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Content in Vascular Smooth Muscles
Denver Research Institute (P.A.W., J.E.-B.R.), Denver Veterans Affairs Medical Center and Department of Medicine (P.A.W., A.N., J.E.-B.R.), Division of Endocrinology, University of Colorado Health Sciences Center, Denver, Colorado 80220; and National Cancer Institute (C.V.), National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Jane E.-B. Reusch, M.D., Denver VA Medical Center, Room 9C-120, 1055 Clermont Street, Denver, Colorado 80220. E-mail: . jane.reusch{at}uchsc.edu
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Abstract |
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receptor gene (PDGFR). Adenovirus-mediated expression of constitutively active CREB mutants decreases PDGFR mRNA, PDGFR protein, and PDGFR promoter-luciferase reporter activity in cultured SMCs. Expression of dominant negative CREB protein, A-CREB, increases PDGFR protein content and the PDGFR-promoter activity in SMCs. Active CREB prevents activation of PDGFR promoter-luciferase reporter activity by CCAAT/enhancer-binding protein-
(C/EBP), shown to mediate IL-1ß stimulation of PDGFR expression. Exposure of cultured SMCs to high glucose or reactive oxidant stress, which decrease CREB protein content and activity, increases PDGFR protein content and promoter activity. Expression of active CREB blunts reactive oxidant stress-induced PDGFR accumulation in SMCs. Loss of CREB protein in aortic walls of rats with streptozotocin-induced diabetes is accompanied by an increase in PDGFR content. In Ob/Ob mice (which demonstrate reduced aortic wall CREB content vs. Ob/- controls), treatment with the peroxisomal proliferator-activated receptor 
rosiglitazone increases CREB content and decreases PDGFR content in the aortic wall. Thus, both in vitro and in vivo loss of CREB content and activity and subsequent accumulation of PDGFR may contribute to SMC activation during diabetes. |
Introduction |
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(PDGFR) and enhance the release of PDGF-AA from SMCs (4, 5, 6, 7). Thus, cytokines augment PDGF action in the vasculature by increasing both PDGFR ligand and receptor. Induction of PDGFR expression in an angioplasty model of vascular injury was recently reported (8).
PDGFR was recently reported to play a critical role in susceptibility of vascular SMCs to proliferation (9, 10). PDGFR is up-regulated by CCAAT-enhancer binding protein- (C/EBP) and down-regulated by the peroxisomal proliferator-activated receptor (PPAR) ligand troglitazone (9, 11, 12). The cAMP response element-binding protein (CREB) negatively regulates PDGFR expression. Our laboratory recently reported down-regulation of PDGFR by CREB in SMCs as part of a cDNA array analysis (13). In that same report, we demonstrated that overexpression of active CREB was able to blunt PDGF-stimulated SMC proliferation and migration. In additional studies we observed a decrease in CREB content in the vasculature of rodents with insulin resistance and streptozotocin diabetes (14). Finally, we observed a restoration of CREB content in the vascular stroma of Ob/Ob mice treated with the PPAR ligand rosiglitazone (14). Thus, PDGFR expression is regulated at the transcriptional level in health and disease.
Changes in the content and function of PDGFR have been associated with vascular pathology. These pathologies include genetic hypertension in spontaneously hypertensive rats (SHRs), atherogenesis, and inflammatory responses in the vasculature, particularly in response to inflammatory cytokines such as IL-1ß. Recent studies indicate that the C/EBP may be involved in up-regulation of PDGFR gene expression and protein content in response to IL-1ß (9, 10, 11, 12). Treatment of SMCs with the PPAR agonist troglitazone inhibits IL-1ß stimulation of PDGFR gene expression (12). Increased expression of PPAR has been observed in the vasculature in response to vascular insults. Treatment of atherosclerosis-prone animals with PPAR ligands decreases plaque burden (15). Thus, increased PPAR expression is thought to limit excessive vascular remodeling in atherosclerosis (16, 17, 18, 19, 20).
