3-Hydroxy-3-methylglutaryl CoA reductase inhibitors up-regulate transforming growth factor-beta signaling in cultured heart cells via inhibition of geranylgeranylation of RhoA GTPase

Abstract

Transforming growth factor-beta (TGFbeta) signaling has been shown to play a role in cardiac development as well as in the pathogenesis of cardiovascular disease. Prior studies have suggested a relationship between cholesterol metabolism and TGFbeta signaling. Here we demonstrate that induction of the cholesterol metabolic pathway by growth of embryonic chicken atrial cells in medium supplemented with lipoprotein-depleted serum coordinately decreased the expression of the TGFbeta type II receptor (TGFbetaRII), TGFbeta(1), and TGFbeta signaling as measured by plasminogen activator inhibitor-1 (PAI-1) promoter activity. Inhibition of the cholesterol metabolic pathway by the hydrophobic 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors, simvastatin and atorvastatin, reversed the effect of lipoprotein-depleted serum and up-regulated TGFbetaRII expression, whereas the hydrophilic HMGCoA reductase inhibitor, pravastatin, had no effect. Simvastatin stimulated the expression of TGFbetaRII, TGFbeta(1), and PAI-1 at the level of transcription. Experiments using specific inhibitors of different branches of the cholesterol metabolic pathway demonstrated that simvastatin exerted its effect on TGFbeta signaling by inhibition of the geranylgeranylation pathway. C3 exotoxin, which specifically inactivates geranylgeranylated Rho GTPases, mimicked the effect of simvastatin on PAI-1 promoter activity. Cotransfection of cells with a PAI-1 promoter-reporter and a dominant-negative RhoA mutant increased PAI-1 promoter activity, whereas cotransfection with a dominant-active RhoA mutant decreased PAI-1 promoter activity. These data support the conclusion that TGFbeta signaling is regulated by RhoA GTPase and demonstrate a relationship between cholesterol metabolism and TGFbeta signaling. Our data suggest that in patients treated with HMGCoA reductase inhibitors, these agents may exert effects independent of cholesterol lowering on TGFbeta signaling in the heart.

Figure 1

Regulation of TGFβRII expression by the cholesterol metabolic pathway. Embryonic chicken atrial cells were cultured in media with either FCS or LPDS and treated with various HMGCoA reductase inhibitors each at 10 μM for 16 h. Lane 1, FCS; lane 2, LPDS; lane 3, LPDS plus pravastatin (Pra); lane 4, LPDS plus atorvastatin (Ato); lane 5, LPDS plus simvastatin (Sim). Thirty micrograms of crude cell extract were subjected to SDS/PAGE followed by immunoblotting with a TGFβRII antibody. Data are typical of four similar experiments.

Figure 2

Coordinate regulation of TGFβRII and TGFβ₁ promoter activity by control of the cholesterol metabolic pathway. Cells were transfected with either pTGFβRII-500/36-Lux (■) or phTGFβ5-Lux (□) plus pCMVβgal. After recovery, cells were incubated for 16 h in media with either FCS, LPDS, or LPDS plus simvastatin (20 μM). Luciferase activity was normalized to β-galactosidase activity. Data are plotted as the mean ± SEM of three independent experiments.

Figure 3

Up-regulation of TGFβRII and TGFβ₁ expression by inhibition of the geranylgeranylation pathway. (A) Embryonic chicken atrial cells were transfected with either pTGFβRII-500/36-Lux or phTGFβ5-Lux plus pCMVβgal. Cells were allowed to recover followed by a 16-h incubation in media supplemented with LPDS and the various inhibitors. Lane 1, control; lane 2, 20 μM simvastatin; lane 3, 50 μM TMD; lane 4, 10 μM FTI-277; lane 5, 10 μM GGTI-298. Luciferase activity was normalized to β-galactosidase activity. Data are plotted as the mean ± SEM of three independent experiments. (B) Cells were grown in media with LPDS and treated with various inhibitors as described above. Thirty micrograms of crude cell extract were analyzed for the expression of TGFβRII protein using a TGFβRII antibody. Data are typical of three similar experiments.

Figure 4

GGPP reverses the effect of simvastatin on TGFβRII expression. (A) Embryonic chicken atrial cells were transfected with pTGFβRII-500/36-Lux plus pCMVβgal. After recovery, cells were incubated for 16 h in media with LPDS plus: lane 1, no additions; lane 2, 10 μM simvastatin; lane 3, 10 μM simvastatin plus 10 μM FPP; lane 4, 10 μM simvastatin plus 10 μM GGPP. Luciferase activity was normalized to β-galactosidase activity. Data are plotted as the mean ±SEM of four independent experiments. (B) Cells were grown in media supplemented with LPDS and treated as described above. Thirty micrograms of crude cell extract were analyzed by immunoblotting for the expression of TGFβRII protein. Data are typical of three similar experiments.

Figure 5

Regulation of TGFβ signaling by control of the cholesterol metabolic pathway. Embryonic chicken atrial cells were transfected with p3TP-Lux plus pCMVβgal. After recovery, cells were incubated for 16 h in media supplemented with either (A) FCS, LPDS, or LPDS plus various concentrations of simvastatin (0–10 μM); (B) LPDS and either: no additions; 10 μM simvastatin; 50 μM TMD; 10 μM FTI-277; 10 μM GGTI-298; 10 μM simvastatin plus 10 μM FPP; 10 μM simvastatin plus 10 μM GGPP. Luciferase activity was normalized to β-galactosidase activity. Values are the mean ± SEM of four independent experiments.

Figure 6

Regulation of TGFβ signaling via the geranylgeranylation pathway. (A) Cells were transfected with p3TP-Lux plus pCMVβgal. After recovery, cells were incubated for 16 h in media with: lane 1, FCS alone; lane 2, FCS plus 10 μM GGPP; lane 3, LPDS alone. Luciferase activity was normalized to β-galactosidase activity. Data are plotted as the mean ± SEM of three independent experiments. (B) Cells were transfected with either pTGFβRII-500/36-Lux or p3TP-Lux plus pCMVβgal. After recovery, cells were cultured for 16 h in media supplemented with LPDS plus: lane 1, control; lane 2, 50 μg BSA/ml; lane 3, 50 μg C3 exotoxin/ml. Luciferase activity was normalized to β-galactosidase activity. Values are the mean ± SEM of three independent experiments.

Figure 7

Regulation of TGFβ signaling by RhoA GTPase. (A) Embryonic chicken atrial cells were cotransfected with p3TP-Lux, pCMVβgal, and either pCDNA3, pRK5 myc-RhoA L63, pCDNA3 myc-RhoA N19, or pEFmyc-C3. After recovery, cells were cultured for 16 h in media with LPDS. Luciferase activity was normalized to β-galactosidase activity. Values are the mean ± SEM of three independent experiments. (B) Thirty micrograms of crude cell extract from cells were analyzed for the expression of RhoA mutants and C3 toxin by immunoblotting using a myc antibody. Data are typical of three similar experiments.