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Am J Physiol Heart Circ Physiol 290: H538-H546, 2006. First published October 14, 2005; doi:10.1152/ajpheart.00240.2005
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17{beta}-Estradiol attenuates PDGF signaling in vascular smooth muscle cells at the postreceptor level

Kai Kappert,1 Evren Caglayan,1 Michael Huntgeburth,1 Anselm T. Bäumer,1 Jan Sparwel,1 Manuela Uebel,1 and Stephan Rosenkranz1,2

1Klinik III für Innere Medizin der Universität zu Köln and 2Center for Molecular Medicine of the University of Cologne (CMMC), Cologne, Germany

Submitted 3 February 2005 ; accepted in final form 13 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17{beta}-estradiol (E2) on {beta}-PDGF receptor ({beta}-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 µM–0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 ± 15.8% and 79.4 ± 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter {beta}-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the {beta}-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-{gamma}, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the {beta}-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.

rac; methoxyestradiol; platelet-derived growth factor; atherosclerosis

THE LATER ONSET and lower incidence of cardiovascular disease in women is thought to result from vasoprotective effects of estrogens in premenopausal women. Consistent with this concept, decreased neointima formation after balloon injury in the presence of estrogens has been demonstrated in vivo (1, 10). Furthermore, in vitro studies have shown direct atheroprotective effects of estrogens at the cellular level (4, 14, 22, 24, 27, 43), which may in part explain their beneficial effects on cardiovascular risk and the reduction of ischemic events such as myocardial infarction, stroke, and peripheral artery disease. Although some protective effects of estrogens such as changes of plasma lipid profiles, inhibition of platelet and monocyte aggregation, decreased extracellular matrix synthesis, suppression of MAPK activation, downregulation of vascular angiotensin receptors, and inhibition of angiotensin II-induced liberation of reactive oxygen species have been described (9, 22, 26, 27, 37), the molecular/cellular mechanisms of estrogen-mediated atheroprotective effects are not completely understood.

Platelet-derived growth factor (PDGF) is a major contributor to atherosclerosis and restenosis (29). PDGF ligand and receptors (PDGFRs) are significantly upregulated in human atherosclerotic plaques (34), and inhibition of PDGFR signaling by specific antibodies or tyrosine kinase inhibitors potently inhibits neointima formation in various models (12, 23). Recent studies demonstrated that of the various PDGF isoforms and receptor subtypes, {beta}-PDGFR-mediated signals are particularly important for vascular remodeling and neointima formation after vascular injury (21, 23).

Activation of the {beta}-PDGFR induces cell cycle progression and migration of vascular smooth muscle cells (VSMCs), which critically contribute to the formation and progression of atherosclerotic plaques. These events are preceded by the ligand-induced tyrosine phosphorylation of the receptor and its association with Src homology region 2-containing signaling enzymes including Src, Src homology region 2-containing phosphatase (SHP)-2, RasGAP, p85, and PLC-{gamma}. These signaling molecules selectively associate with phosphorylated tyrosine residues within the cytoplasmic domain of the receptor, and their activation leads to the induction of highly specific signal relay cascades. Downstream mediators of the {beta}-PDGFR include ERK, Akt, and small G proteins including rho and rac-1, which ultimately mediate PDGF-dependent cellular responses such as cell cycle progression, migration, and survival (31). Because animal models have provided evidence for reduced neointima formation due to 17{beta}-estradiol (E2) treatment (1, 28), we hypothesized that E2 may attenuate PDGFR signaling and PDGF-induced cellular events, like proliferation and chemotaxis, and thereby protect the vessel wall from atherogenesis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. VSMCs were isolated from rat thoracic aorta (male Wistar Kyoto; 6–10 wk old; Charles River Wega, Sulzfeld, Germany) by enzymatic dispersion as previously described (6). Animal studies were approved by the appropriate review committee. Cells were grown in a 5% CO2 atmosphere at 37°C in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1% nonessential amino acids (100x), and 10% FCS. Experiments were performed with cells from passages 5–12.