We have recently reported that high levels of CREB activity in SMCs result in a decrease in PDGFR mRNA content (13). In this report, we demonstrate that CREB activity regulates PDGFR content directly in SMCs by suppressing promoter-regulated transcription.
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Materials and Methods |
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Experiments in insulin-resistant animal models.
Aortas from female ob/ob and lean ob/- littermate control mice (The Jackson Laboratory, Bar Harbor, ME) were obtained in collaboration with Dr. Boris Draznin (Denver, CO). Ob mice were given ad libitum access to food and killed at 12 wk of age. Blood was collected by cardiac puncture and samples were analyzed for insulin and glucose concentrations. Aortic samples from ob/ob and ob/- animals were obtained from the laboratory of Dr. Boris Draznin; unrelated data on these animals have been previously published (21). For the rosiglitazone treatment studies, 40 male ob mice were purchased from The Jackson Laboratory at 6 wk of age. Mice with blood glucose greater than 15 mmol/liter were randomized to treatment via gavage with either 20 mg/kg per day rosiglitazone or vehicle. Glucose and body weight were assessed at baseline and d 4 and d 8. Animals were killed at d 11.
Materials
Fetal bovine serum (FBS), glutamine, and penicillin/streptomycin were purchased from Gemini Bio Products, Inc. (Cabassas, CA). Trypsin/EDTA, lipofectamine, and Plus Reagent were purchased from Life Technologies, Inc. (Grand Island, NY). CREB-specific antisera were purchased from New England Biolabs, Inc. (Beverly, MA). Antisera specific for PDGFR was purchased from R\|[amp ]\|D Systems (Minneapolis, MN). The PDGFR promoter-reporter construct containing 5' regulatory regions of the PDGFR gene driving expression of the gene encoding firefly luciferase was kindly provided by Dr. Paul Joosten (University of Nijmegen, Nijmegen, The Netherlands). A plasmid containing the cDNA encoding the constitutively active CREB mutant DIEDML-CREB (DCREB) was graciously provided by Dr. Richard Goodman (Vollum Institute, Oregon Health Sciences University, Portland, OR). Cells expressing luciferase and ß-galactosidase following transfection with promoter-reporter plasmid constructs were extracted using diluted 5x reporter lysis buffer obtained from Promega Corp. (Madison, WI). Reagents for assays of luciferase and ß-galactosidase activity in cell extracts were purchased from PharMingen (San Diego, CA). MEM Eagle’s and all other reagents were purchased from Sigma (St. Louis, MO).
Cell culture
Aortic smooth muscle cells were isolated by media explant from bovine and rat aortas. Cells were passaged in culture and used for experiments between passages 2 and 5. Cells were maintained in Modified Eagle’s Medium containing penicillin and streptomycin, 2 mM glutamine, and 10% FBS. For most of the experiments presented, bovine cells were used, and results were similar in all cases in rat SMCs. Before experiments, cells were incubated in serum-reduced medium (MEM + 0.1% FBS) for 48 h. In experiments involving different glucose concentrations, glucose was added to MEM + 0.1% FBS at final concentrations of 5 mM (low glucose) and 25 mM (high glucose, HG) for 72 h before assessment. Similarly, hydrogen peroxide and glucose oxidase were added at varying concentrations (0–20 µM or 22 mU/ml, respectively) for 72 h before assay.
