology of diabetes-associated calcific vasculopathy is poorly understood. Medial calcification occurs via a mineralization process that does not form cartilage, with calcium deposition occurring in the collagenous extracellular matrix associated with matrix vesicles adjacent to arterial VSMCs 1The abbreviations used are: VSMC, vascular smooth muscle cells; AP1, activator protein 1; BMP2, bone morphogenetic proteins 2; CMV, cytomegalovirus immediate early promoter; 2-DG, 2-deoxyglucose; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; Gal4RE, Gal4 response element; GAPD, glyceraldehyde-3-phosphate dehydrogenase; G4DBD, Gal4 DNA-binding domain; LDLR, low density lipoprotein receptor; LUC, luciferase gene; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MMP1, matrix metalloproteinase 1; MUT, duplex oligodeoxynucleotide mutant; oligo, oligonucleotide; 3-OMG, 3-O-methylglucose; OPN, osteopontin; pFRLUC, Gal4-responsive LUC reporter plasmid; RSK2, ribosomal subunit kinase 2; USF, upstream stimulatory factor; RSV, Rous sarcoma virus; RT, reverse transcription (5Tanimura A. McGregor D.H. Anderson H.C. J. Exp. Pathol. 1986; 2: 261-273PubMed Google Scholar). Initially thought to be benign, medial calcification has emerged as one predictor of cardiovascular mortality (2Niskanen L. Siitonen O. Suhonen M. Uusitupa M.I. Diabetes Care. 1994; 17: 1252-1256Crossref PubMed Scopus (138) Google Scholar, 3Lehto S. Niskanen L. Suhonen M. Ronnemaa T. Laakso M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 978-983Crossref PubMed Scopus (486) Google Scholar). The mechanism whereby medial calcification conveys excess mortality are unknown, but vascular stiffening prevents the compensatory vasodilatation that decreases afterload and myocardial oxygen consumption with exertion, thus exacerbating myocardial ischemia from concomitant atherosclerotic coronary disease (6Hickler R.B. Clin. Cardiol. 1990; 13: 317-322Crossref PubMed Scopus (108) Google Scholar, 7Lehmann E.D. Hopkins K.D. Rawesh A. Joseph R.C. Kongola K. Coppack S.W. Gosling R.G. Hypertension. 1998; 32: 565-569Crossref PubMed Scopus (146) Google Scholar, 8Cohn J.N. Am. J. Hypertens. 2001; 14 (suppl.): 258S-263SCrossref Google Scholar, 9Blacher J. Guerin A.P. Pannier B. Marchais S.J. London G.M. Hypertension. 2001; 38: 938-942Crossref PubMed Scopus (1239) Google Scholar, 10Demer L.L. Circulation. 1991; 83: 2083-2093Crossref PubMed Scopus (90) Google Scholar). We recently reported the development and initial characterization of a murine model of diet-induced, insulin-resistant diabetic calcific vasculopathy (11Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Feeding LDLR −/− mice high fat diets induces insulin-resistant diabetes and dyslipidemia, with associated mineral deposition in the fibrosa of the valve leaflets and the arterial wall. As initially proposed by Demer and colleagues (12Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (905) Google Scholar, 13Parhami F. Tintut Y. Patel J.K. Mody N. Hemmat A. Demer L.L. Z. Kardiol. 2001; 90 Suppl. 3: 27-30PubMed Google Scholar) from studies of atherosclerotic plaques, our data suggest that an active osteogenic program is initiated during aortic calcification by the dysmetabolic stimuli of diabetes that promote medial calcific vasculopathy (11Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Aortic expression of the bone matrix protein osteopontin (OPN) is up-regulated and is detected in several aortic cell types including activated macrophages of atheroma, vascular smooth muscle cells of the tunica media, and adventitial and valvular fibrosal cells (11Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 14Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar). OPN is a multifunctional protein produced by osteoblasts during skeletal development and in VSMCs, monocytes, and T-cells in response to biological stressors (14Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar, 15O'Brien E.R. Garvin M.R. Stewart D.K. Hinohara T. Simpson J.B. Schwartz S.M. Giachelli C.M. Arterioscler. Thromb. 