GATA-6 Is Involved in PPARγ-Mediated Activation of Differentiated Phenotype in Human Vascular Smooth Muscle Cells
Objective— Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor superfamily involved in the growth and differentiation of many cell types. Although the activation of PPARγ in human vascular smooth muscle cells (VSMCs) inhibits the growth of these cells, the precise mechanism of this effect is unknown. PPARγ-mediated growth inhibition of VSMCs is associated with the induction of the differentiated phenotype. A zinc finger transcription factor, GATA-6, has been implicated in the maintenance of the differentiated phenotype in VSMCs.
Methods and Results— The administration of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a naturally occurring PPARγ ligand, and troglitazone, a thiazolidinedione derivative, induced the expression of smooth muscle myosin heavy chain and smooth muscle α-actin, highly specific markers for differentiated VSMCs. Stimulation of proliferative VSMCs with PPARγ ligands also increased the activity of the transfected wild-type smooth muscle myosin heavy chain promoter but not that of the mutant promoter, in which a GATA-6 binding site was mutated. Compatible with the role of GATA-6, both 15d-PGJ2 and troglitazone upregulated the DNA binding activity of GATA-6 in proliferative VSMCs.
Conclusions— The activation of PPARγ-dependent pathways induces the differentiated phenotype in proliferative VSMCs, and this induction is mediated, in part, through a GATA-6–dependent transcriptional mechanism.
- peroxisome proliferator-activated receptor-&ggr
- vascular smooth muscle cells
- phenotypic modulation
The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily. PPARγ, the most intensively studied isoform, has been implicated in such diverse pathways as lipid and glucose homeostasis, control of cellular proliferation, and differentiation.1,2⇓ The activation of PPARγ with a ligand, such as a class of antidiabetic, insulin-sensitizing agents known as thiazolidinediones,3 or eicosanoid derivatives, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2),4 results in its heterodimerization with the retinoid X receptor and the binding of their complex to the PPAR response element (PPRE) of target genes.5 Since its discovery, PPARγ has been shown to be expressed in monocytes/macrophages, the heart, vascular smooth muscle cells (VSMCs), endothelial cells, and atherosclerotic lesions.6,7⇓ Recently, it has been shown that thiazolidinediones inhibit the development of atherosclerosis in LDL receptor–deficient mice8 and apolipoprotein E–knockout mice9 and of restenosis in rat models of balloon angioplasty.10 In vitro, the thiazolidinediones and 15d-PGJ2 inhibit the proliferation of VSMCs through a PPARγ-dependent pathway.7,10⇓ However, the precise mechanisms of this growth inhibition are unknown at present.
In contrast to normal medial VSMCs, the proliferating VSMCs within thickened arterial intimas observed in atherosclerosis express low levels of proteins that are characteristic of differentiated VSMCs.11,12⇓ These proteins include smooth muscle isoforms of contractile proteins, such as smooth muscle myosin heavy chain (SM-MHC) and smooth muscle α-actin (SM-α-actin).11 Although the mechanisms involved in specifying the proliferative/synthetic or differentiated/contractile VSMC phenotype are largely unknown, the recent observations suggest the role of a zinc finger transcriptional protein, GATA-6, as an important regulator of VSMC phenotype.13–15⇓⇓ We have recently demonstrated that GATA-6 binds a conserved GATA-like motif within the rat SM-MHC promoter and activates this promoter in a sequence-specific manner. In addition, this motif is required for the upregulated expression of the SM-MHC gene during differentiation of VSMCs.16 For all of these reasons, we are interested in investigating the role of GATA-6 in PPARγ-mediated growth inhibition/differentiation of VSMCs. To achieve this, we examined whether PPARγ ligands such as 15d-PGJ2 and troglitazone activate the transcription of a smooth muscle–specific contractile protein, and if so, whether GATA-6 is involved in this process.
