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(Circulation Research. 2001;89:1058.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Platelet-Derived Growth Factor Promotes the Expression of Peroxisome Proliferator-Activated Receptor in Vascular Smooth Muscle Cells by a Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

Mingui Fu, Xiaojun Zhu, Qian Wang, Jifeng Zhang, Qing Song, Hui Zheng, Wataru Ogawa, Jie Du, Yuqing E. Chen

From the Cardiovascular Research Institute (M.F., X.Z., Q.W., J.Z., Q.S., H.Z., Y.E.C.), Morehouse School of Medicine, Atlanta, Ga; Second Department of Internal Medicine (W.O.), Kobe University School of Medicine, Japan; and Renal Division (J.D.), Emory University School of Medicine, Atlanta, Ga.

Correspondence to Dr Yuqing E. Chen, Cardiovascular Research Institute, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA 30310. E-mail echen{at}msm.edu

	Abstract

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Vascular diseases such as atherosclerosis are characterized by abnormal accumulation of vascular smooth muscle cells (VSMCs) within the intimal lining. The intimal VSMCs exhibit an increased expression of peroxisome proliferator-activated receptor (PPAR), and the administration of pharmacological PPAR agonists attenuates vascular lesion formation. The factors that regulate PPAR expression in the vasculature are poorly defined. Here we report that platelet-derived growth factor (PDGF) upregulates PPAR by the phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathway. Using Northern-blotting and Western-blotting analyses, we observed that the levels of PPAR mRNA and protein were increased by 2- to 3.5-fold in human aortic smooth muscle cells (HASMCs) treated with PDGF (20 ng/mL). This was abolished by preincubation of HASMCs with a PI3-kinase inhibitor (LY294002, 50 µmol/L), and partially inhibited by a MEK1 inhibitor (U0126, 10 µmol/L), but not affected by a p38 kinase inhibitor (SB202190, 10 µmol/L). In addition, overexpression of the dominant-negative p85 subunit of PI3-kinase or Akt proteins blocked the PDGF-induced PPAR expression. Taken together, our results suggest that PDGF induces PPAR expression in VSMCs by a PI3-kinase/Akt signaling pathway. The characterization of factors and signaling pathways that modulate PPAR expression in VSMCs may have important implications for understanding the pathogenesis of vascular diseases.

Key Words: platelet-derived growth factor • peroxisome proliferator-activated receptor • phosphatidylinositol 3-kinase • signaling pathway • vascular smooth muscle cells

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptors (PPARs), including , , and ß/, are a family of ligand-activated nuclear transcriptional factors^1,2 that form heterodimers with retinoid X receptors (RXR), bind to the PPAR responsive element (PPRE), and thereby regulate target gene expression. PPAR is found predominantly in adipose tissue, where it plays a crucial role in adipocyte differentiation, fat storage, and glucose homeostasis.³ Recent studies have documented that PPAR is also present in all of the following critical vascular cells: endothelial cells, vascular smooth muscle cells (VSMCs), and monocytes/macrophages.^4,5 This finding suggested that PPAR may play a critical role in vascular biology. Indeed, it has been reported that PPAR is highly expressed in cells within the atherosclerotic plaque and the neointima after balloon injury.⁶ Thiazolidinediones, a class of antidiabetic drugs that are specific ligands of PPAR, inhibit neointima formation after balloon injury⁷ and the development of atherosclerosis in LDL receptor-deficient mice.⁸ Studies in vitro also demonstrate that PPAR agonists inhibit VSMC proliferation and migration.^9,10 It is postulated that the increase in PPAR expression noted in vascular lesion may function as an endogenous inhibitor of vascular disease. However, the factors that regulate PPAR expression in vascular cells remain poorly defined.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant that functions as an important mediator in the pathogenesis of vascular disease.^11,12 In many pathological conditions, PDGF expression within vasculature is increased by a local production from endothelial cells and VSMCs and local secretion by platelets. Stimulation of the PDGF receptors on VSMCs can activate several signaling pathways, including those mediated by p38, MEK1/MAPK, and phosphatidylinositol 3-kinase (PI3-kinase), which transduce the signal into the nucleus and stimulate the proliferation and migration of VSMCs.^13,14

In the present study, we tested the hypothesis that PDGF regulates PPAR expression in human and rat VSMCs. Indeed, our findings indicate that PDGF upregulates PPAR expression in VSMCs by a PI3-kinase/Akt signaling pathway.