1994; 14: 1648-1656Crossref PubMed Google Scholar). As an extracellular matrix protein, OPN controls cell migration mediated by αvβ3 integrin receptors (14Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar, 16Panda D. Kundu G.C. Lee B.I. Peri A. Fohl D. Chackalaparampil I. Mukherjee B.B., Li, X.D. Mukherjee D.C. Seides S. Rosenberg J. Stark K. Mukherjee A.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9308-9313Crossref PubMed Scopus (153) Google Scholar). Via signaling functions dependent upon phosphorylation, OPN inhibits mineral deposition (17Wada T. McKee M.D. Steitz S. Giachelli C.M. Circ. Res. 1999; 84: 166-178Crossref PubMed Scopus (401) Google Scholar). OPN also functions as a proinflammatory cytokine produced by stimulated monocytes and T-cells (14Denhardt D.T. Noda M. O'Regan A.W. Pavlin D. Berman J.S. J. Clin. Invest. 2001; 107: 1055-1061Crossref PubMed Scopus (917) Google Scholar), augmenting monocyte/macrophage activation via up-regulation of interleukin-12 and suppression of interleukin-10 (18Ashkar S. Weber G.F. Panoutsakopoulou V. Sanchirico M.E. Jansson M. Zawaideh S. Rittling S.R. Denhardt D.T. Glimcher M.J. Cantor H. Science. 2000; 287: 860-864Crossref PubMed Scopus (970) Google Scholar). Very recently, Mori and co-workers (19Takemoto M. Yokote K. Yamazaki M. Ridall A.L. Butler W.T. Matsumoto T. Tamura K. Saito Y. Mori S. Ann. N. Y. Acad. Sci. 2000; 902: 357-363Crossref PubMed Scopus (30) Google Scholar) extended our work (11Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) to analyses of human diabetic calcific vasculopathy. They identified that immunoreactive OPN was in fact up-regulated in medial vascular smooth muscles cells in arteries of diabetic patients (19Takemoto M. Yokote K. Yamazaki M. Ridall A.L. Butler W.T. Matsumoto T. Tamura K. Saito Y. Mori S. Ann. N. Y. Acad. Sci. 2000; 902: 357-363Crossref PubMed Scopus (30) Google Scholar). Highly relevant to arterial vasculopathy of diabetes (20Christian R.C. Harrington S. Edwards W.D. Oberg A.L. Fitzpatrick L.A. J. Clin. Endocrinol. Metab. 2002; 87: 1062-1067Crossref PubMed Scopus (62) Google Scholar), Thompson and co-workers (21Li G. Chen Y.F. Kelpke S.S. Oparil S. Thompson J.A. Circulation. 2000; 101: 2949-2955Crossref PubMed Scopus (49) Google Scholar) have demonstrated that the adventitial mesenchymal cell population is migratory, moving into the tunica media and neointima in response to biomechanical injury. A role is thus proposed for OPN in the migration of VSMCs and adventitial mesenchymal progenitors in the diseased aorta. Therefore, the enhanced expression of OPN in the arterial vasculature in response to diabetes is likely to participate in disease progression, regulating macrophage functions, adventitial cell migration, and calcium deposition (17Wada T. McKee M.D. Steitz S. Giachelli C.M. Circ. Res. 1999; 84: 166-178Crossref PubMed Scopus (401) Google Scholar). The goal of this study was to characterize transcription factor complexes that confer basal and regulated OPN gene expression in aortic VSMCs relevant to the pathobiology of diabetic calcific vasculopathy. We have identified that OPN gene expression is differentially regulated by glucose and 2-deoxyglucose (2-DG) in cultured primary aortic mesenchymal cells that express VSMC α-actin. We cloned the 2-kb mouse OPN promoter (−1976 to +78, numbered relative to the start site of transcription) (22Miyazaki Y. Setoguchi M. Yoshida S. Higuchi Y. Akizuki S. Yamamoto S. J. Biol. Chem. 1990; 265: 14432-14438Abstract Full Text PDF PubMed Google Scholar) and evaluated the activity in A7r5 rat aortic VSMCs, a rodent cell line that faithfully reproduces the gene expression protein elicited by aortic VSMCs in vivo (23Solway J. Seltzer J. Samaha F.F. Kim S. Alger L.E. Niu Q. Morrisey E.E., Ip, H.S. Parmacek M.S. J. Biol. Chem. 1995; 270: 13460-13469Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 24Chang P.S., Li, L. McAnally J. Olson E.N. J. Biol. Chem. 2001; 276: 17206-17212Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Glucose (≥5 mm) selectively up-regulates activity of the OPN promoter. By contrast, mannitol, an osmotic control, and 3-O-methyl glucose, a nonmetabolized glucose an