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J. Biol. Chem., Vol. 277, Issue 46, 44485-44496, November 15, 2002

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Osteopontin Transcription in Aortic Vascular Smooth Muscle Cells Is Controlled by Glucose-regulated Upstream Stimulatory Factor and Activator Protein-1 Activities^*

Miri Bidder^§, Jian-Su Shao, Nichole Charlton-Kachigian, Arleen P. Loewy, Clay F. Semenkovich^§, and Dwight A. Towler^§¶

From the Divisions of  Bone and Mineral Diseases and ^§ Endocrinology, Diabetes, and Metabolism, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, June 22, 2002, and in revised form, August 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 OPN promoter-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Individuals with diabetes are characteristically afflicted with medial artery calcification (1-4). 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 VSMCs¹ (5). Initially thought to be benign, medial calcification has emerged as one predictor of cardiovascular mortality (2, 3). 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 (6-10).

We recently reported the development and initial characterization of a murine model of diet-induced, insulin-resistant diabetic calcific vasculopathy (11). 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 (12, 13) 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 (11). 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 (11, 14).

OPN is a multifunctional protein produced by osteoblasts during skeletal development and in VSMCs, monocytes, and T-cells in response to biological stressors (14, 15). As an extracellular matrix protein, OPN controls cell migration mediated by _v3 integrin receptors (14, 16). Via signaling functions dependent upon phosphorylation, OPN inhibits mineral deposition (17). OPN also functions as a proinflammatory cytokine produced by stimulated monocytes and T-cells (14), augmenting monocyte/macrophage activation via up-regulation of interleukin-12 and suppression of interleukin-10 (18). Very recently, Mori and co-workers (19) extended our work (11) 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 (19). Highly relevant to arterial vasculopathy of diabetes (20), Thompson and co-workers (21) 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 (17).

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 -actin. We cloned the 2-kb mouse OPN promoter (1976 to +78, numbered relative to the start site of transcription) (22) 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 (23, 24). 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 the OPN 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 OPN promoter 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents, Antibodies, and Cell Culture-- 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 from Amersham Biosciences. A7r5 cells (ATCC CRL-1444) were maintained as described previously (25, 26). Primary aortic adventitial mesenchymal cells were obtained from aortic explants essentially as detailed previously (27) 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 (25). 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).

Cell Culture under Conditions of Varying Glucose and 2-Deoxyglucose Concentrations-- 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 mM glucose 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 (25, 28).² 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 (29), 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.

Real-time Fluorescence RT-PCR Measurements of OPN and Msx2 Gene Expression and Northern Blot Analyses-- 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 mM 2-deoxyglucose, 4 mM 2-deoxyglucose, 30 mM glucose, or 30 mM glucose + 2 mM 2-deoxyglucose. At the end of the treatment period, total RNA was extracted as described previously (30). 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 (31). Primer Express 1.0 software was used to design amplimers from murine OPN and Msx2 cDNA 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 murine Msx2 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 using OPN 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 (30, 32).

Immunohistochemical Staining of Cultured Cells-- Cultures of mouse aortic adventitial cells and A7r5 rat aortic VSMCs were evaluated for expression of OPN and VSMC -actin by immunohistochemistry using the streptavidin-biotin-immunoperoxidase method (33). A mouse monoclonal antibody to human VMSC -actin was obtained from a commercial source (product code CBL 171, clone asm-1, Cymbus Biotechnology). The osteopontin antibody utilized was MPIIIB10 (1), which has been used previously to localize vascular OPN expression in calcified aortic specimens (34). 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).

Eukaryotic Expression Constructs, OPN Promoter-Reporter Constructs, and Transient Transfection Assays-- 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 the KpnI/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 (35). 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 (36). 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 (22), Genbank^TM accession no. D14816) was obtained by PCR using mouse genomic DNA as a template, applying techniques described previously (37). OPN promoter deletions, point mutants, and heterologous promoter constructs were generated using methods as detailed (38). RSVLUC (38) and CMVLUC (39) 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 -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 -galactosidase activities were measured as detailed previously (38, 40). Statistical analyses were carried out as previously described (39).

Electrophoretic Mobility Gel Shift Assays, Supershift Assays, Immunoprecipitation, and Western Blot Analyses-- Crude extracts for gel shift analyses were prepared from control, glucose, and/or 2-DG treated cells (see above) as detailed previously (26). The radiolabeled OPN 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 (26, 41).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

OPN Gene Expression in Cultured Aortic VSMC Cells Responds to Glucose and to Pharmacological Manipulation of Cellular Glucose Metabolism-- 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 -actin in adventitial cells, mural VSMCs, and valvular fibrosal cells (11). 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 (23, 24). As in A7r5 VSMCs (Fig. 1A), immunohistochemical analysis of primary aortic mesenchymal cells (Fig. 1D) demonstrates robust staining of >80% of cells with VSMC-specific -actin, indicative of smooth muscle phenotype (42, 43). 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 (33) 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. 2A, treatment of primary cultures of aortic mesenchymal VSMCs up-regulated OPN mRNA accumulation by ~2-fold. By contrast, treatment with 2-DG dose dependently and selectively down-regulated OPN mRNA accumulation by 80% (Fig. 2B; GAPD normalized) as quantified by fluorescence RT-PCR analyses. The Msx2 gene was regulated in culture in a completely different manner; glucose suppressed Msx2 mRNA accumulation, and 2-DG actually augmented Msx2 expression 3-fold (Fig. 2B), 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. 2C; 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 (11, 15), 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 (29).

View larger version (104K): [in this window] [in a new window]   Fig. 1.   Expression of immunoreactive VSMC -actin and osteopontin in cultured aortic mesenchymal cells. Cultures of A7r5 aortic VSMCs (A-C) and primary murine aortic mesenchymal cells (D-F) were fixed with methanol:acetone and immunostained for VSMC -actin (A and D, -Act) or OPN (B and E). Note that although cell morphology differs, both A7r5 aortic VSMCs and primary aortic mesenchymal cell cultures express VSMC -actin (A and D) and OPN (B and E). C and F, negative controls (CONT).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Regulation 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 32 media supplemented with vehicle (Control; 0 mM), 30 mM glucose, or 2 mM 2-DG for 24 h as described under "Experimental Procedures." Note that glucose treatment up-regulates OPN mRNA accumulation 2-fold (GAPD normalized), 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 glucose-free medium, 10% FCS supplemented either with vehicle (water) or the indicated concentrations of the solutes glucose and/or 2-DG. RNA was prepared from A7r5 VSMCs treated with vehicle (lane 1), 2 mM 2-DG (lane 2), 4 mM 2-DG (lane 3), 30 mM glucose (lane 4), or 2 mM 2-DG with 30 mM glucose (lane 5) and probed for OPN and GAPD expression.