	Materials and Methods

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Cell Culture
Human aortic smooth muscle cells (HASMCs) were purchased from Clonetics and were cultured in smooth muscle cell growth medium-2 containing (in ng/mL) human basic fibroblast growth factor 2, human epidermal growth factor 0.5, and amphotericin-B 50; 5% FBS; 50 µg/mL gentamicin; and 5 µg/mL bovine insulin (all purchased from Clonetics). For all experiments, early-passage (passages 5 to 7) HASMCs were grown to 80% to 90% confluence and made quiescent by serum starvation (0.4% FBS) for at least 24 hours. Each inhibitor examined was added 30 minutes before the addition of human recombinant PDGF-BB (Sigma). Cells from the rat aortic smooth muscle cell line A7r5, obtained from American Type Culture Collection (ATCC), were cultured in DMEM containing 10% FBS (Life Technologies). The A7r5 cells stably overexpressing a constitutively active Akt construct (Akt/A7r5) and the control GFP (GFP/A7r5) were kindly provided by Dr Gary Gibbons (Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Ga).

Northern Blot Analysis
Total RNA (20 µg) isolated from each condition using acid-guanidinium thiocyanate was subjected to electrophoresis through 1% formaldehyde-agarose gels. After transferring to nylon membranes (Bio-Rad), the RNA was cross-linked to the membrane by an UV cross-linker (Stratagene). ³²P-labeled cDNA probes were generated by using a random primer labeling system (Gibco-BRL). Blots were prehybridized, hybridized, and washed once with 1x SSC at 65°C, and once with 0.1x SSC, 1.0% SDS (wt/vol), at 65°C for 20 minutes. The lane-loading differences were normalized using the GAPDH cDNA probe.

Analysis of PPAR mRNA Half-Life
PPAR mRNA half-life experiments were carried out using HASMCs. The cells were exposed to vehicle or PDGF (20 ng/mL) for the indicated periods before mRNA stability measurements. Transcription was inhibited by the addition of actinomycin D (5 µg/mL). Northern blotting analysis was performed as described above.

Western Blot Analysis
Total cell lysates (50 µg) were subjected to SDS-PAGE and electrotransferred to nitrocellulose membrane (Bio-Rad). After blocking in 20 mmol/L Tris-HCl (pH 7.6) containing 150 mmol/L NaCl, 0.1% Tween 20, and 5% (wt/vol) nonfat dry milk, blots were incubated with specific antibodies against PPAR (Santa Cruz Biotechnology), PPAR2 (Affinity Biotech), or p85 subunit of PI3-kinase (Upstate Biotechnology) for 1 hour at room temperature. The specificity of each antibody has been well documented by the manufacturers and confirmed by other published reports.^6,15 The blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), and immunoactivity was visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) per the manufacturer’s instructions.

Adenovirus Preparation and Infection
Adenovirus was prepared as previously described.¹⁶ For generation of recombinant adenovirus encoding dominant-negative p85 of PI3-kinase (named Ad-p85DN), a mutated form of p85 subunit I in which the inner SH2 domain is deleted¹⁷ was cloned into pCMVTrack (an adenovirus shuttle vector) and cotransformed with AdEasy (an adenovirus backbone) into Escherichia coli. The virus DNA from in vivo recombination was isolated, digested, and used for transfection of packing cell HEK293. A transfection mix was prepared by adding 4 µg of PacI linearized plasmid DNA and 20 µL of LipofectAMINE (Life Technologies) to 500 µL of OptiMEM (Life Technologies) according to the manufacturer’s instructions. The resulting virus was amplified for three to four cycles, and at this point, viral titers were high enough to use for gene-transfer experiments. The viruses were purified by CsCl gradient and final yields were generally 10¹¹ to 10¹² plaque-forming units (pfu)/mL. In this study, the VSMCs were infected with adenovirus vectors at 5 pfu/cell. The cells were subjected to experiments from 24 to 48 hours after infection.