analog, have no effect. We identified that nucleotides −83 to −46 relative to the transcription initiation site encompass information necessary and sufficient for high level transcriptional activity in VSMCs. This 38-bp fragment of theOPN promoter confers basal and glucose metabolism-dependent transcription onto an unresponsive heterologous minimal promoter. A CCTCATGAC motif at −80 to −72 that is recognized by USF1, USF2, c-Fos, and c-Jun supports OPNpromoter activity in VSMCs. These complexes are regulated by glucose. Both USF1 binding and protein accumulation are reciprocally regulated during the manipulation of cellular glucose metabolism with glucose and 2-DG. Co-transfection studies demonstrate that USF (USF1:USF2) and AP1 (cFos:cJun) support OPN transcription and that the transactivation functions of c-Fos and USF1 are specifically enhanced by glucose. Thus, basal and regulated protein-DNA interactions at the CCTCATGAC motif at −80 to −72 participate in OPN gene expression responses in VSMC during glucose-dependent initiation of diabetic vasculopathy. USF and AP1 contribute to the glucose-dependent regulation of arterial VSMC OPN expression in diabetes. Tissue culture supplies and custom synthetic oligodeoxynucleotides were obtained from Invitrogen. Reagents for protein preparation were obtained from Pierce, Sigma, and Fisher. Radionucleotides were obtained fromAmersham Biosciences. A7r5 cells (ATCC CRL-1444) were maintained as described previously (25Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 349-366Crossref PubMed Scopus (322) Google Scholar, 26Newberry E.P. Willis D. Latifi T. Boudreaux J.M. Towler D.A. Mol. Endocrinol. 1997; 11: 1129-1144Crossref PubMed Scopus (84) Google Scholar). Primary aortic adventitial mesenchymal cells were obtained from aortic explants essentially as detailed previously (27Diglio C.A. Grammas P. Giacomelli F. Wiener J. Lab. Invest. 1989; 60: 523-531PubMed Google Scholar) but using C57Bl/6 mice in lieu of rats. Cultures were passaged in DMEM with 4.5 g/liter glucose, 4 mm glutamine, and 10% fetal calf serum (and penicillin-streptomycin) to maintain the phenotype (25Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 349-366Crossref PubMed Scopus (322) Google Scholar). About 16 confluent 10-cm-diameter tissue culture dishes were obtained/eight 2-cm segments of murine aortae. Antibodies were purchased from Santa Cruz Biotechnology; specific antibodies include USF1 (sc-8983 or sc-229), USF2 (sc-861), c-Fos (sc-253 or sc-52), FosB (sc-7203), Fra1 (sc-183), Fra2 (sc-171), c-Jun (sc-44 or sc-45), phospho-c-Jun (sc-822), JunD (sc-74), Smad 1/5 (sc-6031), Oct1 (sc-232), Oct2 (sc-233), Oct4 (sc-9081), Gli1 (sc-6152 or sc-6153), and CTCF (sc-5916) or BTEB2 (sc-12998). A7r5 aortic VSMCs were maintained with DMEM containing 1 g/liter (5.5 mm) glucose. Treatment of A7r5 cells with either glucose or mannitol was performed in glucose-free DMEM, 10% glucose-free FCS (dialyzed against glucose-free DMEM) supplemented with either sterile water (0.1% v/v, vehicle; 0 mm glucose) or carbohydrates (5 mm glucose or mannitol, 50 mm glucose or mannitol, 100 mmglucose or mannitol as indicated). The nonmetabolized derivative of glucose, 3-O-methylglucose (3-OMG) was added as a control. Unlike A7r5 cells, primary aortic VSMCs do not adapt well to ex vivo culture under completely glucose-free conditions even with 4 mm glutamine provided as an alternative carbon source (25Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 349-366Crossref PubMed Scopus (322) Google Scholar,28Hall J.L. Matter C.M. Wang X. Gibbons G.H. Circ. Res. 2000; 87: 574-580Crossref PubMed Scopus (80) Google Scholar). 2M. Bidder, J.-S. Shao, N. Charlton-Kachigian, A. P. Loewy, C. F. Semenkovich, and D. A. Towler, our unpublished observations. Therefore, to permit a direct comparison of OPN mRNA accumulation between A7r5 and primary aortic cell cultures, cohorts were cultured in the presence of 2-DG, a competitive inhibitor of cellular glucose uptake and intracellular glucose metabolism (29Mueller W.M. Gregoire F.M. Stanhope K.L. Mobbs C.V. Mizuno T.M. Warden C.H. Stern J.S. Havel P.J. Endocrinology. 