PDGFR promoter-luciferase promoter transactivation
Early passage cultures (P1-P5) of either bovine or rat aortic SMCs were plated in 6-well culture dishes at a density of 1.4 x 105 cells/cm2. Cells were subsequently maintained for 18 h in growth medium, consisting of MEM (Sigma) containing 1x nonessential amino acids, 0.4 mM glutamine, and 10% FBS (Gemini Bio-Products, Inc.). Transfection was performed using Lipofectamine Plus transfection reagent (Life Technologies, Inc./BRL) as described by the manufacturer. In addition to specific chimeric PDGFR promoter-luciferase plasmid constructs, SMCs were cotransfected with a constitutively expressed ß-gal reporter plasmid construct. Expression vectors encoding mutant CREB proteins (constitutively active DCREB and dominant negative ACREB) were included in cotransfection experiments. The Lipofectamine Plus:DNA mixture in growth medium was left on cells for 3 h, and cells were allowed to recover in growth medium overnight. SMCs were subsequently serum starved in 1x MEM (containing 1x nonessential amino acids and 0.4 mM glutamine) for 24 h. Treatments involving glucose (5 mM and 25 mM), glucose oxidase (22 µM), and hydrogen peroxide (10 or 20 µM) were performed in 1x MEM for durations of 4–24 h, and cells were subsequently extracted in 1x reporter lysis buffer (Promega Corp.) for analysis of reporter gene expression. Luciferase reporter activity was corrected for differences in transfection efficiency, cell number, and extract recovery, using ß-gal activity determined in the same cellular extract. All results are expressed as luminescence as arbitrary light units/ß-gal activity in the sample extract.
Adenovirus-mediated protein expression in vascular SMCs
SMCs were infected using recombinant, replication-deficient adenovirus under the regulation of the cytomegalovirus immediate early promoter regulating the ectopic expression of cDNA-encoding mutant CREB proteins. These mutant CREB proteins included constitutively active CREB proteins, VP16-CREB, or DCREB, the dominant negative CREB A-CREB, as well as ß-gal. Efficacy and effectiveness of these adenovirus constructs has been assessed and reported in prior publications from this laboratory (13, 14).
For experiments, bovine or rat SMCs were plated at a density of 1.4 x 105 cells/cm2 in 6-well culture dishes and maintained in growth medium for 24 h. SMCs in 35-mm culture dishes were treated with purified adenovirus (adVP16-CREB or adßgal) at the previously described moi per cell for 24 h (13, 14). Cells were fed fresh serum-reduced medium lacking virus and incubated an additional 24 h. Protein extraction for Western blot analysis was performed at this time.
Western blot analysis for CREB and PDGFR content
At the end of experimental treatments, SMC cultures were washed once with PBS and wells scraped in 1x Laemmli sample buffer. Protein concentrations of samples were assessed by Bradford protein assay. For assessment of CREB protein content, 40 µg cellular protein were resolved on SDS-PAGE. Analysis of PDGFR protein content was performed following resolution of 40 µg extracted proteins on SDS-PAGE. Resolved proteins were electrophoretically transferred to nylon membranes, and equivalence of protein loading was assessed by staining of membrane-bound proteins by Ponceau stain. CREB and PDGFR contents were evaluated immunologically using commercially available primary antisera (New England Biolabs, Inc. and R\|[amp ]\|D Systems, respectively). Alkaline phosphatase-coupled secondary antibodies along with subsequent enhanced chemiluminescence were used to acquire quantitative signals. Both Ponceau stained membranes (for protein loading) and autoradiographic films (for specific protein signals) were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA). In figures, results are expressed as arbitrary densitometric units with statistical differences (P > 0.05) as determined by t test.
Atlas cDNA array analysis
SMCs were cultured and infected with either adßgal or adVP16CREB, as described above. Total RNA was extracted from cells using TRIzol Reagent (Life Technologies, Inc./BRL). Single-strand cDNA probes were generated from total RNA using 32P-dATP and reagents and protocols provided by CLONTECH Laboratories, Inc. (Palo Alto, CA). These probes were used for hybridization with separate rat cDNA array membranes using protocols and reagents provided by the manufacturer (CLONTECH Laboratories, Inc.). Arrays were subjected to autoradiography at -80 C using Lightning Plus screens (Kodak, Rochester, NY). Scanned arrays were analyzed using Atlas Image software (CLONTECH Laboratories, Inc.), comparing relative intensities of specific cDNA spots, which were corrected for differences in the relative intensities of housekeeping genes between membranes before analysis. Three separate array analyses were performed with probes generated from RNA resulting from two different experiments. Results for cDNAs depicted below are the mean of these three separate determinations. Data are presented as mRNA content in adVP16CREB-treated cells relative to mRNA content in adßgal-treated cells. These results are reiterated (13) to emphasize specific changes in PDGFR mRNA content in these experiments.