A plasmid pSM-MHC chloramphenicol acetyltransferase CAT, consisting of the bacterial CAT driven by 1346 bp of the rat SM-MHC gene promoter,17 was a generous gift from Dr Gray K. Owens (University of Virginia, Charlottesville). Plasmid constructs pwtSM-MHCluc and pmutSM-MHCluc contain firefly luciferase reporter cDNA driven by the wild-type 836-bp rat SM-MHC promoter or by the corresponding promoter in which the GATA-6 binding site is mutated, respectively.16 pRSVCAT and pRSVluc contain the reporter gene driven by Rous sarcoma virus long-terminal repeat sequences.18 The expression plasmid encoding human GATA-6 (phGATA-6) and that encoding human PPARγ (phPPARγ) were kindly donated by Dr Kenneth Walsh (Boston University, Boston, Mass)13 and Dr Krishna Chatterjee (University of Cambridge, Cambridge, UK),19 respectively. pPPREluc, containing 3 copies of the PPRE (GTCGACAGGGGACCAGGACAAAGGTCACG-TTCGGGAGTCGAC) in direct orientation upstream of the basal reporter construct tk-luc, was provided by Dr Kazuhiko Umesono (Kyoto University, Kyoto, Japan).5 Plasmids were purified by anion exchange chromatography (Qiagen), quantified by the measurement of OD260, and examined on agarose gels stained with ethidium bromide before use.
Human aortic VSMCs were obtained from Kurabo Industries Ltd and cultured with 5% fetal bovine serum in the presence of growth factors as previously described.16 To induce the differentiated phenotype, VSMCs were cultured in low-serum media (1% fetal bovine serum) in the absence of growth factors.
Measurement of DNA Synthesis and Assay for Cellular Viability
The incorporation of the thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), was measured to determine the effects of 15d-PGJ2 or troglitazone on DNA synthesis. VSMCs (passage 6) were plated in a 96-well microplate and cultured in low-serum media (1% fetal bovine serum) in the absence of growth factors for 48 hours to induce the differentiated phenotype. Thereafter, these cells were stimulated with growth factors and 5% fetal bovine serum to reenter the cell cycle in the presence or absence of 15d-PGJ2 or troglitazone for 18 hours. The cells kept as the differentiated phenotype were used as a control. BrdU was then added, and the incubation was continued for an additional 6 hours. Subsequent BrdU incorporation assays were carried out according to the protocols supplied by the manufacturer (Biotrak, Amersham Pharmacia Biotech).
A colorimetric assay based on the mitochondrial activity of living cells to cleave the tetrazolium ring and reduce the tetrazolium salt, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Wako Pure Chemicals Industries, Osaka), to formazan was used to quantify cell viability as described previously.20
Immunocytochemistry and Western Blotting
Immunocytochemical staining was performed by the use of an anti–SM-MHC monoclonal antibody (Sigma Chemicals) indirect immunoperoxidase method as described previously.18 For Western blotting, we used monoclonal antibodies (SM-MHC and β-actin obtained from Sigma Chemicals; SM-α-actin from Dako Laboratories). Immunoblots were quantified by densitometry by using NIH Image 1.62 as described previously.18
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from VSMCs as described previously.16 Double-stranded wild-type oligonucleotide (wt SM-MHC GATA) from rat SM-MHC upstream sequences (−827/−795 relative to the transcription start site) and mutant oligonucleotide (mut SM-MHC GATA) of corresponding length, in which the GATA-6 binding site was mutated, were prepared as previously described.16 An oligonucleotide containing consensus PPRE (5′-CAAAAC-TAGGTCAAAGGTCA-3′),5 mutant PPRE (5′-CAAAACTAG-CACAAAGCACA-3′), or the consensus Sp1 site was purchased from Santa Cruz Biotechnology. Electrophoretic mobility shift assays (EMSAs) were carried out as described previously.18
Transfection and Luciferase/CAT Assays
VSMCs (passages 6) were transfected with 2 μg of DNA in a 60-mm plate by using lipofectAMINE PLUS (Life Technologies, Inc) as previously described.16 The lysates from these cells were subjected to assays for luciferase and CAT activities as described previously.18
Data are presented as mean±SE. Statistical comparisons were performed by unpaired 2-tailed Student’s t tests or ANOVA with Scheffe’s test where appropriate, with P<0.05 indicating significance.