Basal OPN Promoter Activity in A7r5 Aortic VSMCs Is Dependent upon an Intact CCTCATGAC Motif at Nucleotides 80 to 72 Relative to the Transcription Initiation Site-- A7r5 cells are an immortalized line derived from rat aortae that faithfully recapitulates transcriptional regulation of aortic VSMCs in vivo. These cells are readily grown and transfected, thus making A7r5 an excellent cell culture system for detailed studies of promoter regulation in aortic VSMCs (23). As noted, like primary aortic mesenchymal cell cultures, this clonal line expresses VSMC -actin and OPN. We wished to identify the promoter elements that regulate OPN gene transcription in aortic VSMCs under basal conditions and in response to the changes in glucose concentrations potentially relevant to diabetic vasculopathy. Therefore, we cloned the 2-kb murine OPN promoter fragment 1976 to +78 (numbering as per Yamamoto and colleagues (22)) upstream of the LUC reporter gene. We then generated a systematic series of 5'-deletion constructs, and evaluated promoter activity after transient transfection of A7r5 cells by quantifying cellular luciferase enzyme activity as described under "Experimental Procedures." As shown in Fig. 3, basal activity was dependent upon three regions: 636 to 135, a region between the vitamin D-responsive enhancer at 764 to 747 (44) and the proximal promoter; 107 to 83, encompassing an E-box and Sp1 regulatory region (45); and a new region between 83 to 61 relative to the transcription initiation site. Notably, under these conditions, the Runx and Ets cognates between 135 to 107 necessary for supporting activity in osteoblasts (46) were not required for activity in A7r5 aortic VSMCs. Basal activity of the proximal OPN promoter was greatly dependent upon elements encompassed between nucleotides 83 to 61. Although 83 OPNLUC (83 to +78) was highly active, 61 OPNLUC (61 to +78) exhibited transcriptional activity only slightly above background signal. Inspection of the promoter sequence between 83 and 61 revealed an 11-bp region (CCTCATGACAC) encoding three potential elements: a CCTC motif for recognition by the zinc finger factors such as CTCF and the Kruppel family (47, 48); a CCTCATGA motif resembling both atypical E-box (CCTNNTG) (49) and Oct (CTCATGA) cognates (50); and an overlapping TCATGAC representing a potential AP1 cognate (51) for recognition by Fos:Jun or ATF:Jun dimers (Fig. 4). To assess the contributions of each element to basal activity, dinucleotide and trinucleotide thymidine mutations were introduced into the wild type sequence (CCTCATGACACA) to map the regions and elements contributing to basal promoter activity (mutant 1, TTTCATGACAC; mutant 2, CCTCATTATAC; mutant 3, CTCATGATTT; see summary in Fig. 4). These point mutations were introduced in the context of 83 OPNLUC (83 to +78) and compared with the activity of the wild type promoter fragment (CCTCATGACACA). As shown in Fig. 3B, the introduction of thymidine mutations at nucleotides 80 and 79 (MUT#1; Figs. 3B and 4) and nucleotides 72 to 70 (MUT#3, Figs. 3B and 4) decreased activity by ~40%. Moreover, a dithymidine transition at nucleotides 74 and 72 (MUT#2, Fig. 3B) reduces basal promoter activity by ~70%. Thus, OPN promoter activity in A7r5 aortic VSMCs is dependent upon the intact CCTCATGACAC motif in the OPN promoter region 80 to 70.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Basal activity of the OPN promoter in A7r5 aortic VSMCs is supported by promoter regions 636 to 135 and 107 to 61 dependent upon an intact CCTCATG motif at nucleotides 80 to 75. A series of OPN promoter-luciferase reporter constructs were transiently transfected into A7r5 aortic VSMCs, and promoter activity was quantified by assaying luciferase activity. CMV promoter-driven -galactosidase was included to control for transfection efficiency as outlined under "Experimental Procedures." A, data are presented as the mean ± S.D. of OPN promoter activity observed, with references to the basal activity of 107 OPNLUC (OPN promoter 107 to +78). The promoter region 636 to 135, lacking the vitamin D response element at 764 to 747, significantly contributes to basal OPN promoter activity in A7r5 VSMCs. Note, however, that in the proximal promoter 5'-deletion of 107 to 61 results in an ~15-fold reduction in basal activity with approximately half of the basal activity conferred by the region 107 to 83 and half by the region 83 to 61. B, a series of thymidine mutations was introduced into the proximal OPN promoter fragment 83 to +78 and analyzed for transcriptional activity after transient transfection of A7r5 VSMCs. Mutations were introduced into the CCTCATGACACA motif at 80 to 69 (See Fig. 4) that mapped elements supporting basal activity. Note that thymidine substitutions in these region all diminished basal OPN promoter activity; MUT #2 (Fig. 4) was most severe, decreasing basal activity by 70% relative to the wild type promoter.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Sequence of the proximal OPN promoter region 85 to 49 and mutants analyzed. Putative cognates for protein-DNA interactions in the OPN promoter in this region are indicated. See text for details. WT, wild type.

