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J. Biol. Chem., Vol. 276, Issue 52, 48950-48955, December 28, 2001

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15-Deoxy-^12,14-prostaglandin J₂ and Thiazolidinediones Activate the MEK/ERK Pathway through Phosphatidylinositol 3-Kinase in Vascular Smooth Muscle Cells^*

Kotaro Takeda, Toshihiro Ichiki, Tomotake Tokunou, Naoko Iino, and Akira Takeshita

From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan

Received for publication, September 10, 2001, and in revised form, October 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptor (PPAR)  belongs to the nuclear receptor superfamily of ligand-dependent transcription factors. Recent results have shown that the ligands for nuclear receptors have rapid effects so called "nongenomic" effects, which are observed within minutes after stimulation. We examined whether 15-deoxy-^12,14-prostaglandin J₂ (15-d-PGJ2) had rapid effects on cultured vascular smooth muscle cells. Phosphorylation of ERK and c-fos mRNA expression were determined by Western and Northern blot analyses, respectively. PPAR agonists 15-d-PGJ2 and thiazolidinediones such as pioglitazone and troglitazone elicited rapid activation of ERK within 15 min and induced c-fos mRNA expression within 30 min, whereas the PPAR agonist bezafibrate failed to activate ERK. 15-d-PGJ2-induced expression of c-fos mRNA was blocked by PD98059 or U0126, two ERK kinase inhibitors, suggesting that the MEK/ERK pathway mediates 15-d-PGJ2-induced c-fos gene expression. Furthermore, pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, inhibited 15-d-PGJ2-induced ERK activation and c-fos mRNA expression, suggesting that PI3-kinase is involved in the process. An electrophoretic mobility shift assay showed that 15-d-PGJ2 enhanced AP-1 binding activity to AP-1 consensus sequence in a time-dependent manner. 15-d-PGJ2 increased thymidine incorporation in a PI3-kinase-dependent manner. Taken together, our findings show that 15-d-PGJ2 and thiazolidinediones activate the MEK/ERK pathway through PI3-kinase and lead to c-fos mRNA expression and DNA synthesis. These findings indicate a novel regulatory mechanism of gene expression by 15-d-PGJ2 and thiazolidinediones.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptor (PPAR)¹ belongs to steroid/thyroid hormone nuclear receptor transcription factor superfamily, and three isoforms, designated , , , have been identified (1). PPAR is highly expressed in adipocytes and activated macrophages and is involved in fatty acid metabolism, adipocyte differentiation (2), and inhibition of macrophage activation (3). PPAR is also expressed in vascular smooth muscle cells (VSMCs) (4). PPAR is activated by a natural ligand, 15-deoxy-^12,14-prostaglandin J₂ (15-d-PGJ2) (5), and synthetic ligands, thiazolidinediones (TZDs) (6) such as pioglitazone (Pio) and troglitazone (Tro). Ligand-activated PPAR forms a heterodimer with retinoid X receptor, binds to a specific DNA sequence, PPAR response element (PPRE), and activates target gene transcription (1). These effects are referred to as "genomic" action, which requires relatively long periods.

A growing body of evidences suggests that steroid hormones have more rapid effects so-called "nongenomic" effects, which may be mediated by membrane-bound steroid receptors (7). Estrogen is well characterized for the rapid nongenomic action. Estrogen induces an increase in the intracellular cGMP level and the release of nitric oxide within a few minutes of administration in endothelial cells (8). Estrogen also activates extracellular signal-regulated protein kinase (ERK) within 15 min (9). These time courses are too rapid to encompass the classical genomic mechanism mediated by estrogen response element-dependent gene transcription. Consistent with these findings, bovine serum albumin-conjugated estrogen, which is impermeable to cell membrane, has been also reported to activate the ERK pathway in endothelial cells (10) and in a neuroblastoma cell line (11). These findings suggest that nongenomic actions of estrogen via novel membrane-bound estrogen receptor play a pivotal role in mediating intracellular signals.