DNA synthesis. PDGF-dependent cell cycle progression was measured by a 5-bromodeoxyuridine (BrdU)-incorporation assay, as described previously (32). Briefly, cells were synchronized for at least 12 h, and PDGF-BB was added to cells for 24 h at the indicated concentrations in the absence or presence of E2 or 2-methoxyestradiol (2-ME). Preincubation with E2 or 2-ME was carried out for 24 h, and—where indicated—the estrogen receptor (ER) antagonist ICI-182780 (10 µM) was given to cells 30 min before E2. BrdU incorporation was measured according to the manufacturer’s specifications (Roche, Mannheim, Germany), with an incorporation time of 16 h.

Migration. PDGF-dependent chemotaxis was assayed with a 48-well modified Boyden chamber (NeuroProbe, Baltimore, MD) and polyvinylpyrrolidone-free polycarbonate filters (8-µm pores; Poretics, Livermore, CA) as previously described (30). In brief, the lower wells of the chamber were filled with DMEM supplemented with 10 ng/ml PDGF-BB or vehicle in the presence or absence of various concentrations of E2, as indicated. The filters were coated with 50 mg/ml rat type I collagen (Collaborative Biomedical Products, Bedford, MA) and fixed atop the bottom wells. Twenty thousand VSMCs per well were allowed to migrate for 5 h. The cells on the upper surface of the filter were gently removed, and the cells on the lower surface were fixed and stained with Diff-Quick (Baxter Healthcare, Miami, FL). Chemotaxis was quantified by counting the number of cells on the lower surface of the filter in one representative high-power field in each well, using a grid containing 100 nonoverlapping fields. Experiments were carried out with n = 6–8 wells per condition and performed at least twice.

Cytotoxicity. Cytotoxicity was measured by lactate dehydrogenase (LDH) release from cells with the CytoTox-One homogeneous integrity assay (Promega, Mannheim, Germany), as described previously (19). Briefly, cells were synchronized by serum deprivation and then stimulated for the indicated time periods with or without different concentrations of E2. The assay was carried out according to the manufacturer’s instructions. Calculations were performed according to the following definition: % cytotoxicity = 100 x [(experimental – culture medium background)/(maximum LDH release – culture medium background)].

Immunoprecipitation and Western blot analyses. Quiescent VSMCs were left resting or stimulated with 50 ng/ml PDGF-BB for the indicated time intervals in the presence or absence of E2. The {beta}-PDGFR was immunoprecipitated as previously described (5), except that immune complexes were bound to protein A-Sepharose, followed by standard immunoblotting procedures using antisera that recognize phosphotyrosine (PY20/4G10), {beta}-PDGFR (97A), p85, PLC-{gamma}, RasGAP (69.3), or SHP-2. To evaluate the expression and activation of rac-1, a mouse monoclonal IgG-antibody (Upstate, Lake Placid, NY) was used, and expression of ER-{beta} was detected by a rabbit polyclonal antibody (PA1-310, Dianova, Hamburg, Germany).

rac-1 glutathione S-transferase-p21-activated kinase pull-down assay. A glutathione S-transferase (GST)-p21-activated kinase (PAK) CRIB domain (PAK-CD) fusion protein (Rac Activation Assay Kit, Upstate, Lake Placid, NY) containing the rac-1 binding region from human PAK1B was used to determine rac-1 activity, as described previously (36). The assay was carried out according to the manufacturer’s instructions. VSMCs were grown to 70–80% confluence and synchronized by serum deprivation for 24 h, followed by treatment with E2 at the indicated concentrations for additional 24 h before PDGF-BB stimulation. VSMCs were treated with PDGF-BB (50 ng/ml), washed with ice-cold PBS, incubated for 5 min on ice in lysis buffer, and centrifuged (5 min, 21,000 g, 4°C). Aliquots were taken from the supernatant to compare protein amounts. Equal amounts of supernatant protein were incubated with the GST-PAK-CD fusion protein bound to glutathione-coupled Sepharose beads at 4°C for 30 min. The beads and proteins bound to the fusion protein were washed three times in an excess of lysis buffer, eluted in sample buffer, and then analyzed for bound rac-1 by immunoblotting.