1998; 139: 551-558Crossref PubMed Scopus (354) Google Scholar), in the presence of glucose-free DMEM with undialyzed FCS (final glucose concentration ∼0.5 mm glucose) to pharmacologically induce acute and reproducible extremes in cellular glucose tone. Experiments designed to examine the reversibility of 2-DG actions by glucose were carried out in the presence or absence of 2 mm 2-DG. Primary aortic mesenchymal cells obtained from wild type adult C57Bl/6 mice were passaged once and then plated at 80% confluence in 10-cm tissue culture dishes. Subsequently, cells were treated for 24 h in 10% undialyzed FCS with glucose-free DMEM (∼0.5 mm residual glucose from the FCS) or in media supplemented with 2 mm2-deoxyglucose, 4 mm 2-deoxyglucose, 30 mmglucose, or 30 mm glucose + 2 mm2-deoxyglucose. At the end of the treatment period, total RNA was extracted as described previously (30Towler D.A. Rutledge S.J. Rodan G.A. Mol. Endocrinol. 1994; 8: 1484-1493Crossref PubMed Scopus (122) Google Scholar). An Applied Biosystems GeneAmp 5700 sequence detection system using Sybr Green dye binding to PCR product was used to quantify osteogenic mRNA accumulation via fluorescence RT-PCR (31Bustin S.A. J. Mol. Endocrinol. 2000; 25: 169-193Crossref PubMed Scopus (3059) Google Scholar). Primer Express 1.0 software was used to design amplimers from murine OPN and Msx2cDNA sequences. Amplimers for OPN are: 5′-GTA TTG CTT TTG CCT GTT TGG-3′ and 5′-TGA GCT GCC AGA ATC AGT CAC T-3′. Amplimers for murineMsx2 are: 5′-TCC CAG CTT CTA GCC TTG GA-3′ and 5′-CAG CCC GCT CTG CTAT GG-3′. Commercially available primers from Applied Biosystems were used to quantify GAPD expression for normalization in RT-PCR (TaqMan Rodent GAPDH Control Reagent, PE Biosytems, Palo Alto, CA). Standard curves were generated usingOPN and Msx2 DNA standards. Data are presented as the mean ± S.D. results from three independent replications of two independent experiments. Total cellular RNA was isolated from VSMCs and Northern blot analysis carried out as described previously (30Towler D.A. Rutledge S.J. Rodan G.A. Mol. Endocrinol. 1994; 8: 1484-1493Crossref PubMed Scopus (122) Google Scholar,32Newberry E.P. Latifi T. Towler D.A. Biochemistry. 1999; 38: 10678-10690Crossref PubMed Scopus (84) Google Scholar). Cultures of mouse aortic adventitial cells and A7r5 rat aortic VSMCs were evaluated for expression of OPN and VSMC α-actin by immunohistochemistry using the streptavidin-biotin-immunoperoxidase method (33Mark M.P. Prince C.W. Gay S. Austin R.L. Bhown M. Finkelman R.D. Butler W.T. J. Bone Miner. Res. 1987; 2: 337-346Crossref PubMed Scopus (69) Google Scholar). A mouse monoclonal antibody to human VMSC α-actin was obtained from a commercial source (product code CBL 171, clone asm-1, Cymbus Biotechnology). The osteopontin antibody utilized was MPIIIB10 (1Niskanen L.K. Suhonen M. Siitonen O. Lehtinen J.M. Uusitupa M.I. Atherosclerosis. 1990; 84: 61-71Abstract Full Text PDF PubMed Scopus (66) Google Scholar), which has been used previously to localize vascular OPN expression in calcified aortic specimens (34Bini A. Mann K.G. Kudryk B.J. Schoen F.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1852-1861Crossref PubMed Scopus (140) Google Scholar). This mouse monoclonal was developed by Michael Solursh and Ahnders Franzen (Dept. of Biological Sciences, University of Iowa, Iowa City) and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health, and maintained by the Department of Biological Sciences, University of Iowa. Immunostaining of cultured cells was performed using the DAKO Animal Research Kit with peroxidase histochemistry (DAKO Corp., Carpinteria, CA) as per the manufacturer's protocol, using diaminobenzidine with H2O2 as the chromagen substrate. Microscopic images of immunohistochemically stained cells were captured with a Nikon Coolpix 990 3.3 megapixel digital camera mounted to a Nikon Eclipse TS100 inverted microscope (Melville, NY). Mouse USF1 cDNA was obtained by PCR amplification (which also introduced convenient 5′- and 3′ linkers) from commercially available murine heart cDNA (Clontech