The Proximal OPN Region Is Differentially Regulated by Glucose and 2-Deoxyglucose in A7r5 Aortic VSMCs-- To determine whether the aortic VSMC OPN gene expression responses observed with pharmacological manipulation of cellular glucose uptake and metabolism reflect, in part, changes in OPN promoter activity, we examined the effects of 2-DG and glucose treatment on OPN promoter-luciferase reporter activity in transiently transfected A7r5 VSMCs. As shown in Fig. 5A, glucose treatment up-regulated 2-kb OPN promoter activity by ~3-fold. Induction is observed with both 5 and 50 mM glucose. Induction was specific for glucose, because mannitol exposure at identical concentrations, an osmotic control, elicits no activation. Moreover, the transcriptional response to glucose was promoter-specific, because the CMV (Fig. 5, A and D) and RSV promoters (Fig. 5C; see below) were not significantly responsive. Induction is dependent upon glucose metabolism, because 3-OMG, a nonmetabolized glucose analog, does not recapitulate or inhibit induction of 2-kb OPNLUC (Fig. 5B). Moreover, competitive inhibition of intracellular glucose metabolism with 2-DG decreases OPN promoter activity, and inhibition was partially reversed with treatment with 60 mM glucose (Fig. 5B). Again, the effects of short term treatment with 2-DG was promoter-specific, because the CMV promoter is not inhibited (data not shown and Fig. 5D; see below) The effects of the systematic series of 5'-truncated OPN promoter-LUC constructs that we described above were tested for induction by glucose in A7r5 cell to map this response. As occurs with 1976 OPNLUC, 636 OPNLUC (not shown), 135 OPNLUC, and 83 OPNLUC (83 to +78; Fig. 5C) were all induced by glucose treatment; 50 mM glucose was used because this provided the maximal response observed (Fig. 5A). By contrast, 61 OPNLUC, possessing the CCAAT box, TATA box, and initiator region element of the OPN gene (61 to +78), was unresponsive (Fig. 5B). To confirm that the proximal region of the OPN promoter confers transcriptional regulation in response to manipulation of cellular glucose tone, we placed the 39-bp OPN fragment 83 to 45 upstream of the RSV minimal promoter. OPN-(83/45) encompasses the CCTCATGACAC motif identified above as necessary for robust basal activity in the critical region between 83 and 61 and the CCAAT box at 53 to 49 (bottom strand). As shown in Fig. 5C, OPN-(83/45)RSVLUC activity was 20-fold greater than that of the minimal RSVLUC in A7r5 VSMCs. Consistent with our deletion analyses, the OPN promoter fragment 83 to 45 conferred glucose responsiveness upon the unresponsive RSV minimal promoter (TATA box and initiator region only; Fig. 5C). Moreover, as shown in Fig. 5D, the OPN promoter fragment 83 to 45 conferred 2-DG inhibition as well as glucose induction upon the unresponsive RSV minimal promoter. Glucose treatment again stimulated transcriptional activity ~2-fold; 2-DG suppressed activity by 60%, and 50 mM glucose reversed 2-DG suppression (Fig. 5D). Again, activities of the RSV and CMV promoters were not inhibited by 2-DG in A7r5 VSMCs (Fig. 5, C and D). Thus, the OPN promoter region 83 to 45 assembles transcriptional complexes that support OPN promoter activity in A7r5 aortic VSMCs; this region participates in both basal transcriptional activity and regulation in response to alterations in cellular glucose exposure and cellular metabolism.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   OPN promoter elements encoded by nucleotides 83 to 61 convey transcriptional responses to glucose and 2-deoxyglucose. A, A7r5 aortic VSMCs were transiently transfected with the 2-kb OPN promoter-luciferase reporter (1976 to +78) as described in the legend to Fig. 3. Two days after transfection, cells were treated for 24 h with the indicated concentrations of glucose or mannitol, and promoter activity was measured by assaying luciferase as described in the legend to Fig. 3. OPN promoter activity is specifically up-regulated by glucose but not by mannitol (osmotic control). The CMV (A) and RSV (C and D) promoters are not significantly regulated. Induction is dependent upon glucose metabolism; the nonmetabolized glucose analog 3-OMG is inactive (B), and 2-DG specifically inhibits glucose-dependent activation. Deletion analyses map the response to 83 to 61 (C) confirmed in heterologous promoter assays. The OPN promoter region 83 to 45 confers basal activity responsive to manipulation of cellular glucose homeostasis to the unresponsive RSV promoter (D). See text for details.

VSMC Protein-DNA Interactions at the CCTCATGAC Motif-- To initiate characterization of the DNA-protein interactions assembled by the OPN proximal promoter, we performed electrophoretic mobility gel shift assays. Duplex synthetic oligodeoxynucleotides corresponding to OPN promoter fragment 85 to 65 were radiolabeled with ³²P and OPN promoter DNA binding activity in A7r5 extracts from cells cultured as described previously (26, 37). As shown in Fig. 6, three closely migrating complexes (labeled A, B, and C) were resolved and visualized after autoradiography (lane 1). (A much more rapidly migrating, variably visualized complex of low specificity is also observed occasionally; this is commonly seen with other oligos as well in this protocol (26)). Cold homologous duplex oligo competed for formation of all three complexes (Fig. 6, lanes 1-3). The cognate for AP1 binding in the collagenase (MMP1) promoter (26) abrogated formation of complex C (Fig. 6, lanes 4-6) and diminished complex A, indicating the presence of AP1-like protein-DNA interactions in these two complexes. However, complex B was unaffected by the AP1 oligo, indicating a different binding specificity (Fig. 6, lanes 4-6). To derive the connections between the OPN promoter sequences between 80 and 70 necessary for robust basal promoter activity in A7r5 cells and in the assembly of specific A7r5 protein-DNA interactions, duplex oligonucleotides containing mutations corresponding to OPN promoter mutants MUT #1, MUT #2, and MUT #3 (Fig. 4) were examined as cold competitors. A duplex oligo possessing the CCTT transition of MUT #1, which decreases OPN activity by ~40% (Fig. 5B) and selectively disrupts the putative E-box (Fig. 4; see below), can still compete for complexes A and C but not complex B (Fig. 6, lanes 7-9). By contrast, a duplex oligo containing the MUT #2 alterations, which most severely lesions basal OPN promoter activity (Figs. 3B and 4), did not compete for any of the three complexes formed (Fig. 6, lanes 10-12; competition studies with cold MUT #2 consistently augmented total binding activity, data not shown). Like the wild type sequence, MUT #3 competed for formation of all complexes (Fig. 6, lanes 13-15), indicating that at the excess oligo concentrations used in competition assays, the intact cognate CCTCATGAC was sufficient to compete for assembly of complexes. Thus, protein-DNA interactions necessary for formation of complexes A, B, and C on OPN promoter region 80 to 72 are necessary to support robust basal OPN promoter activity in A7r5 myoblasts. Point mutations that perturbed the TCATGAC AP1-like interactions alter recognition by complex C, a DNA-protein interaction specifically disrupted by competition with an authentic AP1 cognate. Formation of complex A was also dependent upon sequences in this element that overlapped the AP1 cognate, because nucleotide substitutions that targeted this region precluded activity in competition assays. Complex B DNA-protein interactions, which overlap yet are distinct from the AP1-like interactions, required an intact E-box-like motif; immunological probing confirmed the presence of distinct protein-DNA complexes (see below). DNA-protein interactions altered by MUT #2 simultaneously exert the most significant deleterious effects on formation of specific DNA-protein complexes (Fig. 6, lanes 10-12) and reductions in OPN promoter activity (Fig. 3B).