Nucleofection. Dominant-negative rac-1 (N17) was expressed with Nucleofector technology. The electroporations were performed with the human aortic smooth muscle cell Nucleofector kit (Amaxa Biosystems, Cologne, Germany), which is effective in rat VSMCs (41), according to the manufacturer’s instructions. In brief, VSMCs were harvested and resuspended in a Nucleofector solution at a concentration of 106 cells/ml. The following constructs were transfected: insertless vector (pcDNA3) as a negative control and pRK5-myc-rac-N17 (dominant-negative rac-1) (22). Five micrograms of plasmid DNA and 100 µl of the cell suspension were placed in a 0.4-cm cuvette and mixed. After electroporation, the samples were transferred into six-well plates and incubated at culture conditions for 48 h, before migration assays were performed. Transfection efficiency for nucleofection in VSMCs is ~75%.

Materials and antibodies. PDGF-BB was purchased from Promo Cell (Heidelberg, Germany). Antiphosphotyrosine antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA; PY20) and Upstate Biotechnology (Lake Placid, NY; 4G10). The antibody against SHP-2 was obtained from Transduction Labs (Lexington, KY), PLC-{gamma} and rac-1-antibodies were from Upstate Biotechnology, and ER-{beta} antibodies were from Dianova. Antibodies against the {beta}-PDGFR (97A), RasGAP (69.3), and the p85 subunit of phosphatidylinositol 3-kinase were a kind gift from Andrius Kazlauskas and Alex Toker (Harvard Medical School, Boston, MA). Clostridium sordellii lethal toxin (LTX) was kindly provided by K. Aktories (University of Freiburg, Freiburg, Germany). E2 [1,3,5(10)-estratriene-3,17{beta}-diol] and 2-ME were purchased from Sigma (Munich, Germany), and ICI-182780 was from Tocris (Bristol, UK).

Statistics. All data are expressed as means ± SE. Statistical analysis was evaluated by nonparametric analysis, and P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
E2 attenuates PDGF-dependent cell cycle progression and chemotaxis in VSMCs. DNA synthesis was measured by BrdU incorporation. Stimulation of VSMCs with PDGF-BB led to a concentration-dependent increase of BrdU uptake to a maximum of 2.4-fold at 20 ng/ml (P < 0.01). Therefore, the subsequent experiments were carried out with 20 ng/ml PDGF-BB. Pretreatment of VSMCs with E2 for 24 h led to a concentration-dependent decrease of PDGF-induced cell cycle progression. A significant inhibition of PDGF-dependent BrdU uptake was observed at concentrations of 0.3 µM and higher (Fig. 1A).



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  		Fig. 1. A: effects of 17{beta}-estradiol (E2) on PDGF-induced DNA synthesis. Cells were synchronized by serum deprivation for at least 12 h, pretreated with various concentrations of E2 for 24 h as indicated, and stimulated with PDGF-BB (20 ng/ml) for 24 h. 5-Bromodeoxyuridine (BrdU) incorporation was measured after 16 h. DNA synthesis rates are expressed as the relative % of BrdU uptake compared with untreated control cells. Shown are means ± SE from at least 3 independent experiments. #P < 0.01 vs. untreated control cells; **P < 0.01 and *P < 0.05 vs. PDGF. B: effects of E2 on PDGF-induced chemotaxis. Synchronized VSMCs were placed into Boyden chemotaxis chambers and allowed to migrate for 5 h. Chemotaxis was induced by adding PDGF-BB (10 ng/ml) with or without E2 in the lower chamber. Each experiment was performed with n = 6–8 wells per condition. Data are expressed as fold increase compared with control cells. Shown are means ± SE from at least 3 independent experiments. #P < 0.01 vs. control cells; **P < 0.01 and *P < 0.05 vs. PDGF.

 
Stimulation of VSMCs with PDGF-BB (10 ng/ml) resulted in a significant induction of cell migration compared with nonstimulated cells (P < 0.01). When PDGF was administered in the presence of E2, PDGF-dependent chemotaxis was inhibited in a concentration-dependent manner (Fig. 1B).

Inhibition of proliferation and chemotaxis is mediated by ER. To examine whether the observed effects of E2 on PDGF-dependent migration and proliferation were mediated via ERs, VSMCs were incubated with E2 in the presence of the dual ER antagonist ICI-182780 (10 µM). Cotreatment with ICI-182780 prevented the inhibitory effects of E2 on PDGF-dependent migration and proliferation, indicating that these effects are mediated via ERs (Fig. 2).