Northern blot analysis of RNA from adenovirus-infected SMCs
SMCs in culture were infected with recombinant adenoviruses encoding constitutively active CREB (VP16CREB or DCREB) and ß-gal control for 6 d. Total RNA was extracted from cells using Trizol reagent (Bio-Rad Laboratories, Inc.). Isolated total RNA was quantified spectrophotometrically, and 40 µg total RNA from each group was subjected to formaldehyde-agarose (1%) gel electrophoresis. RNA was transferred to nylon membranes (Immobilon P, Millipore Corp., Bedford, MA). Probe was generated from a human PDGFR cDNA (kindly provided by Dr. L. Claesson-Welsh, Uppsala, Sweden) in the presence of 32P-dATP, which was hybridized to the membrane-bound RNA at 42 C overnight. The membrane was subsequently washed and subjected to autoradiography using Lightning-Plus screen (DuPont, Wilmington, DE) for 24 h. The autoradiogram was subjected to densitometric analysis as described above for Western blot analyses, with arbitrary densitometric values for PDGFR mRNA-specific bands corrected for densitometric values for corresponding 28S rRNA bands obtained from the agarose gel.
Harvest of aortic tissue from animals
Following 8 wk of diabetes during which animals that had been injected had elevated blood glucose (>400 mg/dl), the animals were killed with 45 mg/kg sodium pentobarbital and aorta tissue removed. Tissue was frozen in liquid nitrogen until analysis.
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Results |
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protein content was decreased in cells expressing constitutively active CREB (13). This suggested one mechanism whereby CREB could decrease proliferative capacity. To better characterize the impact of CREB on PDGFR expression, we infected SMCs with viruses encoding two isoforms of active CREB and another encoding dominant negative CREB. Expression of ACREB increased PDGFR expression, whereas constitutively active CREB isoforms (DCREB or VP16 CREB) decreased PDGFR expression (Fig. 1
). Thus, loss of CREB function results in increased PDGFR expression.
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mRNA in SMCs. Cells were infected with recombinant adenoviruses expressing either constitutively active VP16CREB or DCREB or ß-gal as a control. Overexpression of either VP16CREB or DCREB reduced the content of PDGFR mRNA in SMCs relative to the content of rRNA (Fig. 2B) relative to the content following ß-gal adenovirus infection.
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expression at the level of transcription protein content could be regulated at the levels of transcription, mRNA stability, or translation. We next examined the impact of CREB on mRNA content and a heterologous PDGFR promoter-luciferase plasmid construct. Increased CREB activity as the result of expression of constitutively active CREB (VP16-CREB or DCREB) led to a significant loss of PDGFR mRNA content (Fig. 2
, A and B). To determine the impact of CREB activation on PDGFR gene expression, cells were cotransfected with the PDGFR luciferase construct and plasmids expressing either ACREB or active CREB (DCREB). ACREB augmented the PDGFR-promoter activity, but DCREB decreased PDGFR-promoter activity (Fig. 2C). Hence, CREB activity regulates PDGFR expression.
C/EBP is known to enhance PDGFR expression. In light of the contradictory effects of CREB and C/EBP on PDGFR gene expression, we examined their impact on receptor expression in cotransfection experiments. As expected, cotransfection of SMCs with PDGFR promoter-luciferase constructs and C/EBP leads to enhanced activity of the PDGFR promoter (Fig. 3). However, inclusion of an active DCREB expression vector in the transfection mix suppresses C/EBP-mediated activation of the PDGFR promoter (Fig. 3). Expression of active CREB prevents activation of PDGFR promoter-luciferase reporter activity by C/EBP. These studies suggest competition for regulation of the PDGFR gene by C/EBP and CREB.