Marathon-Ready cDNA; Clontech, Palo Alto, CA) and subcloned into theKpnI/BamHI sites of pcDNA3 (Invitrogen). In addition, coupled in vitro transcription/translation (Promega, Madison, WI) was used to verify protein production using techniques detailed previously (35Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar). The same techniques were used to develop the eukaryotic expression constructs for c-Fos and c-Jun in pcDNA3. The expression vector for USF2 (kindly provided by Dr. M. Sawadogo) has been described previously (36Luo X. Sawadogo M. Mol. Cell. Biol. 1996; 16: 1367-1375Crossref PubMed Scopus (118) Google Scholar). PATHDETECT vectors for eukaryotic expression of Gal4DBD fusion proteins (pFACMV) and the Gal4RE-LUC reporter (pFRLUC) were purchased from Stratagene. pFACMV was used to assemble the eukaryotic expression plasmid for Gal4DBD-USF1. The synthesis of 1976 OPNLUC (mouse OPN promoter fragment −1976 to +78 in pGL2 Basic; numbering as per Yamamoto and colleagues (22Miyazaki Y. Setoguchi M. Yoshida S. Higuchi Y. Akizuki S. Yamamoto S. J. Biol. Chem. 1990; 265: 14432-14438Abstract Full Text PDF PubMed Google Scholar), GenbankTM accession no. D14816) was obtained by PCR using mouse genomic DNA as a template, applying techniques described previously (37Towler D.A. Bennett C.D. Rodan G.A. Mol. Endocrinol. 1994; 8: 614-624Crossref PubMed Scopus (88) Google Scholar). OPN promoter deletions, point mutants, and heterologous promoter constructs were generated using methods as detailed (38Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). RSVLUC (38Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and CMVLUC (39Rifas L. Towler D.A. Avioli L.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7549-7554Crossref PubMed Scopus (41) Google Scholar) promoter-reporter constructs have been described previously. All constructs were sequenced to verify fidelity (Applied Biosystems Prism Dye Terminator Kit, Foster City, CA). A7r5 aortic VSMCs were transfected using LipofectAMINE (Invitrogen). CMV β-galactosidase (300 ng) was included as an internal control for transfection efficiency. One day after transfection, cultures were rinsed with glucose-free DMEM and then fed with glucose-free DMEM, 10% dialyzed FCS serum supplemented with glucose, mannitol, 3-OMG, or 2-DG as indicated. After 3 days, cellular luciferase and β-galactosidase activities were measured as detailed previously (38Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 40Newberry E.P. Boudreaux J.M. Towler D.A. J. Biol. Chem. 1997; 272: 29607-29613Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Statistical analyses were carried out as previously described (39Rifas L. Towler D.A. Avioli L.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7549-7554Crossref PubMed Scopus (41) Google Scholar). Crude extracts for gel shift analyses were prepared from control, glucose, and/or 2-DG treated cells (see above) as detailed previously (26Newberry E.P. Willis D. Latifi T. Boudreaux J.M. Towler D.A. Mol. Endocrinol. 1997; 11: 1129-1144Crossref PubMed Scopus (84) Google Scholar). The radiolabeledOPN promoter duplex oligo fragments (see Fig. 4) were used to detect DNA-protein interactions by gel shift and supershift as described. Western blots were carried out with chemiluminescent immunodetection (Tropix, Bedford, MA) as described previously (26Newberry E.P. Willis D. Latifi T. Boudreaux J.M. Towler D.A. Mol. Endocrinol. 1997; 11: 1129-1144Crossref PubMed Scopus (84) Google Scholar,41Bidder M. Loewy A.P. Latifi T. Newberry E.P. Ferguson G. Willis D.M. Towler D.A. Biochemistry. 