View larger version (97K): [in this window] [in a new window]   Fig. 6.   The OPN promoter region 85 to 65 assembles three protein-DNA complexes. Extracts were prepared from A7r5 cells grown under standard (25 mM glucose) conditions, and DNA binding activities recognizing the radiolabeled OPN promoter region 85 to 65 were detected by gel shift assay as described under "Experimental Procedures." DNA binding specificity was evaluated by competition with cold homologous oligo (lanes 1-3), the interstitial collagenase (MMP1) AP1 cognate (lanes 4-6), or three OPN promoter mutants (lanes 7-9, 10-12, and 13-15) corresponding to the mutants outlined in Fig. 4. Lanes 1, 4, 7, 10, and 13, no cold competitor; lanes 2, 5, 8, 11, and 14, indicated cold competitors at 10-fold molar excess; lanes 3, 6, 9, 12, and 15, indicated cold competitors at 30-fold molar excess. Three closely migrating specific complexes are visualized in the gel shift (lane 1). exposure times were shortened to facilitate visualization of all three complexes. Complexes C and A (to a lesser extent) both are reduced by competition with the AP1 cognate, indicating AP1-like DNA-protein interactions in these two complexes. Further note that complex B is not disrupted by this oligo (lanes 4-6) nor by OPN promoter fragment MUT #1 (lanes 7-9), which contains mutations in the 5' portion of a putative E-box (Fig. 4). None of these complexes were inhibited by competition with the OPN promoter fragment mutant 2 (lanes 10-12) possessing thymidine substitutions that simultaneously destroys both the E-box and AP1 cognates in this region. As observed for the wild type oligo (lanes 1-3) mutant 3 was able to compete for formation of all three complexes (lanes 13-15). See "Results" for details.

Immunological Supershift Analyses Demonstrate the Presence of USF and AP1 Factors in A7r5 DNA Binding Activities That Recognize and Regulate the OPN CCTCATGAC Motif-- As noted above, in addition to the TCATGAC AP1 motif, inspection of the OPN promoter region 85 to 65 revealed an overlapping CCTCATG motif at 80 to 74 that resembles that of the CANNTG motif of classical E-box cognates. The competition studies indicate that complex B specifically recognizes the CCTCATG motif in the OPN promoter. Because (a) the E-box binding factor USF1 has been shown to recognize other distinct elements in the OPN promoter in VSMCs (45), and (b) USF1 and USF2 have been shown to mediate hepatic glucose responses (52, 53), we immunologically probed the OPN promoter binding complex B with anti-USF antibodies in gel supershift assays as described under "Experimental Procedures." For these experiments (Figs. 7), gel shifts were carried out with radiolabeled OPN-(85/65) duplex oligo and simultaneously in the presence of cold AP1 cognate to eliminate band C; this permitted unobscured visualization of band B responses to immunological probing. Because this region of the OPN promoter also contains potential cognates (CCTC, CTCATGA) for recognition by zinc finger and octamer factor, respectively, we immunologically probed for these specific protein-DNA interactions as well. As shown in Fig. 7, in this systematic screen, only the anti-USF1 antibody almost completely disrupted the formation of complex B (Fig. 8A, lanes 25-26). By contrast, antibodies against octamer factors (Fig. 7A, lanes 16-17, 19-20, 22-23), the irrelevant FLAG motif (lanes 28-29), or zinc finger factors BTEB2 (lanes 10-11), Gli (lanes 13-14), or CTCF (lanes 7-8) factors that would potentially recognize the CCTC motif had no effect on complex B formation. Anti-Fos (Fig. 7A, lanes 1-2) and anti-Jun (lanes 4-5) antibodies had weak effects on formation of the USF complex. Notably, anti-USF1 did not perturb formation of the AP1 binding complex forming on this region of the OPN promoter, indicating the specificity of the immunoreagent (not shown). No nonspecific interaction of antibody with radiolabeled probe was observed (Fig. 7A, lanes 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30). Complex disruption without supershift was noted with the anti-USF1 antibody (Fig. 7A). Therefore, to provide additional evidence for endogenous USF recognition of this OPN element, we probed the complex with polyclonal antibodies directed against two completely different domains of USF1. Both antibodies directed to a USF1 internal domain (H86; Fig. 7B, lanes 1-3) and USF1 C-terminal domain (C20; Fig. 7B, lanes 4-6) inhibited complex B formation on OPN-(85/65). USF1 is known to form a heterodimer in cells with the related family member USF2 (54, 55). To provide further support that this complex B was indeed USF, we tested the effects of anti-USF2 antibody on complex formation. As shown in Fig. 7B, lanes 7-9, like anti-USF1, anti-USF2 antibody disrupts formation of complex B. These antibodies had no effect on the formation of DNA-protein complexes on the AP1 cognate (not shown), indicating specificity of the immunological USF1 reagents. Cold competition studies again confirm that this very complex that is perturbed by anti-USF antibodies recognized the CCTCATG motif in the OPN promoter, as suggested in the data for Fig. 6. (Again, these gel shifts were carried out in the presence of cold AP1 oligo to permit robust visualization of complex B.) Unlike the wild type oligo sequence (Fig. 7B, lanes 10-12), oligos with MUT #1 (lanes 13-15) or MUT #2 (lanes 16-18) alterations that perturb the CCTCATG E-box-like motif (Fig. 4) cannot compete for complex B formation. By contrast, in MUT #3. (An alteration does not alter this E-box-like cognate; Fig. 4) can still compete for formation of complex B (Fig. 7B, lanes 19-22), consistent with data presented above (see Fig. 6). Thus, the A7r5 protein-DNA complex B assembled by the proximal OPN promoter region 85 to 65 is a heterodimer of USF1 and USF2 that recognizes the CCTCATG motif at nucleotides 80 to 74.