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protein content. Our interest in CREB’s impact on gene expression stems from recent work in our laboratory indicating that CREB regulates migration and proliferation of SMCs and that its content is diminished by exposure to conditions accompanying diabetes and oxidant stress. Prior work from this laboratory (14) indicates that exposure to HG, 25 mM, which increases intracellular ROS, or hydrogen peroxide (10 µM or 20 µM) results in decreased CREB expression. We examined the impact of HG (Fig. 4A) or oxidant stress (Fig. 4B) on PDGFR expression in SMCs. As expected, both manipulations (which decrease CREB content) led to increased PDGFR expression. To test whether the loss of CREB was important for enhanced PDGFR expression, cells overexpressing either control or active adenoviral CREB were exposed to ROS. Expression of constitutively active DCREB through infection of cultures with a recombinant DCREB adenovirus blunts increases in PDGFR protein content resulting from reactive oxidant species (Fig. 4B). These studies indicate that enhanced PDGFR expression is permitted by loss of CREB function or CREB interference with ROS generation.
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levels are increased in animal models of insulin resistance and diabetes) (14). One common mechanism of vascular injury in these two models is accumulation of ROS. We predicted that PDGFR would be up-regulated by both insulin resistance and STZ- induced diabetes. Indeed, STZ diabetes increased PDGFR content in the aortic wall (Fig. 5B). We next assessed whether treatment of insulin resistance with rosiglitazone, an intervention that increases CREB content (14), would decrease PDGFR expression. Ob/Ob mice have decreased CREB content in the vascular stroma, which is restored by short-term treatment with rosiglitazone (14). Rosiglitazone treatment of Ob/Ob mice decreased PDGFR expression in the aortic wall (Fig. 6). No change in PDGFRß content was observed following rosiglitazone therapy. These data suggest that an inverse relationship exists in vivo between CREB content and PDGFR expression.
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Discussion |
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. We also show that CREB decreases PDGFR expression. These studies expand on our previous reports that CREB can restrain SMC proliferation and migration. One of the likely mechanisms whereby a transcription factor could affect proliferative capacity is the regulation of growth factor receptor expression. In these studies we demonstrate an inverse correlation between CREB and PDGFR expression. This is an important physiological response because up-regulation of PDGFR expression is a common response to vascular injury that could be a target for therapeutic intervention.
PDGFR and its ligand PDGF-AA are important because they are both synthesized in SMCs, and release of PDGF-AA can result in an autocrine loop in SMCs. This loop appears to be activated following exposure to cytokines such as IL-1ß, which is released by macrophages that accumulate at sites in the vasculature following injury and during the progression of atherosclerosis (2). As such, it can be speculated that PDGFR expression and activation may act to support SMC activation following an initial injury to the vessel wall. A recent report wherein C/EBP overexpression led to increased SMC proliferative response to endogenous PDGF (potentially the result of enhanced PDGFR expression) illustrates the physiological importance of PDGFR expression and regulation in vivo (10). Because CREB loss also appears to be involved in the etiology of SMC activation, the connection between CREB loss and PDGFR accumulation may delineate a critical component of the mechanism by which persistent SMC activation contributes to vessel wall pathologies.
PDGFR is present at low levels under control conditions and can be up-regulated by atherogenic stimuli. Increased PDGFR content is observed in SMC from genetically SHRs relative to SMCs from their Wistar-Kyoto rat controls (11). Likewise, the progression of atherosclerosis that appears, in part, to be mediated by release of infiltrating macrophages and their released cytokines (such as IL-1ß) also result in increased expression of PDGFR. Indeed, the proliferative response of SMCs to IL-1ß appears to be mediated indirectly through stimulation of PDGF-AA release, increased PDGFR expression, and binding of PDGF-AA to these receptors on SMCs (5). The effects of IL-1ß on PDGFR expression have been attributed to increased expression of C/EBP and its binding to the PDGFR promoter (11). Indeed, increased expression of PDGFR in the SMCs of SHRs has been attributed to increased transcription mediated by a single C/EBP-binding element in the promoter (11). The observation in this report that C/EBP stimulation of the PDGFR promoter can be prevented by active CREB supports contradictory roles for these two transcription factors, the mechanism of which is the focus of future work in this laboratory.