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  		Fig. 2. Dependence of E2 effects on estrogen receptors (ERs). Coincubation of cells with the pure ER antagonist ICI-182780 (ICI; 10 µM) reversed the inhibitory effect of E2 (10 µM) on PDGF-induced cellular responses. A: E2-induced inhibition of PDGF (20 ng/ml)-dependent DNA synthesis determined by BrdU incorporation was reversed by ICI-182780. Data are expressed as relative % of BrdU uptake compared with untreated control cells and represent means ± SE from at least 3 independent experiments. #P < 0.05 vs. control cells; *P < 0.05 vs. PDGF. B: E2-induced inhibition of PDGF (10 ng/ml)-dependent vascular smooth muscle cell (VSMC) migration was reversed by ICI-182780. Data are expressed as relative % of migrated cells compared with PDGF-treated cells and represent means ± SE from at least 2 independent experiments. #P < 0.05 vs. control cells; *P < 0.05 vs. PDGF.

 
Estrogens do not alter PDGFR expression and activation. E2 may attenuate PDGF-induced cellular responses by altering {beta}-PDGFR expression or ligand-induced receptor activation or by affecting signaling pathways at the postreceptor level. {beta}-PDGFR expression was analyzed by Western blotting of total cell lysates prepared from VSMCs that had been incubated with various concentrations of E2 for up to 48 h. No alterations of receptor levels were observed throughout the observation period (Fig. 3, A and B).



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  		Fig. 3. A: expression levels of {beta}-PDGF receptor ({beta}-PDGFR) after indicated times of E2 pretreatment. Quiescent cells were lysed, and total cell lysates were subjected to immunoblotting. RasGAP was used as a lysate control. B: densitometric quantification of receptor expression blots shown in A, C, and E: effects of E2 on {beta}-PDGFR tyrosine phosphorylation and ligand-induced binding of receptor-associated signaling molecules. Quiescent VSMCs were left resting (–) or stimulated with PDGF-BB (50 ng/ml; +) for 5 min in the presence of various concentrations of E2 for 24 h as indicated. {beta}-PDGFR immunoprecipitates were subjected to immunoblotting analysis using antisera recognizing the {beta}-PDGFR, phosphotyrosine (P-Y, C), Src homology region 2-containing phosphatase (SHP-2), RasGAP, p85, and PLC-{gamma} (E). A: similar amounts of receptor were present in the samples: the mature, glycosylated species (top) and the immature form of the receptor (bottom) are shown. C: phosphotyrosine content of the receptor. E: ligand-induced binding of {beta}-PDGFR-associated signaling enzymes after pretreatment with various concentrations of E2 for 24 h. D: densitometric quantification of ligand-induced receptor phosphorylation. F: densitometric quantification of ligand-induced association of signaling molecules bound to the activated {beta}-PDGFR [n = 6 (RasGAP, SHP-2, PLC-{gamma}) or 4 (p85)]. Even though some variations in binding patterns were observed, no significant E2 concentration-dependent alterations were detected. #P < 0.05 vs. PDGF.

 
To further study the effects of E2 on {beta}-PDGFR activation, ligand-induced tyrosine phosphorylation of the {beta}-PDGFR was measured in the presence of various concentrations of E2. To this end, VSMCs were pretreated with E2 for 24 h and subsequently stimulated with PDGF-BB (50 ng/ml). The cells were then lysed, the {beta}-PDGFR was immunoprecipitated, and the immunoprecipitates were subjected to Western blot analysis, using antisera against phosphotyrosine or the {beta}-PDGFR. Treatment of VSMCs with PDGF-BB significantly increased the phosphotyrosine content of the {beta}-PDGFR. As shown in Fig. 3, C and D, E2 did not alter the ligand-induced tyrosine phosphorylation of the {beta}-PDGFR.

In addition to monitoring ligand-induced {beta}-PDGFR auto-/transphosphorylation, we performed coimmunoprecipitations to examine the interaction of the activated {beta}-PDGFR with receptor-associated signaling molecules such as SHP-2, RasGAP, PLC-{gamma}, and p85. Quiescent VSMCs were preincubated with various concentrations of E2 for 24 h, the cells were then stimulated with PDGF-BB, and coimmunoprecipitations were performed against SHP-2, RasGAP, PLC-{gamma}, or p85. These studies revealed that E2 does not affect the association of {beta}-PDGFR-associated signaling molecules with the activated receptor (Fig. 3, E and F).