View larger version (55K): [in this window] [in a new window]   Fig. 7.   Identification of USF1 and USF2 in the protein-DNA complexes bound by the proximal OPN promoter region 85 to 65. Gel shifts were carried out as described in the legend to Fig. 6 but in the presence of cold AP1 cognate from the MMP1 promoter (26) to preclude AP1 binding to the OPN promoter fragment 85 to 65 and facilitate visualization of complex B. A, A7r5 extracts analyzed by gel shift after preincubation with 3 µg of the indicated antibodies. Lanes 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28, basal binding. Lanes 2, 5, 8, 11, 14, 17, 20, 23, 26, and 29, binding in the presence of the indicated antibody. Note that the anti-USF1 antibody (lane 26) markedly diminished formation of this protein-DNA complex. Anti-Fos anti-body also slightly reduced complex formation as well. No nonspecific interaction of antibody with radiolabeled probe was observed (lanes 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30). B, antibodies directed to the mid-region (H86, lanes 1-3) or C terminus (C20, lanes 4-6) of USF1 inhibit formation of complex B, as do antibodies against USF2 (lanes 7-9). Competition assays (lanes 10-22) confirm the binding specificity of this USF1:USF2 complex is that of complex B. See Fig. 4 for competitor sequences.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Identification of c-Fos and phospho-c-Jun in protein-DNA complexes bound by the proximal OPN promoter region 85 to 65. Immunological probing of protein-DNA complexes assembled with specific Fos and Jun family members. Autoradiogram exposure times that reveal the significant supershifted complexes (e.g. lanes 2, 17, and 21) obliterate the resolution of complexes A, B, and C. Lanes 1, 4, 7, 13, 16, and 19, basal binding; lanes 2, 5, 8, 11, 14, 17, and 20, binding in the presence of the indicated antibodies. Note that an anti-cFos antibody disrupts and partially supershifts the complex (lane 2). Notably, a small amount of basal binding activity remains after treatment with anti-c-Fos antibody, reflecting the residual USF binding activity unperturbed by c-Fos antibody (lane 2). Antibodies to other Fos family members did not affect binding (lanes 4-12). Antibody to c-Jun reduced basal binding (lane 14). Moreover, antibody to the activated phospho-c-Jun supershifted the complex (lane 17). A weaker supershift is observed with antibody to JunD (lane 20). None of the antibodies interacts with the radiolabeled OPN probe in the absence of extract (lanes 3, 6, 9, 12, 15, 18, and 21).

A7r5 Aortic VSMC DNA Binding Activities Containing c-Fos, c-Jun, and JunD Recognize the OPN Promoter Region 85 to 65-- The competition studies presented above identified that bands C and A represent complexes assembled by the OPN promoter region 85 to 65 contain factors recognizing AP1 cognates. To provide additional evidence for AP1 protein-DNA interactions and to identify the specific VSMC AP1 complexes binding the OPN promoter, we immunologically probed these complexes as described previously under "Experimental Procedures." As shown in Fig. 8, antibody to c-Fos markedly diminished and partially supershifted complexes formed on radiolabeled OPN-(85/65) (Fig. 8, lanes 1-2). Autoradiogram exposure times that reveal the significant supershifted complexes (e.g. Fig. 8, lanes 2, 17, and 21) obliterate the resolution of complexes A, B, and C. Notably, a small amount of basal binding activity remained after treatment with anti-c-Fos antibody, presumably reflecting the residual USF binding activity unperturbed by c-Fos antibody (Fig. 8, lane 2). By contrast, antibodies to other Fos family members had no effect on complex formation (Fig. 8, lanes 4-12). AP1 family members are typically heterodimers of specific Fos and Jun proteins (51). As shown in Fig. 8, antibodies to c-Jun and phospho-c-Jun partially reduced and supershifted the complex (Fig. 8, lanes 13-17). A subset of the AP1 complexes assembled by the OPN promoter contain JunD, revealed again by immunological supershift (Fig. 8, lanes 19-20). None of the antibodies interacted nonspecifically with the radiolabeled oligos (Fig. 8, lanes 3, 6, 9, 12, 15, 18, and 21). Antibodies to CTCF, BTEB2, Gli1, Oct1, Oct2, and Oct4 do not perturb or supershift any of the complexes assembled by the OPN promoter 85 to 65 (not shown). Thus, AP1 binding factors recognize and regulate the OPN promoter region 85 to 65. c-Fos, active (phosphorylated) c-Jun, and JunD are present in the A7r5 aortic VSMC complexes assembled by this region of the OPN promoter that supports basal and glucose-dependent activity in A7r5 cultures.

The OPN Promoter USF Binding Activity in A7r5 Aortic VSMCs Is Regulated by Glucose and 2-DG-- We next examined the effects of glucose and 2-DG treatment on formation of the USF:OPN and AP1:OPN protein-DNA interactions. Extracts were prepared from A7r5 aortic VSMCs treated with either vehicle (control; 0 mM glucose), 5 mM glucose, or 5 mM mannitol as described under "Experimental Procedures." As shown in Fig. 9A, 5 mM glucose treatment increased the formation of the AP1 and USF binding activities that regulate OPN promoter. However, 5 mM mannitol treatment has no effect (USF binding again visualized in presence of excess cold AP1 oligo; see above). The influence of 2-DG in the presence of 5 or 30 mM glucose was also examined. As shown in Fig. 9B, treatment with 2-DG did not significantly alter total AP1 binding (Fig. 9B, upper panel). However, treatment with 2-DG markedly down-regulated the USF binding activity recognizing the OPN promoter region 85 to 65 in the presence of 5 mM glucose (Fig. 9B, lanes 3-6 versus lanes 1-2, lower panel; USF binding again was visualized in the presence of excess cold AP1 oligo). Moreover, the addition of 30 mM glucose completely prevented the down-regulation of USF binding to the OPN promoter by 2-DG (Fig. 9B, lower panel, lanes 9-10). Thus, glucose and 2-DG regulate USF and AP1 binding activities, recognizing the proximal OPN promoter region 85 to 65 in A7r5 VSMCs.