Phosphatidylinositol 3 (PI3)-kinase, a heterodimer of an adapter subunit (p85) and a catalytic subunit (p110), is activated by many growth factors (12). PI3-kinase phosphorylates the D-3 position of the inositol ring of PI to generate phosphatidylinositol 3-phosphate (13). One of the effector molecules downstream from PI3-kinase is Akt/PKB, a serine/threonine kinase (14). The signaling cascade from PI3-kinase to Akt mediates cell proliferation and the cell survival signal (15). It has been reported that activation of the ERK kinase (MEK) ERK pathway is regulated through PI3-kinase by several stimuli (16-18). We have demonstrated in the present study that PPAR agonists such as 15-d-PGJ2, Pio, and Tro rapidly activated the MEK/ERK pathway and led to c-fos mRNA induction in VSMCs. We have also demonstrated that PI3-kinase was responsible for ERK activation by 15-d-PGJ2. To our knowledge, this is the first report to reveal that PPAR agonists activate the MEK/ERK pathway in VSMCs. PPAR agonist-induced ERK activation provides a novel regulatory mechanism of gene expression by PPAR agonists in VSMCs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- 15-d-PGJ2 was purchased from Cayman Chemical Co. Pio and Tro were provided by Takeda Chemical Industries and Sankyo Pharmaceutical Co., respectively. Bezafibrate was provided by Kissei Pharmaceutical Co. 15-d-PGJ2, Pio, and Tro were dissolved in dimethyl sulfoxide (Me₂SO). PD98059 and wortmannin were obtained from Research Biochemicals Int. U0126 was obtained from Promega Corp. SB203580 is a generous gift from SmithKline Beecham Pharmaceuticals. Bovine serum albumin, phorbol 12-myristate 13-acetate (PMA), and actinomycin D were purchased from Sigma. Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Gibco BRL DuPont NEN. [-³²P]dCTP, [-³²P]ATP, and [³H]thymidine were purchased from PerkinElmer Life Sciences. Other chemical reagents were purchased from WAKO Pure Chemical unless mentioned specifically.

Cell Culture-- VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats and maintained as described previously (19). Between 4 and 9 passages were used for the experiments.

Western Blot Analysis-- VSMCs were lysed in a sample buffer (50 mmol/liter NaCl, 30 mmol/liter sodium pyrophosphate, 50 mmol/liter NaF, 5 mmol/liter EDTA, 10 mmol/liter Tris, pH 7.4, 1% Triton X-100, 1 mmol/liter PSMF, 0.2 unit/ml aprotinin, 10 mmol/liter pepstatin A, and 25 mmol/liter leupeptin). The lysates were subjected to 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The membrane was probed with antibodies against phospho-ERK1/2, ERK1/2, phospho-Akt, and Akt (New England Biolabs) and enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). Phosphorylation of ERK is reported to correlate with kinase activity (20). Therefore, we used phosphorylation of ERK as a measure for its activation.

Northern Blot Analysis-- Total RNA was prepared by an acid guanidinium-phenol-chloroform extraction method, and Northern blot analysis was performed as described previously (21). The radioactivity of hybridized band of c-fos mRNA and 18 S rRNA was quantified by a MacBAS Bioimage Analyzer (Fujifilm).

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared from cultured VSMCs, and an electrophoretic mobility shift assay was performed as described previously (22). A DNA probe (5'-CGCTTGATGAGTCAGCCGGAA-3') that encodes consensus AP-1 sequence was labeled with [-³²P]ATP. Five µg of nuclear extracts were incubated with 1 × 10⁵ cpm of labeled DNA probe for 30 min at room temperature and electrophoresed on 5% acrylamide gel. Fifty-fold molar excess of unlabeled AP-1 was added as a competitor. After electrophoresis, gels were dried and exposed on x-ray films (Kodak).