Estrogens do not alter cell viability and ER-{beta} expression in VSMCs. To rule out that the inhibitory effects of E2 on PDGF-dependent cellular effects in VSMCs were due to cytotoxicity, we performed cell viability assays. A significant decrease of VSMC viability could not be demonstrated in cells treated with E2 up to a concentration of 1 µM and an incubation time of 72 h (Fig. 4A).



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  		Fig. 4. A: effect of E2 on viability of VSMC cultures. Cell viability was analyzed by lactate dehydrogenase release. VSMCs were treated with E2 at the indicated concentrations for 24, 48, and 72 h. Data are expressed as % of viable cells calculated as described in METHODS and represent means ± SE from 1 representative experiment performed in triplicate. Three independent experiments revealed similar results. B: expression levels of ER-{beta} after indicated times of E2 pretreatment. Quiescent cells were lysed, and total cell lysates were subjected to immunoblotting. RasGAP was used as a lysate control.

 
Long-term exposure of VSMCs with E2 might result in altered ER expression. However, significant differences of ER-{beta} were not induced by E2 up to 72 h of incubation (Fig. 4B).

E2 attenuates PDGF-dependent rac-1 activity. Key downstream mediators of the {beta}-PDGFR include ERK, Akt, and rac-1. Immunoblotting of total cell lysates prepared from ±PDGF-stimulated VSMCs with phospho-specific antibodies demonstrated that E2 does not significantly alter PDGF-dependent activation of ERK and Akt (not shown). In light of a recent study demonstrating that estrogen attenuates the angiotensin II-induced liberation of reactive oxygen species by affecting small G proteins (22), we focused on rac-1. To investigate the effects of E2 on PDGF-induced rac-1 activation, rac-1 activity was measured with GST pull-down assays. Stimulation of VSMCs with PDGF-BB (50 ng/ml) led to a transient activation of rac-1, which was maximal at 5 min (Fig. 5A). Therefore, we evaluated whether E2 would alter PDGF-induced rac-1 activation at this time point. rac-1 activity was significantly inhibited by pretreatment with E2 in a concentration-dependent manner (Fig. 5B). To investigate whether E2 regulates rac-1 expression, we analyzed cell lysates from synchronized VSMCs that had been incubated with various concentrations of E2 for up to 72 h. As demonstrated in Fig. 5C, a concentration-dependent decrease of rac-1 expression was observed on E2 stimulation.



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  		Fig. 5. A: PDGF-BB-induced rac-1 activation. Synchronized cells were either left resting or stimulated with PDGF-BB (50 ng/ml) for the indicated time periods, followed by glutathione S-transferase (GST) pull-down assays. B: effects of E2 on PDGF-dependent rac-1 activation. VSMCs were synchronized by serum deprivation, incubated for 24 h with the indicated concentrations of E2, and subsequently stimulated with PDGF-BB (50 ng/ml, 5 min). C: effects of E2 on rac-1 protein expression. Cells were incubated for the indicated time periods with various concentrations of E2, lysed, and subjected to immunoblotting using antibodies against rac-1 and RasGAP (lysate control).

 
Recent studies suggested that E2 metabolites rather than E2 itself are responsible for antiproliferative effects (4, 43). To evaluate whether E2 metabolites are responsible for decreased rac-1 expression and cell cycle progression, we additionally exposed VSMCs to 2-ME. Interestingly, 2-ME induced effects on rac-1 expression and PDGF-dependent cell cycle progression identical to those of E2 (Fig. 6), indicating that the attenuation of rac-1 expression/PDGF signaling by E2 is at least in part mediated by its metabolites.