View larger version (38K): [in this window] [in a new window]   Fig. 9.   Glucose regulates AP1 and USF DNA binding activities in A7r5 aortic VSMCs. A, extracts were prepared from A7r5 cells treated with vehicle, 5 mM glucose, or 5 mM mannitol at the indicated concentrations for 1 day (independent duplicates), and DNA binding activities recognizing the OPN promoter region 85 to 65 were identified by gel shift assay. Upper panel, total binding activity, reflecting primarily AP1 (see Fig. 9); lower panel, USF binding activity assessed as described in the legend to Fig. 8. Note that glucose up-regulates both activities, whereas 5 mM mannitol does not. B, regulation of binding by 2-DG. Upper panel, total AP1 binding activity (see Fig. 9); lower panel, USF binding activity. Note that 2-DG down-regulates USF binding activity recognizing the OPN promoter (lanes 3-6) and that 30 mM glucose (Glc) reverses the suppression of USF binding by 2-DG treatment (lanes 7-8). See "Results" for details.

USF Protein Accumulation Is Regulated in Cultured Aortic VSMCs by Glucose and 2-DG-- USF binding was markedly sensitive to glucose and 2-DG treatment. We wished to identify whether these changes in USF binding activity reflect changes in the accumulation of USF1 and/or USF2 protein levels. Therefore, we performed Western blot analyses of extracts prepared from A7r5 cells treated with either vehicle (0 mM glucose) or 5 mM glucose or mannitol. As shown in Fig. 10A, treatment of cells with glucose up-regulated USF1 but not USF2 protein accumulation (independent duplicates). By contrast, mannitol exerted little if any effect on USF protein accumulation. The effect of 2-DG in the presence of 5 (basal) or 30 mM glucose was also examined independently (Fig. 10B). As shown, 2-DG weakly down-regulated USF1 protein levels in A7r5 VSMCs (Fig. 10B, lanes 2 and 3 versus 1); by contrast, 30 mM glucose up-regulated USF1 protein accumulation and completely prevented the 2-DG-mediated decrements in protein accumulation (Fig. 10B, lanes 4-5). USF2 levels are not regulated by manipulation of glucose tone in A7r5 VSMCs (Fig. 10B), indicating specificity for regulation of USF1 protein accumulation. Similar results were obtained in cultures of primary aortic mesenchymal cells (not shown). Thus, USF1 protein accumulation is specifically responsive to manipulation of cellular glucose tone in A7r5 aortic VSMCs and primary mouse aortic VSMCs, consistent with effects on total OPN USF binding activity. Regulation of the OPN-(80/72) promoter USF binding activity in culture A7r5 aortic VSMCs by 2-DG and glucose reflects in part the regulation of USF1 protein accumulation.

View larger version (42K): [in this window] [in a new window]   Fig. 10.   Regulation of USF1 protein levels in A7r5 cells by glucose. A, A7r5 aortic VSMCs were cultured in glucose-free DMEM with 10% dialyzed FCS supplemented either with vehicle or the indicated concentrations of glucose or mannitol (independent duplicates) as described in the legend to Fig. 9. Cell extracts were prepared and assays for USF protein accumulation by Western blot analyses. Note that glucose up-regulates USF1 but not USF2 protein accumulation. Further note that 5 mM mannitol does not regulate USF protein levels. B, extracts were prepared from A7r5 VSMCs treated with vehicle (lane 1), 2 mM 2-DG (lane 2), 4 mM 2-DG (lane 3), 30 mM glucose (lane 4), or 2 mM 2-DG with 30 mM glucose (lane 5). USF1 and USF2 protein levels were assessed by Western blot. Note that glucose increases USF1 protein levels (lane 4 versus 1) and prevents 2-DG suppression (lane 5 versus 2). USF2 levels are not significantly regulated.

The OPN Promoter Is Regulated in Transient Co-transfection Assays by USF1 and AP1 (c-Fos:c-Jun) in A7r5 Aortic VSMCs-- To examine the effects of AP1 (c-Fos:c-Jun) and USF (USF1:USF2) on OPN promoter activity, we transiently co-transfected eukaryotic expression vectors for c-Fos, c-Jun, USF1, and USF2 with the OPNLUC reporter in A7r5 aortic VSMCs. As shown, both USF (Fig. 11A) and AP1 (c-Fos:c-Jun; Fig. 11B) significantly augmented OPN promoter activity in A7r5 cells. USF regulation is complex, because mutation of the composite cognate at 80 to 72 did not completely prevent OPNLUC activation (not shown); this likely reflects the well described capacity of USF to activate transcription both via E-box elements and also via the pyrimidine-rich initiator region element (Inr; CYCAYNYN) (56-58) common to a number of promoter start sites (59, 60) including the OPN gene (22). Synergy for OPN promoter activation between AP1 and USF was not observed (Fig. 11A). USF dominated and limited AP1-dependent activation of the OPN promoter (Fig. 11), and a 10-fold greater expression of AP1 with USF resulted in transcriptional squelching, suggesting the sequestration of an unidentified, rate-limiting co-regulator by these factors (not shown; see "Discussion"). AP1 (c-Fos and c-Jun) transactivates the proximal OPN promoter 83 to +78, and activation was completely abrogated by mutation of the CCTCATGAC element in the OPN promoter region 80 to 72 (MUT #2; Fig. 11B). Thus USF and c-Fos:c-Jun both regulate the proximal OPN promoter and function to support OPN gene transcription in A7r5 rat aortic VSMCs.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   USF and AP1 (c-Fos:c-Jun) up-regulate basal OPN promoter activity in A7r5 aortic VSMCs. A7r5 cells were co-transfected with 83 OPNLUC and eukaryotic expression vectors for USF subunits (USF1, USF2) or AP1 subunits (c-Fos, c-Jun) as indicated. Empty expression vector adjustment was used to maintain constant DNA concentrations in all transfections. A, transfection of USF (USF1/USF2) significantly up-regulates proximal OPN promoter activity in A7r5 cells. No additive or synergistic effects were observed with AP1 expression. B, AP1 expression (c-Fos, c-Jun) significantly up-regulates basal OPN promoter activity. Mutation of the CCTCATGAC motif to CCTCATTAT (MUT #2) at 80 to 72 completely abrogates the AP1 response. See "Results" for details.