PI3-Kinase Assay
To determine whether PI3-kinase is an essential signaling molecule mediating PDGF-induced PPAR expression, we generated an adenovirus (Ad-p85DN) of the dominant-negative mutant 85-kDa subunit of PI3-kinase. To test the function of Ad-p85DN, we measured the PI3-kinase activity of this mutated p85 as previously described.¹⁵

Transient Transfection and Luciferase Assays
Transient transfection was performed with 1 µg of total DNA per well of six-well plates and LipofectAMINE (Gibco-BRL) according to the manufacturer’s instructions (DNA:LipofectAMINE ratio, 1:3). Pilot studies have documented a transfection efficiency of 15% to 25% in rat VSMCs (A7r5, ATCC) using this approach. Briefly, A7r5 cells achieving 70% confluence in six-well plates were cotransfected with 800 ng of an expression vector containing the reporter gene luciferase driven by a 3.0-kb PPAR1 promoter¹⁸ (a gift from Dr J. Auwerx, IGBMC, C.U. de Strasbourg, France), and 200 ng of the expression vector containing the reporter ß-gal driven by the cytomegalovirus promoter (Clontech) was used as the control for transfection efficiency. Twenty-four hours after transfection, the cells were washed twice with PBS and subsequently cultured for 24 hours in serum-free medium. After incubating with PDGF at 20 ng/mL for 6 hours, the cells were prepared for luciferase activity measurement using the reporter luciferase assay kit (Promega Co). The luciferase activity was measured by a luminometer (Victor II, Perkin Elmer) and normalized by ß-gal activity.

Statistical Analysis
Each experimental condition was tested in triplicate, and each experiment was repeated a minimum of three times. Statistical analyses were performed by ANOVA or unpaired two-tailed Student test. Data are presented as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PDGF Induces PPAR Expression in HASMCs
Although it was reported that PPAR expression is upregulated in the neointima,⁶ little is known about the mechanism. To test whether PDGF regulates PPAR gene expression in HASMCs, we examined the effect of PDGF on PPAR gene expression in HASMCs using Northern blotting analysis for mRNA expression and Western blotting analysis for the protein level. In response to 20 ng/mL PDGF stimulation, the level of PPAR mRNA was increased by 2.8-fold, and the PPAR protein was increased 2.2-fold (Figures 1A and 1B).

View larger version (31K): [in this window] [in a new window]   Figure 1. PDGF induces PPAR expression in HASMCs. Top, Northern blotting (A) or Western blotting (B) analyses reveal that PPAR expression is induced by PDGF (20 ng/mL) stimulation for 2 hours (A) or 24 hours (B) in HASMCs. Bottom, Average values normalized by GAPDH (A) or ß-actin (B) of three independent experiments (**P<0.01, compared with vehicle treatment). C, PDGF stimulation activates PPAR1 promoter. A 3.0-kb PPAR1 promoter/luciferase reporter construct and a ß-gal expression vector were cotransfected into rat VSMCs (A7r5). Forty-eight hours after transfection, cells were stimulated with vehicle or PDGF at 20 ng/mL for 24 hours. Luciferase activities normalized by ß-gal activity are expressed in relative units to no PDGF stimulation, which was designated 1 (mean±SEM, n=3, **P<0.01). D, PPAR1 is expressed in HASMCs. Western blotting analysis was performed using a PPAR common antibody (Santa Cruz Biotechnology, sc-7169, 1:500 diluted), which can detect both PPAR1 and PPAR2, and a PPAR2-specific antibody (Affinity BioReagents, PA1-824, 1:500 diluted). Protein samples were isolated from rat white fatty tissue (lane 1) and from HASMCs treated with vehicle (lane 2) or PDGF (lane 3).

To determine whether PDGF-induced PPAR gene expression in VSMCs is at the transcriptional level, we transfected an expression vector containing the luciferase reporter gene driven by a 3.0-kb PPAR1 promoter into rat VSMCs (A7r5, ATCC). PDGF stimulation for 24 hours increased the luciferase activity by 2.2-fold (Figure 1C). These results indicated that PDGF stimulates PPAR gene transcription in VSMCs.