This is the first report that PDGFR is up-regulated in diabetes mellitus and insulin resistance. The only reports regarding PDGFR expression in diabetes demonstrate increased PDGFRß expression in glomeruli in diabetes (22). Importantly, PDGFR up-regulation is responsive to treatment with the PPAR ligand rosiglitazone (22). Previous observations imply that PDGFR activation may play a role in supporting SMC activation and the progression of pathology in SMCs. Indeed, SMC-targeted overexpression of C/EBP in transgenic mice leads to increased proliferation of SMCs under both basal and PDGF-stimulated conditions (10).
It is interesting to note that PPAR agonists, which have been ascribed the capacity to attenuate SMC activation in animal models of atherosclerosis (16, 17), can suppress C/EBP-mediated transcription of the PDGFR gene (12). Previously published work from this laboratory indicates that rosiglitazone treatment of Ob/Ob insulin-resistant mice restores CREB content toward that seen in control Ob/- mice (14). Here we describe that rosiglitazone-stimulated increases in CREB content in the aortic wall of Ob/Ob mice are accompanied by suppression of PDGFR expression, implying that perhaps CREB also contributes to down-regulation of PDGFR expression by PPAR ligands.
Animal data and preliminary studies in humans indicate that PPAR ligands can limit SMC activation (16, 17) and atherosclerotic plaque formation (15). A number of inflammatory proteins and metaloproteases are regulated in the vasculature by PPAR ligands. Results presented both here and in our previously published work indicate that one of the targets of PPAR activity is to elevate SMC CREB content and decrease PDGFR. It is reasonable to speculate that CREB is an important mediator of the effect of thiazolidinediones in atherosclerosis.
The results presented here reinforce our previously published conclusion (13, 14) that CREB activity serves to stabilize a mature, contractile, nonproliferative phenotype in SMCs. These results provide evidence that (1) CREB loss in response to a wide variety of SMC-activating agents contributes to the proliferation and migration of these cells, and (2) the loss of CREB-mediated suppression of PDGFR expression may be a critical step toward the participation of SMCs in the etiology of vascular dysfunction. Results also suggest that up-regulation of CREB expression may be of key importance for the effect of PPAR on the response of SMCs to PDGF.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACREB, Dominant negative mutant CREB protein; C/EBP, CCAAT/enhancer-binding protein-; CREB, cAMP response element-binding protein; DCREB, constitutively active CREB mutant DIEDML-CREB; FBS, fetal bovine serum; ß-gal, ß-galactosidase; HG, high glucose; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor alpha; ROS, reactive oxidant stress; SHR, spontaneously hypertensive rat; SMC, smooth muscle cell; STZ, streptozotocin.
Received December 19, 2001.
Accepted for publication April 22, 2002.
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References |
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receptor gene via CCAAT/enhancer-binding protein- in vascular smooth muscle cells. J Biol Chem 274:25576–25582 evokes enhanced gene transcription of platelet-derived growth factor -receptor in vascular smooth muscle cells. Circ Res 89:503–508 receptor in cultured vascular smooth muscle cells. J Clin Invest 96:558–567
activation inhibits interleukin-1ß-mediated platelet-derived growth factor- receptor gene expression via CCAAT/enhancer-binding protein- in vascular smooth muscle cells. J Biol Chem 276:12893–12897: a new approach to the macrovascular complications of diabetes. Diabetes Care 24:392–397 activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res 83:1097–1103This article has been cited by other articles:
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