Measurement of DNA Synthesis-- VSMCs were incubated with 15-d-PGJ2 or TZDs for 24 h. The cells were labeled with [³H]thymidine during the final 4 h. After labeling, the cells were washed with phosphate-buffered saline, fixed in 10% trichloroacetic acid, and then washed with a mixture of ethanol and ether (2:1). The cells were lysed in 0.5 NaOH, and incorporated [³H]thymidine was measured by a liquid scintillation counter.

Statistics-- Statistical analyses of the phosphorylated ERK level, the relative c-fos mRNA expression, and thymidine incorporation were performed using one-way analysis of variance (ANOVA) and Fisher's test if appropriate. Data are shown as the mean ± S.E., and p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

15-d-PGJ2 Activated the ERK1/2 Pathway-- VSMCs were incubated with 10 µmol/liter 15-d-PGJ2, and phosphorylation of ERK1/2 was determined by Western blot analysis. Fig. 1A shows that 15-d-PGJ2 significantly increased phosphorylation of ERK1/2. Fig. 1B shows that incubation with varying concentrations of 15-d-PGJ2 for 15 min resulted in a dose-dependent increase in the phosphorylation level of ERK1/2. Agonists for PPAR such as clofibrate and Wy-14,643 were also reported to activate ERK in mouse liver cell (23). However, bezafibrate, one of the PPAR agonists, rather suppressed phosphorylation of ERK in our VSMCs (Fig. 1C).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   15-d-PGJ2 activated ERK1/2 in VSMCs. VSMCs were incubated with 15-d-PGJ2 (10 µmol/liter) for varying time periods as indicated (A). VSMCs were incubated with 15-d-PGJ2 at varying concentrations as indicated for 15 min (B). VSMCs were incubated with bezafibrate (20 µmol/liter) for varying time periods as indicated (C). Phosphorylated ERK1/2 (p-ERK) and total ERK1/2 (ERK) were determined by Western blot analysis using antibodies specific to phosphorylated ERK1/2 or total ERK1/2. Left, a representative result from three independent experiments is shown. Right, the density of specific band was scanned and quantified by a Bioimage Analyzer. The ratio of phosphorylated ERK1/2 to total ERK1/2 is shown. Values (mean ± S.E.) are expressed as fold induction compared with control (n = 3). **, p < 0.01 versus control.

Induction of c-fos mRNA Expression and AP-1 Binding Activity by 15-d-PGJ2-- Activation of ERK leads to immediate early gene expression such as c-fos (24). Thus, we examined whether activation of ERK by 15-d-PGJ2 resulted in c-fos mRNA induction. 15-d-PGJ2 (10 µmol/liter) transiently induced expression of c-fos mRNA after 30 min of exposure (Fig. 2A). Me₂SO used for a vehicle for 15-d-PGJ2 did not affect c-fos mRNA expression (data not shown). Fig. 2B shows that incubation with varying concentrations of 15-d-PGJ2 for 30 min resulted in a dose-dependent increase in c-fos mRNA expression. We performed an electrophoretic mobility shift assay to determine whether 15-d-PGJ2-induced c-fos gene expression led to an increase in the AP-1 binding activity. As shown in Fig. 2C, treatment of 15-d-PGJ2 increased the AP-1 binding activity to the AP-1 consensus sequence in a time-dependent manner. The band is specific for AP-1 sequence, because it was eliminated by the addition of 50-fold molar excess of unlabeled AP-1 probe. These results suggest that 15-d-PGJ2 induces not only ERK activation but also c-fos mRNA expression and AP-1 binding activity. However, the reason that AP-1 binding activity lasted longer than ERK activation and c-fos mRNA induction is not clear at this point.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Induction of c-fos mRNA expression by 15-d-PGJ2 in VSMCs. VSMCs were incubated with 15-d-PGJ2 (10 µmol/liter) for varying time periods as indicated (A) and with 15-d-PGJ2 at varying concentrations as indicated for 30 min (B). Total RNA was isolated, and expression of c-fos mRNA and 18 S rRNA was determined by Northern blot analysis. Upper panel, representative autoradiography of c-fos mRNA and 18 S rRNA is shown. Lower panel, radioactivity of the c-fos bands was counted with a Bioimage Analyzer and normalized with radioactivity of the 18 S rRNA bands. Values (mean ± S.E.) are expressed as fold induction compared with control (n = 3). *, p < 0.05 versus control, **, p < 0.01 versus control. C, VSMCs were incubated with 15-d-PGJ2 (10 µmol/liter) for varying time periods as indicated. Nuclear extracts were prepared and incubated with radiolabeled AP-1 probe for 30 min and electrophoresed. Fifty-fold molar excess of unlabeled AP-1 probe was used as a competitor.