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  		Fig. 6. A: effects of 2-methoxyestradiol (2-ME) on rac-1 protein expression. Cells were incubated for the indicated times with various concentrations of 2-ME, lysed, and subjected to immunoblotting using antibodies against rac-1 and RasGAP (lysate control). B: effects of 2-ME on PDGF-induced DNA- synthesis. Synchronized cells were pretreated with various concentrations of 2-ME for 24 h as indicated and stimulated with PDGF-BB (20 ng/ml) for 24 h. The assay was performed as described in Fig. 1A. Shown are means ± SE from 4 independent experiments. #P < 0.01 vs. untreated control cells; **P < 0.01 and *P < 0.05 vs. PDGF.

 
rac-1 is involved in PDGF-dependent migration of VSMCs. To further investigate whether abolished rac-1 activation in the presence of E2 is responsible for the inhibition of PDGF-dependent cellular responses by estrogen, we monitored the dependence of PDGF-induced VSMC migration on rac-1 activity. rac-1 was inhibited by cotreatment of VSMCs with C. sordellii LTX, which inhibits rac-1 activity by specific glycosylation (2), or by overexpression of rac-N17. As shown in Fig. 7A, cotreatment of cells with LTX (200 ng/ml) significantly reduced PDGF-induced migration to the same extent as E2 (–62.3 ± 3.1%). Consistently, overexpression of rac-N17 reduced chemotaxis toward a PDGF-BB gradient by 41.5 ± 9.5% compared with empty vector-expressing cells (P < 0.05), whereas basal motility of VSMC was not altered (Fig. 7B). Furthermore, E2 did not further reduce PDGF-dependent migration in rac-N17-overexpressing cells, indicating that E2 exerts inhibitory effects on VSMC migration by altering rac-1 activity. These results indicate that rac-1 is a key element in the regulation of PDGF-BB-induced chemotaxis in VSMCs and that E2 potently inhibits PDGF-dependent migration by reducing rac-1 activity.



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  		Fig. 7. A: effects of Clostridium sordellii lethal toxin (LTX) on PDGF-induced migration. LTX (200 ng/ml) was placed in the lower chamber of a Boyden chemotaxis chamber in addition to PDGF-BB (10 ng/ml), and VSMC migration was measured. Data represent means ± SE from 2 independent experiments with n = 7–8 per condition. *P < 0.05 vs. control; #P < 0.05 vs. PDGF. B: effects of dominant-negative rac (rac-N17) overexpression on PDGF-induced migration. Migration of VSMCs that were transfected with rac-N17 or pcDNA3 vector (empty vector) was induced by PDGF-BB (10 ng/ml) alone or in the presence of E2 (1 µM). *P < 0.05 vs. control cells; #P < 0.05 vs. migration of control cells toward 10 ng/ml PDGF-BB alone.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that E2 significantly abolishes PDGF-induced cellular responses in VSMCs, particularly cell cycle progression and migration, without affecting cell viability. Experiments with the specific ER antagonist ICI-182780 revealed that these effects are mediated via ERs. The inhibitory effects of E2 on PDGF signaling were due to neither impaired {beta}-PDGFR expression/activation nor altered recruitment of intracellular signaling molecules to the activated receptor. Instead, we established that the inhibitory effect of E2 occurs at the postreceptor level, as E2 significantly impaired the PDGF-induced activation and expression of rac-1 GTPase. Similar effects were observed with the E2 metabolite 2-ME. Finally, our findings provide evidence that rac-1 activity is required for efficient PDGF-dependent chemotaxis, indicating that E2 abrogates PDGF-induced cellular responses via inhibition of rac-1.

The crucial role of VSMC migration and proliferation within the vascular wall for the formation and subsequent progression of atherosclerotic lesions is well established (33). PDGF was shown to be a major contributor to these processes, and both PDGF ligand and PDGFR are significantly upregulated in atherosclerotic plaques (29). Moreover, inhibition of PDGF signaling potently prevented atherogenesis in various models (21, 23). Here, we demonstrate a significant inhibition of PDGF-induced atherogenic responses in VSMCs by treatment with E2, as shown in chemotaxis and DNA synthesis assays. Consequently, we explored the direct influence of E2 on {beta}-PDGFR expression, ligand-induced tyrosine phosphorylation, and recruitment of receptor-associated signaling enzymes. However, these events were not influenced by E2. Therefore, we next focused on signaling events downstream from the receptor. Previous studies have addressed the involvement of MAPK (p38 and ERK) activation in VSMC chemotaxis (11, 13). Both p38 and p42/44 are activated by PDGFRs and in response to vascular injury (30, 31). Consistent with our findings, Dubey and coworkers (9, 10) previously demonstrated an E2-induced reduction of VSMC migration toward PDGF, which was also reversed in the presence of an ER antagonist. However, less attention has been paid to the precise underlying mechanisms of estrogen-induced PDGF antagonizing effects in VSMCs. In our study, E2 did not significantly affect the PDGF-dependent activation of signaling molecules such as Akt and ERK1/2 (not shown). However, we observed a robust reduction of PDGF-induced rac-1 activation by E2, which time- and concentration-dependently correlated with E2-mediated inhibition of PDGF-dependent cellular responses.