PPAR has two isoforms, PPAR1 and PPAR2, generated by alternative promoters and differential splicing.¹⁸ PPAR2 has 30 additional amino acids at the N-terminus and is reportedly expressed primarily in adipocytes. To date, whether PPAR2 is expressed in VSMCs is controversal.^6,9 To identify which PPAR isoform was regulated by PDGF, we performed Western blotting analyses using an anti-PPAR antibody (Santa Cruz) that cross-reacts with both PPAR1 and PPAR2 and an anti-PPAR2 antibody (Affinity Biotech) that only reacts with PPAR2. The protein from rat white fatty tissue was used as positive control. As shown in Figure 1D, there was only one band around 52 kDa detected by the anti-PPAR antibody in PDGF-induced cell lysates, and no band was detected by the anti-PPAR2 antibody in these samples. With the positive control, two bands (around 52 to 55 kDa) were detected in fat tissue by the anti-PPAR antibody, whereas only one band (55 kDa) was detected by the anti-PPAR2-selective antibody. In addition, PPAR2 mRNA was not detected in HASMCs by reverse transcriptase-polymerase chain reaction assay using the human PPAR2-specific primers as previously described¹⁹ (data not shown). These data suggested that only PPAR1 was regulated by PDGF in VSMCs, and that PPAR2 was undetectable in PDGF-treated or -untreated VSMCs.

Time Course and Dose-Dependent Effect of PDGF on PPAR Gene Expression in VSMCs
HASMCs were treated with 20 ng/mL of PDGF for 0, 0.5, 2, 6, 12, 24, 48, and 72 hours, and then the levels of PPAR mRNA in the cells were determined by Northern blotting analyses. As shown in Figure 2A, the levels of PPAR mRNAs induced by PDGF were dramatically increased at 2 hours, but declined to some extent at 6 hours. At 12 hours, the mRNA levels rose again to achieve the second peak at 24 hours and a sustained level for at least 72 hours.

View larger version (28K): [in this window] [in a new window]   Figure 2. Time course and dose-dependent effects of PDGF-induced PPAR expression. A, HASMCs were incubated with PDGF (20 ng/mL) for different times as indicated. B, Different concentrations of PDGF (0 to 50 ng/mL) were incubated with HASMCs for 2 hours. Levels of PPAR mRNAs were analyzed by Northern blotting analyses. Values for PPAR mRNA normalized by GAPDH level are mean±SEM (n=3, *P<0.05 and **P<0.01).

The dose-response effect of PDGF-induced PPAR expression was documented at 2 hours of PDGF stimulation. The expression of PPAR mRNA was upregulated in a dose-dependent manner, with significant increases observed at a concentration as low as 5 ng/mL. Maximal increases were obtained at a PDGF concentration of 10 ng/mL (Figure 2B). These results revealed that PDGF activates PPAR gene expression in VSMCs.

PDGF Stimulation Does Not Affect the PPAR mRNA Stability in VSMCs
To evaluate whether PPAR mRNA stability contributes to PDGF-induced PPAR gene expression, we examined the half-life of PPAR mRNA in HASMCs. Northern blotting analyses were performed with addition of actinomycin D (5 µg/mL) after 2 hours of PDGF (20 ng/mL) stimulation. In HASMCs, the half-life of PPAR mRNA was 4 hours. There was no significant difference between PDGF-treated and -untreated cells (Figure 3).

View larger version (37K): [in this window] [in a new window]   Figure 3. PDGF stimulation does not affect PPAR mRNA stability in HASMCs. Cells were incubated with or without 20 ng/mL of PDGF for 2 hours and de novo mRNA transcription was inhibited by addition of actinomycin D (5 µg/mL). Total RNA was extracted at 0, 2, and 4 hours after administration of actinomycin D for Northern blotting analyses. A, Representative Northern blot. B, Average values (n=3) at each time point in graph indicate the remaining signal expressed as percentage of initial PPAR mRNA level.