TZDs Induced ERK1/2 Activation and c-fos mRNA Expression-- To determine whether activation of ERK1/2 by 15-d-PGJ2 is mediated by PPAR, we examined the effect of Pio, another PPAR agonist, on ERK1/2 activation. Treatment with Pio (20 µmol/liter) induced phosphorylation of ERK1/2 within 15 min (Fig. 3A). Pio dose-dependently induced phosphorylation of ERK1/2 (Fig. 3B). Pio also induced c-fos mRNA expression within 30 min (Fig. 3C). The expression of c-fos mRNA by Pio was increased dose-dependently (Fig. 3D). To further confirm the involvement of PPAR, we examined the effect of Tro, another type of TZD, on ERK1/2 phosphorylation. Treatment of Tro (20 µmol/liter) for 15 min induced ERK1/2 phosphorylation (Fig. 3E). Tro also induced c-fos mRNA expression after 30 min (Fig. 3F). These results suggest that PPAR may be involved in ERK activation and c-fos mRNA induction by PPAR agonists such as 15-d-PGJ2, Pio, and Tro.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   TZDs induced ERK1/2 activation and c-fos gene expression in VSMCs. VSMCs were incubated with Pio (20 µmol/liter) for varying time periods as indicated (A and C). VSMCs were incubated with Pio at concentrations varying from 5 to 20 µmol/ liter for 15 (B) or 30 min (D). VSMCs were incubated with Tro (20 µmol/ liter) for 15 (E) or 30 min (F). Phosphorylated ERK1/2 (p-ERK) and total ERK1/2 (ERK) were determined by Western blot analysis (A, B, and E), and the expression of c-fos mRNA and 18 S rRNA was determined by Northern blot analysis (C, D, and F). A representative result from three independent experiments is shown.

MEK/ERK Pathway Mediates 15-d-PGJ2-induced c-fos mRNA Expression-- We determined the effect of two different inhibitors of MEK, PD98059 and U0126, on 15-d-PGJ2-induced ERK1/2 phosphorylation. Pretreatment with PD98059 (30 µmol/liter) or U0126 (20 µmol/liter) completely inhibited 15-d-PGJ2-induced ERK1/2 phosphorylation (Fig. 4A). Pretreatment with PD98059 or U0126 also completely suppressed 15-d-PGJ2-induced c-fos mRNA up-regulation (Fig. 4B). However, SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not affect 15-d-PGJ2-induced c-fos mRNA expression (Fig. 4C). These results suggest that activation of the MEK/ERK pathway is responsible for c-fos mRNA induction by 15-d-PGJ2.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   MEK/ERK pathway mediated 15-d-PGJ2-induced c-fos mRNA expression. VSMCs were preincubated with PD98059 (30 µmol/liter) or U0126 (20 µmol/liter) for 30 min. Then the cells were stimulated with 15-d-PGJ2 (10 µmol/liter) for 15 min. Phosphorylated ERK1/2 (p-ERK) and total ERK1/2 (ERK) were determined by Western blot analysis (A). VSMCs were preincubated with PD98059 (30 µmol/ liter) or U0126 (20 µmol/liter) for 30 min (B) or with SB203580 (10 µmol/liter) for 30 min (C). Then the cells were stimulated with 15-d-PGJ2 (10 µmol/liter) for 30 min. The expression of c-fos mRNA and 18 S rRNA were determined by Northern blot analysis (B and C). VSMCs were preincubated with actinomycin D (5 µg/ml) for 30 min. Then the cells were stimulated with 15-d-PGJ2 (10 µmol/ liter) for 15 min. Phosphorylated ERK1/2 and total ERK1/2 was determined by Western blot analysis (D). A representative result from three independent experiments is shown.