rac-1 (21 kDa) is a member of the Rho family of small GTPases. Rho proteins are important regulators of the actin cytoskeleton, serve as transducers between mechanical forces, cell morphology, and gene regulation, and thus regulate cellular motility. rac-1 is responsible for controlling membrane ruffling and the formation of lamellipodia (42). In its active GTP-bound state it plays a major role in the regulation of cell shape, adhesion, motility, and release of reactive oxygen species. Besides growth factors (35), mechanical forces like shear stress have been shown to regulate rac-1 activity (42). Even though rac-1 was identified as a downstream mediator of PDGF-induced chemotaxis in fibroblasts (2, 8), the dependence of PDGF-mediated cellular functions on rac-1 signaling remains largely elusive in VSMCs. Here we show that PDGF-BB leads to a transient conversion of rac-1 to its active form and that rac-1 activity is crucial for PDGF-dependent migration of VSMCs. In the presence of E2, both expression and PDGF-dependent rac-1 activation as well as migration of VSMCs were dramatically reduced. These findings are in accordance with a study by Ryu and coworkers (35) demonstrating that the prevention of rac-1 activation by sphingosine-1-phosphate also resulted in a significant negative regulation of cell migration. The requirement of rac-1 for efficient VSMC motility in our study was demonstrated by the finding that the rac inhibitor LTX significantly inhibited PDGF-directed migration. These data were further confirmed by the fact that overexpression of dominant-negative rac-N17 in VSMCs resulted in a general reduction of PDGF-directed chemotaxis. Importantly, E2 did not lead to a further reduction of PDGF-induced migration in rac-N17-overexpressing cells, whereas it did so in empty vector-expressing cells. These data substantiate the hypothesis that E2 exerts inhibitory properties on PDGF signaling by altering rac-1 activity. Our finding that E2 negatively regulated rac-1 activity in VSMCs is consistent with a recent study demonstrating that estrogen significantly attenuates angiotensin II-induced rac-1 activation and angiotensin II-dependent liberation of reactive oxygen species (22).

The reduction, but not complete inhibition, of PDGF-induced migratory responses by E2 or rac-N17 overexpression is in accordance with previous findings showing that PDGF-directed migration is not entirely abolished by rac-1 inhibition (35). However, complete reversion of PDGF-BB-directed migration in rac-N17-transfected fibroblasts was observed in one study (3). These divergent findings may be explained by cell type-specific effects as well as transfection levels. Considering the fact that PDGF-dependent proliferation and migration are mediated by multiple, partly redundant signaling pathways (30, 40), it is not surprising that inhibition of rac-1 by E2 did not lead to a complete abolishment of migration, particularly because Akt and ERK phosphorylation were not affected.

Our observation that E2 inhibits PDGF-induced cellular responses, which are known to critically contribute to atherogenesis, is in accordance with epidemiologic studies providing evidence that estradiol and hormone replacement therapy (HRT) might protect from atherosclerosis (18), a process involving growth of VSMCs in the vessel wall. Consistent with this, experimental data suggest that estrogens attenuate injury-induced atherogenesis (10, 28). However, in secondary intervention trials no beneficial effects of HRT on the incidence of cardiovascular events were observed (7, 16). The concept of primary prevention is not yet thoroughly tested. Regarding the lack of a cardiovascular benefit in women when HRT is initiated at a time when cardiovascular disease is already present, one current theory emphasizes the concept of beginning HRT before postmenopause and cardiovascular disease have occurred (15, 25). In addition, it has been proposed that differential metabolism of estradiol in VSMCs in women receiving HRT due to genetic differences regarding the major metabolic conversion enzymes, cytochrome P-450 (CYP450) and catechol-O-methyl transferase (COMT), may be responsible for discrepancies in clinical trials (4). Differences of estrogen metabolism in vivo involving CYP450 and COMT are likely to explain the fact that cardioprotective effects of HRT occur only in a subset of postmenopausal women (38, 39). This is supported by the finding that methoxyestradiols such as 2-ME at least in part mediate the cellular effects of E2 (Fig. 6; Refs. 4, 43).On the cellular/molecular level, our findings clearly indicate that E2-metabolites exert vasoprotective effects in vitro.