15d-PGJ2 and Troglitazone Inhibit DNA Synthesis in Proliferative VSMCs
To examine the effect of PPARγ ligands such as 15d-PGJ2 and thiazolidinediones on human VSMC proliferation, we measured BrdU, a thymidine analogue, incorporation in the presence of various concentrations of 15d-PGJ2 or troglitazone after stimulation with growth factors and 5% fetal bovine serum. As shown in Figure 1A, administering 10−9 to 10−7 mol/L of 15d-PGJ2 (Cayman Chemical Co) into proliferative VSMCs resulted in mild but not significant inhibition of BrdU incorporation. The inhibition was significant at concentrations of 10−6, 5×10−6, and 10−5 mol/L. As shown in Figure 1C, administering 10−9 to 10−6 mol/L of troglitazone (a gift from Sankyo Pharmaceutical Co, Tokyo, Japan) into proliferative VSMCs did not significantly inhibit BrdU incorporation, although it appeared to have some inhibitory effects. Significant inhibition was observed at the concentrations of 5×10−6 and 10−5 mol/L.
To rule out the possibility that 15d-PGJ2 and troglitazone induced VSMC death, cell viability was assessed with the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The data in Figures 1B and 1D show that 10−5 mol/L of 15d-PGJ2 or troglitazone induced cell death. However, 15d-PGJ2 or troglitazone in the range of 10−9 to 5×10−6 mol/L had no effect on cell viability, as indicated by equivalent mitochondrial activity in untreated and treated VSMCs of proliferative phenotype.
15d-PGJ2 and Troglitazone Induce the Expression of SM-MHC and SM-α-Actin in Proliferative VSMCs
To explore whether PPARγ ligands induce the differentiated phenotype, we examined the effects of 15d-PGJ2 or troglitazone on the expression of SM-MHC and SM-α-actin, highly specific markers for differentiated VSMCs. Proliferative VSMCs were incubated with 15d-PGJ2 (10−9 to 10−6 mol/L) or troglitazone (10−8 to 10−6 mol/L) for 48 hours. Lysates from these cells were then subjected to Western blotting. Expression levels of smooth muscle–specific proteins were quantified and normalized to the β-actin expression level. As shown in Figure 2A and B, administering 10−8 mol/L of 15d-PGJ2 significantly induced the expression of SM-MHC and SM-α-actin in proliferative VSMCs. This induction did not occur at higher concentrations of 15d-PGJ2. Similarly, as shown in Figure 2C and D, troglitazone significantly induced the expression of SM-MHC and SM-α-actin at a concentration of 10−7 mol/L but not at a concentration of 10−6 mol/L. Administering dimethyl sulfoxide, which dissolves 15d-PGJ2 and troglitazone in their stock solutions, into proliferative VSMCs did not affect the expression of SM-MHC at any concentrations lower than 0.1% (Figure 2E).
15d-PGJ2 Activates PPARγ-Dependent Pathways and Induces the Expression of SM-MHC in Proliferative VSMCs
To determine whether 15d-PGJ2 actually activates PPARγ, we examined the activity of a PPRE-containing promoter. We transfected a luciferase expression vector driven by 3 copies of the PPRE (pPPREluc). As shown in Figure 3A, 10−8 mol/L of 15d-PGJ2 resulted in marked activation of PPRE promoter activity. Administration of prostaglandin F2α (10−8 mol/L; Wako Pure Chemical Industries), which causes inhibitory phosphorylation of PPARγ by stimulating mitogen-activated protein kinase,21 largely blocked this activation. Next, we examined DNA-protein interaction between the consensus PPRE and endogenous PPARγ by EMSAs. Proliferative VSMCs were stimulated with 15d-PGJ2 to activate PPARγ-dependent pathways. Nuclear extracts prepared from these cells were probed with a radiolabeled oligonucleotide containing the consensus PPRE (Figure 3B and 3C). Competition EMSAs in lanes 4 and 5 of Figure 3B revealed that the retarded bands represented PPRE sequence–specific binding. As shown in Figure 3C, compared with the administration of normal rabbit IgG (lane 4), administering anti-PPARγ antibody markedly diminished the activity of these retarded bands. However, the anti-PPARα antibody (lane 3) only slightly diminished the activity. These findings indicated that the retarded bands mainly consisted of PPARγ and might contain a small amount of PPARα. Compatible with the results of transient transfection experiments, the activities of these bands were markedly increased by 15d-PGJ2 stimulation (compare lanes 1 and 2 in Figure 3B). Further administration of prostaglandin F2α almost completely blocked the 15d-PGJ2–stimulated increase in these activities (lane 3 in Figure 3B). These findings suggest that stimulation of proliferative VSMCs with 15d-PGJ2 predominantly activates the DNA binding of PPARγ rather than that of PPARα. In contrast, neither 15d-PGJ2 nor prostaglandin F2α affected the DNA binding activity of Sp1 (Figure 3D).