To confirm that the activation of ERK1/2 by 15-d-PGJ2 is independent of de novo gene transcription such as PPRE-dependent transcription, we examined the effect of actinomycin D (5 µg/ml), an inhibitor of gene transcription, on ERK1/2 phosphorylation. Fig. 4D shows that 15-d-PGJ2 induced ERK1/2 phosphorylation in the presence of actinomycin D, suggesting that activation of ERK1/2 by 15-d-PGJ2 is independent of de novo gene transcription.

PI3-kinase Mediated ERK Activation and c-fos mRNA Induction by 15-d-PGJ2-- It has been reported that activation of the ERK pathway was regulated by PI3-kinase (16-18). We examined whether PI3-kinase mediated 15-d-PGJ2-induced ERK1/2 phosphorylation and c-fos mRNA up-regulation. Pretreatment with wortmannin (100 nmol/liter), an inhibitor of PI3-kinase, inhibited 15-d-PGJ2-induced ERK activation (Fig. 5A) as well as c-fos mRNA induction (Fig. 5B). To determine whether 15-d-PGJ2 activates PI3-kinase, we examined the phosphorylation level of Akt, one of the downstream targets of PI3-kinase, by Western blot analysis. Treatment of 15-d-PGJ2 for 10 min increased the phosphorylation level of Akt (Fig. 5C). These results suggest that 15-d-PGJ2 activates the P13-kinase pathway, and PI3-kinase activation is necessary for ERK activation.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   PI3-kinase was responsible for 15-d-PGJ2-induced ERK activation. VSMCs were preincubated with wortmannin (100 nmol/ liter) for 30 min. Then the cells were stimulated with 15-d-PGJ2 (10 µmol/liter) for 15 min. Phosphorylated ERK1/2 (p-ERK) and total ERK1/2 (ERK) were determined by Western blot analysis (A). VSMCs were stimulated with 15-d-PGJ2 (10 µmol/liter) for 30 min. The expression of c-fos mRNA and 18 S rRNA was determined by Northern blot analysis (B). VSMCs were stimulated with 15-d-PGJ2 (10 µmol/ liter) for 10 min. Phosphorylated Akt (p-Akt) and total Akt (Akt) were determined by Western blot analysis (C). A representative result from three independent experiments is shown.

It has been also reported that protein kinase C (PKC) activates the ERK pathway (25). Thus, we examined the effect of PKC inhibitors on 15-d-PGJ2-induced ERK activation and c-fos mRNA expression. Although pretreatment of PMA for 24 h (PKC depletion) or GF109203X, a PKC inhibitor, for 30 min completely inhibited PMA-induced ERK activation and c-fos mRNA induction (Fig. 6, A and B), these pretreatments did not affect 15-d-PGJ2-induced ERK activation and c-fos mRNA induction (Fig. 6, C and D). These results suggest that PKC is not involved in 15-d-PGJ2-induced ERK activation pathway.

View larger version (51K): [in this window] [in a new window]   Fig. 6.   PKC was not necessary for ERK activation by 15-d-PGJ2. VSMCs were preincubated with PMA (1 µmol/liter) for 24 h or GF109203X (1 µmol/liter) for 30 min. Then the cells were stimulated with PMA (100 nmol/liter) for 5 min (A) or 15-d-PGJ2 (10 µmol/liter) for 15 min (C). Then phosphorylated ERK1/2 (p-ERK) and total ERK1/2 (ERK) were determined by Western blot analysis (A and C), or the cells were stimulated with PMA (100 nmol/liter) (B) or 15-d-PGJ2 (10 µmol/ liter) (D) for 30 min. The expression of c-fos mRNA and 18 S rRNA was determined by Northern blot analysis (B and D). A representative result from three independent experiments is shown.