2000; 39: 8917-8928Crossref PubMed Scopus (17) Google Scholar). Previously, we identified that OPN gene expression is up-regulated in aortic VSMCs, adventitial cells, and macrophages of LDLR−/− mice fed diabetogenic diets. In situ hybridization studies revealed that aortic mesenchymal OPN expression overlaps the pattern of VSMC α-actin in adventitial cells, mural VSMCs, and valvular fibrosal cells (11Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). We wished to establish a cell culture model for studying OPN transcription in aortic mesenchymal cells. Therefore, we studied the expression and regulation of OPN in primary aortic mesenchymal cell cultures and the A7r5 aortic VSMC line, a rat aortic VSMC line that faithfully recapitulates the transcriptional regulatory features of arterial smooth muscle cells (23Solway J. Seltzer J. Samaha F.F. Kim S. Alger L.E. Niu Q. Morrisey E.E., Ip, H.S. Parmacek M.S. J. Biol. Chem. 1995; 270: 13460-13469Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 24Chang P.S., Li, L. McAnally J. Olson E.N. J. Biol. Chem. 2001; 276: 17206-17212Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). As in A7r5 VSMCs (Fig.1 A), immunohistochemical analysis of primary aortic mesenchymal cells (Fig.1 D) demonstrates robust staining of >80% of cells with VSMC-specific α-actin, indicative of smooth muscle phenotype (42Halayko A.J. Solway J. J. Appl. Physiol. 2001; 90: 358-368Crossref PubMed Scopus (235) Google Scholar,43Walsh K. Takahashi A. Z. Kardiol. 2001; 90 Suppl 3: 12-16PubMed Google Scholar). Immunohistochemistry further identified the expression of OPN in both A7r5 cells and primary murine aortic cells; in A7r5 cells, the prominent perinuclear Golgi staining described by others (33Mark M.P. Prince C.W. Gay S. Austin R.L. Bhown M. Finkelman R.D. Butler W.T. J. Bone Miner. Res. 1987; 2: 337-346Crossref PubMed Scopus (69) Google Scholar) was readily apparent. Northern blot and RT-PCR analyses confirmed expression of OPN mRNA in these cells and identified response to alterations in glucose treatment and metabolism. As shown in Fig. 2 A, treatment of primary cultures of aortic mesenchymal VSMCs up-regulatedOPN mRNA accumulation by ∼2-fold. By contrast, treatment with 2-DG dose dependently and selectively down-regulatedOPN mRNA accumulation by 80% (Fig. 2 B;GAPD normalized) as quantified by fluorescence RT-PCR analyses. The Msx2 gene was regulated in culture in a completely different manner; glucose suppressed Msx2mRNA accumulation, and 2-DG actually augmented Msx2expression 3-fold (Fig. 2 B), demonstrating that the acute exposure to 2-DG is not toxic to primary aortic cells. Although expressed at much higher levels, OPN mRNA accumulation was similarly regulated by 2-DG and glucose in A7r5 cells (Fig.2 C; and see below), and 5–50 mm mannitol did not regulate OPN in this VSMC line (data not shown, and see below). Thus, as noted in vivo in the VSMCs and adventitial cells of diseased aortae (11Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 15O'Brien E.R. Garvin M.R. Stewart D.K. Hinohara T. Simpson J.B. Schwartz S.M. Giachelli C.M. Arterioscler. Thromb. 1994; 14: 1648-1656Crossref PubMed Google Scholar), cultured aortic mesenchymal cells of the VSMC lineage express the OPN gene under basal conditions. The aortic mesenchymal cell OPN gene in vitro is responsive to glucose concentrations and pharmacological manipulation of cellular glucose metabolism (29Mueller W.M. Gregoire F.M. Stanhope K.L. Mobbs C.V. Mizuno T.M. Warden C.H. Stern J.S. Havel P.J. Endocrinology. 1998; 139: 551-558Crossref PubMed Scopus (354) Google Scholar).Figure 2Regulation of OPN gene expression in aortic mesenchymal cells by manipulation of cellular glucose homeostasis. A, Northern blot analyses of RNA from cultured primary aortic VSMCs treated with 32media supplemented with vehicle (Control; 0 mm), 30 mmglucose, or 2 mm 2-DG for 24 h as described under “Experimental Procedures.” Note that glucose treatment up-regulatesOPN mRNA accumulation 2-fold (GAPDnormalized), whereas 2-DG suppresses expression slightly. B, primary murine aortic mesenchymal cells were treated with the indicated concentrations of 2-DG or glucose either alone or in combination for 24 h in an independent experiment. OPN mRNA accumulation was quantified by real time fluorescence RT-PCR (normalized to the expression of GAPD mRNA). Note that 2-DG dose-dependently suppresses OPN mRNA accumulation in primary murine aortic cells, whereas glucose increases expression. By contrast, the gene for Msx2 is up-regulated by 2-DG and suppressed by glucose in these same cell cultures.B, A7r5 rat aortic VSMCs were cultured in gluc
Referência(s)