To examine the effect of 15d-PGJ2 and prostaglandin F2α on the endogenous expression of SM-MHC, we carried out immunocytochemical staining. As shown in Figure 4, differentiated VSMCs showed abundant expression of SM-MHC in the cytoplasm, whereas little expression was observed in proliferative VSMCs. Activation of PPARγ by stimulating proliferative VSMCs with 15d-PGJ2 resulted in the induction of SM-MHC. This induction was abolished by further administration of prostaglandin F2α.
GATA-6 Is Required for PPARγ-Stimulated SM-MHC Transcription in Proliferative VSMCs
We examined the roles of GATA-6 and PPARγ-dependent pathways in SM-MHC transcription. NIH3T3 cells, which express neither PPARα nor PPARγ,22 were transfected with pPPREluc or pSM-MHC CAT together with the expression plasmid encoding human PPARγ (phPPARγ) and/or that encoding human GATA-6 (phGATA-6), and these were stimulated with 15d-PGJ2 for 48 hours. As expected, the exogenous PPARγ activated the PPRE activity in NIH3T3 cells in the presence of 15d-PGJ2 (Figure 5A). Interestingly, although expression of either PPARγ or GATA-6 mildly activated the 1346-bp rat SM-MHC promoter, expression of both PPARγ and GATA-6 synergistically activated this promoter (Figure 5B), suggesting that PPARγ signaling converges with GATA-6–dependent transcriptional mechanisms.
Next, to examine the role of GATA-6 in PPARγ-mediated activation of SM-MHC transcription in VSMCs, we transfected a luciferase expression vector driven by the 836-bp wild-type SM-MHC promoter (pwtSM-MHCluc) or the SM-MHC promoter with a mutation in the GATA-6 binding site (pmutSM-MHCluc). As shown in Figure 5C and 5D, the administration of 15d-PGJ2 and troglitazone resulted in the activation of the wild-type SM-MHC promoter. Mutating the GATA-6 binding site abolished the activation induced by 15d-PGJ2 and troglitazone.
PPARγ Ligands Increase the DNA Binding Activity of GATA-6 in Proliferative VSMCs
To determine whether PPARγ ligands modulate the DNA binding activity of GATA-6 in proliferative VSMCs, we performed EMSAs by using an oligonucleotide containing the SM-MHC GATA site as a probe. As shown in lanes 2 and 3 of Figure 6B, competition EMSAs revealed that a retarded band represented GATA sequence–specific binding. As shown in Figure 6C, the activity of this band was diminished by the administration of anti–GATA-6 antibody (lane 2) but not by the anti–GATA-4 antibody (lane 3) or normal rabbit IgG (lane 5). The activity of this band was modestly diminished by the anti-p300 antibody (lane 4). These data indicate that the retarded band contains a significant amount of GATA-6 and p300. As shown in Figure 6A, the intensity of the specific band was increased in nuclear extracts from VSMCs stimulated with troglitazone (lane 2) or 15d-PGJ2 (lane 4) compared with those from saline-stimulated cells (lane 1). Increases in the DNA binding activity of GATA-6 by troglitazone or 15d-PGJ2 were reduced by prostaglandin F2α, a PPARγ-inhibitory compound (lanes 3 and 5). In contrast, troglitazone, 15d-PGJ2, and prostaglandin F2α did not alter the DNA binding activity of Sp1 (Figure 6D).