PDGF Induces PPAR Expression by PI3-Kinase/Akt Signaling Pathway in VSMCs
To investigate the signaling pathways mediating PDGF-induced PPAR expression, we initially focused on defining the roles of PI3-kinase, MEK/ERK, and p38 MAPK. HASMCs were treated with 20 ng/mL PDGF for 2 hours after pretreatment with LY294002 (50 µmol/L, a PI3-kinase inhibitor), SB202190 (25 µmol/L, a p38 kinase inhibitor), or U0126 (10 µmol/L, a MEK inhibitor). As shown in Figure 4, inhibition of PI3-kinase completely blocked the effect of PDGF (P<0.01), whereas inhibition of MEK partially reduced the effect of PDGF by 65±8.5% (P<0.05). However, inhibition of p38 MAPK had no significant effects on PDGF-induced PPAR mRNA expression.

View larger version (48K): [in this window] [in a new window]   Figure 4. The PI3-kinase inhibitor blocks PDGF-induced PPAR expression in HASMCs. Cells were treated with LY294002 (50 µmol/L, a PI3-kinase inhibitor), SB202190 (25 µmol/L, a p38 kinase inhibitor), or U0126 (10 µmol/L, an MEK inhibitor) for 30 minutes before 2-hour PDGF (20 ng/mL) stimulation. PPAR mRNA levels were determined by Northern blotting analyses. Shown are representative Northern blot (top) and quantitative graph of three independent experiments (bottom). Values normalized by GAPDH are mean±SEM (n=3, *P<0.05 and **P<0.01).

To further define whether the PI3-kinase signaling pathway mediates PDGF-induced PPAR gene expression in VSMCs, we generated an adenoviral vector containing a dominant-negative mutant p85 (Ad-p85DN) in which the inner SH2 domain of p85 subunit is deleted. It has been documented that this mutant of p85 subunit cannot activate the p110 subunit of PI3-kinase. Using Western blotting analyses, we confirmed that this p85 dominant-negative protein was overexpressed in HASMCs that were infected with Ad-p85DN (Figure 5D). Additionally, Ad-p85DN blocked the PI3-kinase activity induced by insulin-like growth factor-1 (IGF-1) in HASMCs using thin-layer chromatography (Figure 5E). In contrast, Ad-GFP did not affect PI3-kinase activity induced by IGF-1. These results indicated that Ad-p85DN could function as a dominant-negative inhibitor of the PI3-kinase pathway.

View larger version (38K): [in this window] [in a new window]   Figure 5. Overexpression of dominant-negative p85 of PI3-kinase or protein kinase B (Akt) proteins blocks the PDGF-induced PPAR gene expression. Confluent HASMCs were infected with p85 or Akt dominant-negative adenovirus. Twenty-four hours after adenovirus infection, cells were made quiescent by serum starvation for 24 hours and then stimulated with PDGF at 20 ng/mL for 2 (A) or 24 (C) hours. A, Representative Northern blot. B, Average values of PPAR mRNA normalized by GAPDH are represented as mean±SEM compared with GFP without PDGF stimulation, which was designated 1. (n=3; **P<0.01). C, Levels of PPAR protein were determined by Western blotting analyses using PPAR antibody from Santa Cruz. ß-Actin was used as control for equal loading. D, Overexpression of truncated p85 in HASMCs. Cells were infected by p85 dominant-negative adenovirus (Ad-p85DN) at the indicated multiplicity of infection (pfu/cell). Levels of p85 protein were determined by Western blotting analyses using p85 antibody from Upstate Biotechnology. E, Ad-p85DN blocks IGF-1-induced PI3-kinase activity in HASMCs.

To test the hypothesis that the stimulatory effect of PDGF is mediated by the PI3-kinase/Akt pathway, we selectively blocked this signaling pathway by using Ad-p85DN or ad-AktDN (a dominant-negative adenovirus of Akt from W.O.²⁰). Blockade of the PI3-kinase/Akt pathway using this approach effectively prevented PDGF-induced PPAR gene expression in HASMCs (Figure 5). However, PPAR expression was not affected by the control adenovirus (Ad-GFP) infection in HASMCs (data not shown). Taken together, these results provided the first evidence that PPAR gene expression is regulated by a PI3-kinase/Akt-dependent pathway.