Several studies have explored the involvement of ERs in estrogen-mediated effects in vascular cells (11, 17, 20, 43). Estrogen binds to two distinct ERs, namely, ER-{alpha} and ER-{beta}, which are expressed in all vascular cell types (24). Nonetheless, before pure ER antagonists without agonistic activity became available, it was difficult to reliably judge the involvement of ER in antiatherogenic properties of estrogens in VSMCs and to distinguish between genomic (ER dependent) and nongenomic (ER independent) pathways. Although the present dogma is that estrogen effects are mediated via ERs, current experimental data also suggest alternative mechanisms (4, 43). We aimed to explore the involvement of ERs in E2-mediated effects on PDGF-signaling. In our experiments the ER antagonist ICI-182780 was capable of reversing E2-induced inhibition of PDGF-induced migration and DNA synthesis, suggesting that these effects are mediated via ERs. This is in accordance with other studies demonstrating the potency of this inhibitor to attenuate E2-induced cellular responses (4, 11, 22). In these studies, ICI-182780 was used in a concentration range between 1 and 100 µM, similar to our study (10 µM). Because the molecular structure of ICI-182780 resembles estradiol, it most likely exerts—in addition to its ER-blocking effect—a competitive antagonism concerning the metabolism of E2 via CYP450 to hydroxyestradiol and subsequently further to methoxyestradiol via COMT (4). This may be of importance, because our experimental data are in concert with other investigators (4, 43) and support the hypothesis that estradiol metabolites (methoxyestradiols and hydroxyestradiols) are the key molecules in VSMC growth inhibition, rather than estradiol itself (4). This idea was further supported by recent findings in COMT-deficient cells, which lack methylation capability and therefore fail to metabolize estradiol to its potent metabolite methoxyestradiol. In these cells, estradiol did not inhibit DNA synthesis and proliferation (43). In this context, we demonstrate that 2-ME was equally potent in reducing rac-1 expression and inhibitory PDGF-dependent proliferation as E2, which substantiates the concept of estrogen metabolites playing a significant role in VSMC signaling. However, even though the pharmacological blocking of ER reversed the E2-induced effects in our study, we currently cannot rule out that the inhibition of PDGF-induced rac-1 activation in VSMCs by E2 might also involve non-ER pathways.

In summary, our findings provide evidence that estrogens inhibit PDGF-induced cellular responses in VSMCs by affecting PDGF signaling at the postreceptor level, namely via inhibition of rac-1. Because PDGF-dependent cellular events are known to contribute critically to the atherogenic process, our novel findings offer a molecular explanation for the vasoprotective effects of estrogens.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Köln Fortune Program, Faculty of Medicine, University of Cologne (84/2002 to K. Kappert) and by the Deutsche Forschungsgemeinschaft (Ro 1306/2–1 to S. Rosenkranz).


   ACKNOWLEDGMENTS
 
The technical assistance of Tanja Kübler and Sandra Hübsch and the critical input of Christian Grohé (University of Bonn) are greatly appreciated. The antibodies against the {beta}-PDGFR (97A), RasGAP (69.3), and p85 were kindly provided by Andrius Kazlauskas and Alex Toker (Harvard Medical School, Boston, MA) and C. sordellii LTX by K. Aktories (Freiburg, Germany).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Rosenkranz, Klinik III für Innere Medizin der Universität zu Köln, Kerpener Str. 62, 50924 Köln, Germany (e-mail: stephan.rosenkranz{at}uk-koeln.de)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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