Using 2 different and structurally unrelated PPARγ ligands, troglitazone and 15d-PGJ2, we have demonstrated that PPARγ activation in proliferative VSMCs induced the endogenous expression of SM-MHC and SM-α-actin, highly specific differentiation markers. We confirmed the activation of PPARγ-dependent pathways by demonstrating the induction of PPARγ-specific DNA binding as well as PPRE-dependent transcription. Furthermore, exogenous expression of PPARγ in NIH3T3 cells, which express neither PPARα nor PPARγ, activated the GATA-6–dependent SM-MHC transcription in the presence of 15d-PGJ2. Interestingly, induction of differentiated markers by 15d-PGJ2 and troglitazone decreased at micromolar concentrations of these agents, whereas inhibition of DNA synthesis became evident at these concentrations. At present, the precise mechanisms of this activity are unknown. However, several studies have shown that there is a very poor correlation between proliferation rates in intimal VSMCs and their modified differentiated phenotype in both humans and experimental animal models.11 Thus, our data are compatible with these studies. It has been shown that micromolar concentrations of these agents possess PPARγ-independent activity and directly modify other signaling pathways, such as nuclear factor-κB, extracellular signal–regulated kinase, and phosphatidylinositol 3-kinase/protein kinase B.23–27⇓⇓⇓⇓ Therefore, it is possible that these PPARγ-independent pathways are involved in the decreased expression of smooth muscle–specific contractile proteins at higher concentrations of these agents. Further studies are required on the regulated expression of smooth muscle–specific proteins by PPARγ-dependent and -independent pathways.
This study demonstrated that the 15d-PGJ2–inducible expression of SM-MHC was mediated in part through a transcriptional mechanism and that the GATA-6 binding site within the SM-MHC promoter was required for the induction by PPARγ ligands. In addition, EMSA experiments showed that both troglitazone and 15d-PGJ2 increased the DNA binding activity of GATA-6 in proliferative VSMCs. These findings demonstrate that GATA-6 is involved in the PPARγ-mediated transcriptional activation of the SM-MHC gene in VSMCs. However, neither troglitazone nor 15d-PGJ2 affected the endogenous expression of GATA-6, PPARγ, or p300, an important cofactor of GATA-6 (data not shown). Thus, an increase in the DNA binding activity of GATA-6 might involve its posttranslational modification. It has been shown that other members of the GATA family of transcription factors, namely GATA-1, -2, -3, and -4, exist as phosphoproteins.28,29⇓ Interestingly, Altiok and colleagues30 demonstrated that the activation of PPARγ decreased expression of the serine/threonine phosphatase PP2A and increased a phosphorylated form of transcription factor DP-1. Further studies are needed on the mechanisms by which PPARγ-dependent pathways increase the DNA binding activity of GATA-6.
Recently, we showed that a transcriptional coactivator, p300, interacted with GATA-6 and augmented the GATA-6–dependent transcription of the SM-MHC promoter.16 A p300 protein may function as a bridge between GATA-6 and components of the basal transcription machinery. Interestingly, the PPAR/retinoid X receptor heterodimer also forms a complex with p300.31 It would be interesting to study the relation between p300 and PPARγ-dependent signaling pathways during VSMC differentiation.
Although the data of our present study suggest a requirement of GATA-6 in PPARγ-induced activation of the 836-bp SM-MHC promoter, these data do not rule out other possible mechanisms. For example, we could not find PPRE, at least in the 2.4-kb SM-MHC promoter sequences, by our computer search. However, we cannot rule out the possibility that PPARγ directly binds and activates the SM-MHC promoter through PPRE, which might exist outside these sequences. In fact, smooth muscle–specific expression in vivo requires sequences outside the 2.4-kb SM-MHC promoter.32 Another transcription factor that might be involved in the PPARγ-induced transcription of VSMC differentiation marker genes is serum response factor, a MADS box–containing transcription factor. This factor binds CArG sequences in the SM-MHC promoter and activates this promoter in a sequence-specific manner.17,32⇓ Interestingly, serum response factor is also reported to cooperate with p300 and GATA factors.33 Roles of p300, GATA-6, and serum response factor in PPARγ-induced VSMC differentiation should be further investigated in the context of a longer SM-MHC promoter by a transgenic approach.
Thiazolidinediones are now widely used as insulin-sensitizing agents in patients with diabetes mellitus. These patients often have atherosclerosis complications. Given the inhibitory effects of thiazolidinediones on atherosclerosis in vivo, it will be of particular importance to elucidate the precise mechanisms by which PPARγ-dependent signaling pathways induce the differentiated phenotype in VSMCs.
This work was supported in part by the Advanced and Innovational Research program in Life Science; grants to T.K. and K.H. from the Ministry of Education, Science and Culture of Japan; and grants to M.A. from the Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism. We thank N. Sowa for excellent technical assistance.
Received July 8, 2002; revision accepted January 5, 2003.
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