To further confirm the involvement of Akt in PDGF-induced PPAR expression in VSMCs, we examined the PPAR protein levels in the rat VSMC lines (A7r5) that were stably transfected with a constitutively active Akt versus control cells stably transfected with a GFP construct. The level of the PPAR in Akt/A7r5 is 2.3-fold higher than that in the control GFP/A7r5 VSMCs (Figure 6). These results further confirmed that Akt is involved in the regulation of PPAR expression in VSMCs.

View larger version (33K): [in this window] [in a new window]   Figure 6. Overexpression of Akt in A7r5 cells increases PPAR expression. Same-passage A7r5/Akt and GFP/A7r5 stable cell lines were cultured in DMEM/F12 supplement with 10% FCS. Proteins were prepared after 48 hours of serum starvation. Levels of PPAR were determined by Western blotting analyses. Top, Representative Western blot. Bottom, Average values of PPAR proteins normalized by ß-actin are mean±SEM (n=3, **P<0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptors are a family of nuclear receptors comprising three subtypes of designated isoforms, PPAR, PPAR, and PPAR (also termed PPARß, FAAR, or NUC-1) with different tissue distributions.¹⁶ PPAR has been documented in the regulation of adipocyte differentiation, lipid metabolism, and insulin action.³ Several groups have reported that PPAR is present in rat and human VSMCs,^9,10 and is highly expressed within the atherosclerotic plaque and the neointima after balloon injury.⁶ However, little is known about the regulation of PPAR expression in VSMCs. In this study, we demonstrated that PDGF induced PPAR expression in VSMCs by a PI3-kinase/Akt-dependent signaling pathway.

PDGF-induced PPAR mRNA expression is most likely due to an induction of transcription rather than altering the stability of PPAR mRNA, given that the addition of PDGF failed to change the degradation rates of PPAR mRNA in HASMCs. It is noteworthy that there are two peaks (at 2 and 24 hours) of PDGF-induced PPAR gene expression. What is the mechanism of this biphasic induction? Recently, it was reported that angiotensin II induced cytoplasmic-to-nuclear translocation of the nuclear factor (NF)-B subunits with parallel changes in DNA binding activity in a biphasic manner, which was comparable with the biphasic induction of interleukin-6 stimulated by angiotensin II.²¹ Because the PPAR1 promoter contains NF-B sites (data not shown), we hypothesize that NF-B may be involved in mediating the PDGF-induced PPAR expression in VSMCs. Although systematic deletion mapping of PDGF response elements in the PPAR promoter is necessary to understand this mechanism, that is beyond the scope of the present study.

PDGF is an important regulator that mediates the aberrant behavior of VSMCs in the pathogenesis of vascular diseases.¹² PDGF binding to its receptor on VSMC can activate several signal pathways including p38-, MEK1/ERK-, and PI3-kinase-mediated signal pathways, which transduce the signals into the nucleus and stimulate the proliferation and migration of VSMCs.^13,14 Our experiments have shown that the inhibition of PI3-kinase completely blocked the effect of PDGF-induced PPAR expression in VSMCs. The MEK1 inhibitor partially reduced the effect of PDGF on PPAR mRNA, whereas p38 kinase inhibitors had no effect on this action. Although those pharmacological probes are relatively selective, we also verified these results by using adenoviral vectors with dominant-negative mutant constructs. The concordance between the data using pharmacological probes and the dominant-negative constructs provides the first definitive evidence that PPAR gene expression is regulated by a PI3-kinase-dependent pathway.

It was well known that Akt is a proximal downstream effector of the PI3-kinase pathway.²² Therefore, we examined the involvement of Akt in PDGF-induced PPAR expression. Our results showed that infection with adenovirus containing dominant-negative mutant of Akt also completely inhibited the increase of PPAR expression induced by PDGF in HASMCs. Furthermore, as an additional test of our hypothesis, we confirmed that Akt activation is a sufficient condition for the upregulation of PPAR gene expression. In stably transfected VSMCs with a constitutive upregulation of Akt activity, we observed that the level of PPAR in Akt/A7r5 was 2.3-fold higher than the control VSMC line, GFP/A7r5. Taken together, these results demonstrated that Akt is a key mediator of PDGF-induced PPAR expression in VSMCs.