Transactivation Directed by c-Fos and USF1 Is Augmented by Glucose in A7r5 Aortic VSMCs-- In addition to DNA binding activity, transcriptional activation function can be regulated by signaling cascades that control functionally important protein-protein interactions with the basal transcriptional machinery. We wished to assess whether the transactivation functions AP1 and USF1, the factors recognizing and regulating glucose-responsive OPN proximal promoter, were regulated by glucose, as further evidence for their role in transcriptional responses to glucose in A7r5 cells. Therefore, we performed one-hybrid analyses, assessing the ability of c-Fos, c-Jun, and USF1 to convey glucose responsiveness onto the unresponsive Gal4 DNA-binding domain (G4DBD) using five copies of the Gal4 response element placed upstream of the LUC reporter gene (Gal4RE-LUC or pFRLUC). As shown in Fig. 12A, the Gal4 DNA-binding domain (which lacks a transcriptional activation domain) does not activate transcription driven by the Gal4RE-LUC either in the presence or absence of glucose treatment. By contrast, basal activity was significantly augmented by expression of the Gal4DBD-c-Fos fusion protein. Moreover, transcription was further increased by treatment with 50 mM glucose (Fig. 12A). Of note, Gal4DBD-cJun only weakly up-regulated basal activity and is minimally responsive to glucose. Gal4DBD-USF1 also increased basal activity weakly. However, the transactivation function of Gal4DBD-USF1 is up-regulated 11-fold by 50 mM glucose treatment (Fig. 12A). Transactivation was also augmented at lower glucose concentrations (Fig. 12B). Moreover, as observed in the OPN promoter context, c-Fos and USF1 transactivation were enhanced by 5 mM glucose but not by 5 mM mannitol (Fig. 12B). Thus, transactivation functions of c-Fos and USF1, the DNA binding factors that recognized the glucose-responsive OPN proximal promoter region, are selectively activated by glucose treatment of A7r5 aortic VSMCs.

View larger version (16K): [in this window] [in a new window]   Fig. 12.   Transactivation functions of c-Fos and USF1 are augmented by glucose in A7r5 aortic VSMCs. A7r5 cells were co-transfected with Gal4RE-LUC (pFRLUC) and pFACMV expression vectors encoding the indicated Gal4 DBD (G4DBD) fusion proteins. Transcription driven from Gal4RE-LUC was assayed from cells treated either with vehicle (control) or 50 mM glucose. A, note that glucose specifically augments c-Fos and USF1-dependent transcription. B, further note that 5 mM glucose, but not 5 mM mannitol, induces c-Fos and USF1 transcriptional activation. See "Results" for details,

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

OPN is a multifunctional extracellular protein that mediates attachment and migration of osteoblasts, osteoclasts, macrophages, and mesenchymal VSMCs (14). OPN enhances activation of cells of the monocyte/macrophage lineage via up-regulation of interleukin-12 and suppression of interleukin-10, thus recapitulating a Th1-type cytokine activation profile (14). One cell type derived from the monocyte/macrophage lineage is the osteoclast. Elegant studies by Denhardt, Noda, and colleagues (61) have now identified a role for OPN in osteoclast-dependent bone resorption activated by estrogen deficiency parathyroid hormone treatment, RANKL (receptor activator of NF-B ligand) stimulation (62), or mechanical unloading (63). Thus, in bone, OPN emerges as a crucial regulator of mineralized tissue turnover.

Unlike bone, the role of OPN in the arterial vasculature is poorly characterized, and little is known of its transcriptional regulation in this tissue. The OPN promoter is highly modular (14). The OPN promoter can be organized into four regions: (i) a nuclear receptor and Ras-responsive region (64) encoded by the OPN promoter sequence 1000 to 550; peroxisome proliferator-activated receptor  (65), vitamin D receptor (44), and estrogen receptor-related receptor- responses (66-68) all map to elements in this region; (ii) a second region entraining expression to the osteoblast differentiation program; BMP2-regulated Hoxc8:Smad1 (69) and Runx2:Ets1 (46) transcriptional responses map to the OPN promoter region 220 to 110; (iii) a basal promoter region that maps to nucleotides 107 to +78 relative to the start site (see below); and (iv) an intronic enhancer at +799 to +864 that supports Sox2:Oct4 regulated expression in pre-implantation embryonic development (70). It is likely that the Runx2 and BMP2 regulatory cascades play a crucial role in vascular OPN expression at later stages of calcific vasculopathy (71), i.e. once the progressive, osteogenic regulation of macrovascular calcification has been established as a consequence of responses to initial metabolic or inflammatory insults (20, 71-73). However, we identified that the OPN promoter regions required for Smad responses (220 to 190) and Runx2:Ets recognition (134 to 129; 118 to 113) in osteoblasts are not required for basal or glucose-regulated OPN promoter activity in A7r5 aortic VSMCs.

In our studies, we determined that the proximal OPN promoter region 83 to 45 assembles DNA-protein interactions sufficient for basal and acute glucose-regulated transcription in the VSMC. We show that specific AP1 and USF complexes recognize and regulate the OPN promoter via a CCTCATGAC element in this region. Using rat VSMCs derived from either embryonic or adults sources, Giachelli and co-workers (45) first identified that USF1 binds the E-box cognate CAGGTG present at nucleotides 105 to 100 in the murine OPN gene (see Fig. 4); USF2 association, regulation by altered glucose signaling, and transcriptional responses to transient co-expressed USF1 were not examined in their seminal study. In data not shown, we also identified protein-DNA interactions at this same E-box in A7r5 cells, confirming the results of Giachelli's group (45). Moreover, the decrement in basal activity we note upon comparison of 107 OPNLUC and 83 OPNLUC reflects the contribution of that USF protein:DNA interaction to basal activity. However, using A7r5 cells, a line that faithfully recapitulates in vitro transcriptional regulatory features validated in aortic VSMCs in vivo (23, 24), we identified yet another, more proximal composite element; the CCTCATGAC motif at 80 to 72 in the OPN promoter is identified as supporting expression in A7r5 cells. This more proximal motif contributes to activity in the homologous promoter context, and specific USF1:USF2 and c-Fos:c-Jun complexes recognize and regulate the OPN promoter via this element in VSMCs. Our data demonstrate that this element is a cognate for recognition by an endogenous USF1:USF2 heterodimer in A7r5 aortic VSMCs. The presence of USF in this complex was initially difficult to assess because of the significant amount of AP1 binding to this region; this problem was resolved by selectively inhibiting AP1 binding to the OPN promoter region 85 to 65, achieved by carrying out gel shift assays in the presence of excess heterologous MMP1 AP1 cognate (26). Moreover, we detected an AP1 binding activity consisting primarily of c-Fos:c-Jun that also recognizes this element. We show for the first time that transient expression of USF1 or AP1 (c-Fos:c-Jun) up-regulates OPN promoter activity in aortic VSMCs, directly demonstrating the function of these factors in OPN gene transcription. Perhaps most importantly, we additionally demonstrate that the transactivation functions of c-Fos and USF1 are activated by glucose in A7r5 VSMCs.