Induction of DNA Synthesis by 15-d-PGJ2-- We examined whether 15-d-PGJ2 or TZDs affected DNA synthesis in VSMCs. As shown in Fig. 7, A-C, 15-d-PGJ2 and TZDs increased thymidine incorporation. The increase in thymidine incorporation by 15-d-PGJ2 was suppressed by wortmannin (Fig. 7D), confirming the role for PI3-kinase in 15-d-PGJ2 signaling. However, the inhibition of thymidine incorporation by wortmannin is partial, suggesting that pathways other than the PI3-kinase/ERK pathway are also involved in 15-d-PGJ2-induced DNA synthesis.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   15-d-PGJ2 and TZDs induced thymidine incorporation in VSMCs. Incorporation of [3H]thymidine was measured after stimulation with 15-d-PGJ2 (10-20 µmol/liter) (A), Pio (20-40 µmol/liter) (B), and Tro (20-40 µmol/liter) (C) for 24 h (n = 4). VSMCs were pretreated with wortmannin (Wort; 100 nmol/liter) and stimulated with 15-d-PGJ2 (10 µmol/liter), and [3H]thymidine incorporation was measured (D). Results are expressed as percent of control culture, which was incubated with Me2SO alone. Data are shown as the mean ± S.E. *, p < 0.05 versus control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have demonstrated in the present study that 15-d-PGJ2 induces rapid and transient activation of the MEK/ERK pathway in VSMCs. Pio and Tro, two types of TZDs, also induced rapid ERK activation, suggesting that PPAR may be involved in this process. We also showed that 15-d-PGJ2 and TZDs increased c-fos mRNA expression via activation of the MEK/ERK pathway and that PI3-kinase is responsible for 15-d-PGJ2-induced ERK activation. 15-d-PGJ2-induced DNA synthesis was partially dependent on PI3-kinase.

15-d-PGJ2 and TZDs induced rapid activation of ERK. The time course is too rapid to encompass PPRE-dependent gene transcription. Consistent with this finding, we demonstrated that 15-d-PGJ2 could activate ERK in the presence of actinomycin D, an inhibitor of gene transcription (Fig. 3D). The analysis of the c-fos promoter sequence up to about 1300 bp showed an absence of putative PPRE sequences. These results may suggest that 15-d-PGJ2-induced ERK activation is independent of PPRE-dependent gene transcription. An increasing body of evidence suggests that steroid hormone has rapid nongenomic effects, which may be mediated by membrane-bound receptors (7). It is not clear from our results whether membrane-bound receptor for PPAR agonists is present in VSMCs. Further investigation using bovine serum albumin-conjugated PPAR agonists, which are impermeable to plasma membrane, will address the presence of putative membrane-bound receptors for PPAR agonists. However, bovine serum albumin-conjugated 15-d-PGJ2 or TZDs is not available.

We explored the upstream signaling involved in 15-d-PGJ2-induced ERK activation. PI3-kinase has been reported to activate the MEK/ERK pathway by several stimuli (16-18). We demonstrated that the activation of ERK by 15-d-PGJ2 was completely suppressed by wortmannin, suggesting that this process is dependent on PI3-kinase activation. The finding that 15-d-PGJ2 induced phosphorylation of Akt, a target molecule of PI3-kinase, supports this idea. However, Goetze et al. (25) obtained a contradictory result in which Tro had no effect on Akt phosphorylation in VSMCs. The reason for this discrepancy is not clear at this time. It has been reported that PI3-kinase regulates the activity of Ras (17, 18, 26), upstream molecules of the MEK/ERK pathway. Our findings suggest that activation of PI3-kinase by 15-d-PGJ2 may activate Ras and lead to activation of the MEK/ERK pathway. PKC is reported to activate the ERK pathway (27). However, pretreatment with PMA for 24 h or with GF109203X did not affect 15-d-PGJ2-induced ERK activation or c-fos mRNA induction (Fig. 6). These results suggest that PKC pathway does not play a dominant role in 15-d-PGJ2-induced ERK activation and c-fos mRNA induction in VSMCs.