Although we demonstrated that PDGF-induced PPAR gene expression is mediated by PI3-kinase-dependent pathway, it would be quite interesting to determine whether the PPAR target genes are activated after PDGF stimulation in VSMCs. We examined two well-characterized PPAR target genes, CD36 identified in macrophages²³ and uncoupling protein-2 (UCP-2)²⁴ identified in adipocytes, because the PPAR target genes are not well characterized in VSMCs. Using Western blotting analyses, we found that the level of UCP-2 was increased 2.6-fold after 48 hours of PDGF stimulation in HASMCs (data not shown). However, CD36 was undetectable by both reverse transcriptase-polymerase chain reaction and Western blotting analyses in HASMCs. These results suggested that PPAR might be the mediator of PDGF-induced UCP-2 expression. To further understand the role of PPAR in vasculature, it is necessary to globally define the PPAR target genes in VSMCs. We are currently using a DNA microarray analysis to approach this challenge.

To date, several putative targets of PI3-kinase/Akt pathway have been proposed, among them Bad,²⁵ caspase-9,²⁶ and the transcription factor Forkhead.²⁷ Interestingly, a recent report demonstrated that NF-B is also the target of Akt,²⁸ suggesting that NF-B may be the downstream mediator of Akt in PDGF-induced PPAR expression. To further study the mechanism of PDGF-induced PPAR gene expression in VSMCs, we recently cloned the 5.4-kb human PPAR1 gene promoter (data not shown). Computer sequence analysis revealed that there were several consensus-binding motifs, including the NF-B, GATA1, C/EBP, and AP1 sites in this 5.4-kb PPAR1 gene promoter. To our knowledge, there are no published studies that have analyzed these functional elements in the PPAR1 gene promoter. Therefore, cloning this 5.4-kb PPAR gene promoter will provide a unique resource for us to study the molecular mechanisms that govern the upregulation of PPAR in VSMC during neointima formation.

PPAR exists in at least two isoforms, PPAR1 and PPAR2, and PPAR2 has an NH₂-terminal extension of 30 amino acids. PPAR2 is expressed selectively in adipose tissue, whereas PPAR1 is present in many tissues. Previous reports have identified that PPAR mRNA and protein exist in rat and human aortic VSMCs. However, the expression pattern of PPAR isoforms was not described. In accord with a recent report,⁶ our findings indicate that only PPAR1 is expressed in cultured human aortic VSMCs. Furthermore, we found that PDGF activates the PPAR1 promoter activity as shown in Figure 1C, but not PPAR2 promoter (data not shown).

It is well documented that PDGF is one of the key pathological factors in vascular diseases. Several recent reports suggest that PPAR may function as a protective factor in vascular diseases such as atherosclerosis.^29,30 Therefore, PDGF-induced PPAR expression might provide a feedback mechanism by which PPAR inhibits PDGF-induced VSMC proliferation and migration. The characterization of factors and signaling pathways that modulate PPAR expression in vasculature may have important implications for the pathogenesis of vascular diseases.

Although the regulation of PPAR gene expression was the focus in this study, we realized that the phosphorylation of PPAR by mitogen-activated protein kinase at the N-terminal domain of PPAR inhibits the transcriptional activity in adipocytes.^31,32 In the present study, we have not detected the phosphorylated band of PPAR in HASMCs. However, it will provide a better understanding of the role of PPAR in vasculature to define whether other growth factors or cytokines can induce PPAR expression, and whether phosphorylation of PPAR is involved in its transcriptional activation in VSMCs.

	Acknowledgments

 
This work was partially supported by a starting grant from Morehouse Cardiovascular Research Institute (Enhancement of Cardiovascular and Related Research Areas, NIH/NHLBI 5 UH1 HL03676-02) and an institutional grant (NIH/NIHGMS S06GM08248), as well as by the American Heart Association (to Y.E.C. and J.D.). We thank Dr Gary Gibbons for the useful discussions of this project and the critical review of this manuscript.

Received May 7, 2001; revision received August 30, 2001; accepted September 21, 2001.

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