Valount and co-workers (52-54) have elegantly demonstrated the contributions of USF1 and USF2 to normal, robust hepatic glucose responses by developing and analyzing mice possessing homologous disruption of these two transcription factor genes. In their studies of glucose-dependent control of hepatic gene expression, USF2 appeared capable of compensating in part for USF1 functions; USF1 / mice exhibited significantly delayed yet measurable responses to glucose challenges via the formation of USF2 homodimers (54). Our analyses of aortic OPN gene expression confirm glucose-dependent control of USF OPN promoter binding in VSMC glucose responses as well as regulation of USF protein accumulation. To our knowledge, this is the first demonstration that USF1 protein accumulation, DNA binding activity, and transactivation function are directly responsive to manipulation of cellular glucose uptake and metabolism. Future studies will examine which mechanisms regulate USF activity and protein expression in aortic VSMCs.

Although alterations in USF protein accumulation contribute to the changes in protein-DNA interactions at the OPN promoter in response to glucose manipulation, a glucose-regulated post-translational modification or co-regulatory protein-protein interaction is also likely necessary, as revealed by our one-hybrid studies. It is intriguing to note that the osmotically activated p38 MAPK signaling cascade enhances USF1-dependent transcription (74). However, OPN induction by glucose in aortic VSMCs is not inhibited by p38 MAPK antagonists.² Evidence that an osmotic stress response is not a component of OPN induction in VSMCs is provided by the observations that (a) mannitol cannot recapitulate signaling and (b) the nonmetabolized glucose analog 3-O-methylglucose does not activate transcription. The fact that 2-DG inhibits induction suggests that aortic VSMC OPN expression represents, in part, a transcriptional response entrained to cellular glucose metabolism. The roles of Jun N-terminal kinase, p38 MAPK, and MEK/ERK/RSK2 signaling cascades will be evaluated for their contributions to OPN transcriptional responses in vitro and aortic OPN expression during progression of diabetic vasculopathy in vivo.

The Fos-Jun AP1 heterodimer also augments transcription via the element CCTCATGAC in the proximal OPN promoter, and glucose promotes c-Fos transactivation function. Glucose responses have mapped to AP1 elements in other genes, including the TGF-1 promoter (75). Notably, USFs are members of the hHLH-ZIP family that heterodimerize with each other and with Fos family members. Blanar and Rutter (76) initially cloned USF2 as FIP (Fos-interacting protein) using the Far Western interaction cloning technique (76). USF1 and Fra1 have been shown to physically interact. However, unlike the synergistic activation observed between USF2 and c-Fos, USF1 functionally antagonizes Fra1-dependent transcription (77). We did not observe synergy; in fact we noted squelching of OPN promoter activity when excessive USF and AP1 subunits were co-transfected (not shown). Direct protein-protein interactions between USF and AP1 factors may be responsible. As noted in other composite elements such as the c-Fos promoter SRE (serum response element), such interactions are required to overcome stereological constraints that regulate stable assembly of multiple components on short overlapping DNA cognates (78). Therefore, we favor a model in which transient over-expression of both USF and AP1 compete for a rate-limiting cofactor, functionally akin to SPIN/TFII-I in the SRE complex, which stabilizes complex formation. Sawadogo and colleagues (55) recently provided evidence for a cell type-specific USF co-regulator necessary for transactivation responses. Of note, RSK2-a c-Fos-regulating kinase (79), interacts with CBP (CREB-binding protein) (80), a co-activator for both AP1 and USF, and regulates carbohydrate metabolism in the liver (81). Whether RSK2 participates in glucose-regulated c-Fos and USF function is unknown. Future studies will identify co-regulatory proteins that participate in USF- and AP1- dependent control of OPN gene expression in A7r5 cells and will test whether USF:AP1 heterotetramers or Fos:USF heterodimers preferentially assemble in the context of the endogenous OPN promoter chromatin.

It is interesting to note that Msx2 and OPN gene expression in primary aortic VSMCs cultures are differentially regulated by 2-DG and glucose; 2-DG induced Msx2 expression, and glucose suppressed mRNA accumulation. These data suggest that vascular Msx2 induction by diabetes in vivo (11) is not an immediate response to hyperglycemia. However, Msx2 expression is a direct target of BMP2 signaling (82). We have identified augmented aortic BMP2 expression in our diabetic animal models (not shown) as predicted from the early work of Demer and colleagues (12). Therefore, unlike OPN expression, vascular expression of Msx2 is likely to be secondary to BMP2 induction in response to metabolic insults such as diabetes and hypercholesterolemia that promote calcific vasculopathy (83). This notion remains to be tested directly.

In conclusion, we have ascertained that OPN gene expression in aortic VSMCs is directly responsive to cellular glucose exposure and metabolism. Responses are achieved via up-regulation of (a) USF1 and AP1 (c-Fos) transactivation functions and (b) USF protein-DNA interactions dependent upon USF1 protein accumulation. A CCTCATGAC motif in the proximal OPN promoter assembles this glucose-responsive transcriptional complex. We conclude that the enhanced expression of OPN observed in diabetic arteries occurs in part via hyperglycemia that activates USF- and AP1-dependent transcription of the OPN gene. Because OPN regulates age-dependent arterial intimal-medial thickness and cellular proliferation in vivo (84), strategies that inhibit vascular OPN expression and OPN signaling in vivo may ameliorate diabetic vasculopathy and associated cardiovascular morbidity and mortality.

	FOOTNOTES

^* This work was supported by Grants HL69229 and DK52446 (to D. A. T.) from the National Institutes of Health and by the Barnes-Jewish Hospital Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Washington University Medical Center, Division of Bone and Mineral Diseases, Barnes-Jewish Hospital, North Campus, 216 South Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314-454-7434; Fax: 314-454-5047; E-mail: dtowler@im.wustl.edu.

Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M206235200

² M. Bidder, J.-S. Shao, N. Charlton-Kachigian, A. P. Loewy, C. F. Semenkovich, and D. A. Towler, our unpublished observations.

	ABBREVIATIONS

The 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.

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