PPAR agonists regulate various gene expressions; two different mechanisms have been reported previously (1, 3). First, ligand-activated PPAR nuclear receptor induces gene expression via binding to PPRE in the target gene promoter (1), the so-called classical genomic effect. Second, ligand-activated PPAR nuclear receptor suppresses other gene expression by antagonizing transcriptional factors such as AP-1 and NF-B (3). In the present study, we have demonstrated that PPAR agonists induced c-fos mRNA expression in an ERK-dependent manner. This may be a novel regulatory mechanism of gene expression by PPAR agonists. We also demonstrated that 15-d-PGJ2 enhanced AP-1 binding activity (Fig. 2C). AP-1 regulates a large number of genes associated with developmental, proliferative, and inflammatory processes. It was reported previously that estrogen suppressed gene expression of type I collagen through AP-1 in mesangial cells (28, 29); these authors observed that estrogen stimulated AP-1 activity via ERK activation and that PD98059 inhibited ERK activity as well as suppression of collagen gene expression. It has also been reported that estrogen reduces the synthesis of type I collagen in VSMCs (30). Therefore, PPAR agonists may reduce collagen synthesis through AP-1 activation, which may contribute to the anti-atherogenic effects of PPAR agonists in vascular walls.

Estrogen attenuated superoxide production and phagocytic activity in the MEK/ERK-dependent pathway in microglial cells (31). The exact mechanism by which estrogen-induced ERK activation suppresses microglial cells has not been clearly determined. One possibility is that estrogen may induce the negative feedback mechanism of ERK activation via up-regulation of MAP kinase phosphatase (MKP). It was previously reported that activated ERK induced the expression of MKP-1 (32), which dephosphorylates and inactivates ERK. Therefore, it may be possible that transient MEK/ERK activation by 15-d-PGJ2 or TZDs and subsequent MKP expression may be responsible for the repression of the MEK/ERK signal induced by stimulation with other cytokines or growth factors.

Although 15-d-PGJ2 or TZDs were reported to suppress insulin (26)-, basic fibroblast growth factor (33)-, and angiotensin II (34)-induced proliferation (or DNA synthesis) of VSMCs, the direct effects of these ligands on VSMCs have not been examined. To our surprise, 15-d-PGJ2 and TZDs were weak mitogen for VSMCs. However, it is not clear at this point whether this in vitro finding has a biological significance in vivo, and thus it requires further investigation.

In conclusion, the present study has demonstrated that PPAR agonists rapidly induce ERK activation and c-fos mRNA expression in VSMCs. The rapid kinetics suggest the presence of a nongenomic action of PPAR agonists or a PPAR-independent action of these ligands. The rapid signaling pathway of PPAR agonists may implicate a potential mechanism responsible for the cardiovascular protective effects of PPAR agonists.

	FOOTNOTES

^* This study was supported in part by Grants-in-aid 12877113 and 11770355 for Scientific Research from the Ministry of Education, Science and Culture, Japan.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. Tel.: 81-92-642-5361; Fax: 81-92-642-5374; E-mail: ichiki@cardiol.med.kyushu-u.ac.jp.

Published, JBC Papers in Press, October 30, 2001, DOI 10.1074/jbc.M108722200

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; VSMC, vascular smooth muscle cell; 15-d-PGJ2, 15-deoxy-^12,14-prostaglandin J₂; TZD, thiazolidinedione; Pio, pioglitazone; Tro, troglitazone; ERK, extracellular signal-regulated protein kinase; MAP, mitogen-activated protein; MKP, MAP kinase phosphatase; MEK, MAP kinase/ERK kinase; PMA, phorbol 12-myristate 13-acetate; AP-1, activating protein 1; PKC, protein kinase C.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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