Simvastatin and ROCK Inhibitor Y-27632 Inhibit Myofibroblast Differentiation of Graves' Ophthalmopathy-Derived Orbital Fibroblasts via RhoA-Mediated ERK and p38 Signaling Pathways

Abstract

Transforming growth factor-β (TGF-β)-induced differentiation of orbital fibroblasts into myofibroblasts is an important pathogenesis of Graves' ophthalmopathy (GO) and leads to orbital tissue fibrosis. In the present study, we explored the antifibrotic effects of simvastatin and ROCK inhibitor Y-27632 in primary cultured GO orbital fibroblasts and tried to explain the molecular mechanisms behind these effects. Both simvastatin and Y-27632 inhibited TGF-β-induced α-smooth muscle actin (α-SMA) expression, which serves as a marker of fibrosis. The inhibitory effect of simvastatin on TGF-β-induced RhoA, ROCK1, and α-SMA expression could be reversed by geranylgeranyl pyrophosphate, an intermediate in the biosynthesis of cholesterol. This suggested that the mechanism of simvastatin-mediated antifibrotic effects may involve RhoA/ROCK signaling. Furthermore, simvastatin and Y-27632 suppressed TGF-β-induced phosphorylation of ERK and p38. The TGF-β-mediated α-SMA expression was suppressed by pharmacological inhibitors of p38 and ERK. These results suggested that simvastatin inhibits TGF-β-induced myofibroblast differentiation via suppression of the RhoA/ROCK/ERK and p38 MAPK signaling pathways. Thus, our study provides evidence that simvastatin and ROCK inhibitors may be potential therapeutic drugs for the prevention and treatment of orbital fibrosis in GO.

Keywords: ERK; Graves’ ophthalmopathy; Ras homolog family member A (RhoA); Rho‑associated protein kinase (ROCK); Y-27632; myofibroblast; p38; simvastatin.

Figure 1

Immunocytochemical characterization of transforming growth factor-β (TGF-β)-induced α-smooth muscle actin (α-SMA) expression in Graves’ ophthalmopathy (GO) orbital fibroblasts. Primary cultured GO orbital fibroblasts were stimulated with 3 ng/mL TGF-β1 for 48 h with or without a 1-h pretreatment with different concentrations of simvastatin (1, 5, 10 μM) (A) or ROCK inhibitor Y-27632 (1, 10, 30 μM) (B). α-SMA expression was detected using immunofluorescence staining (green). The bar charts show mean data of relative fluorescence intensity of α-SMA presented as the fold of control. *p < 0.05, **p < 0.01 compared to cells treated with TGF-β1 alone. S = simvastatin; Y = Y-27632.

Figure 2

Effect of simvastatin and ROCK inhibitor Y-27632 on transforming growth factor-β (TGF-β)-induced α-smooth muscle actin (α-SMA) messenger RNA (mRNA) and protein expression in Graves’ ophthalmopathy (GO) orbital fibroblasts. Primary cultured GO orbital fibroblasts were stimulated with TGF-β1 (3 ng/mL) for 48 h with or without a 1-h pretreatment with different concentrations of simvastatin (1, 5, 10 μM) (A, C) or ROCK inhibitor Y-27632 (1, 10, 30 μM) (B, D). The α-SMA mRNA expression was examined using real-time PCR (A, B). The α-SMA protein production was determined using western blot analysis (C, D). Data are presented as mean ± SD from at least three independent experiments. *p < 0.05.

Figure 3

Effects of hydroxymethylglutaryl-coenzyme A (HMG-CoA) downstream intermediates on simvastatin-mediated inhibition of transforming growth factor-β (TGF-β)-induced RhoA, Rho-associated protein kinase 1 (ROCK1), and α-smooth muscle actin (α-SMA) expression. (A) Graves’ ophthalmopathy (GO) orbital fibroblasts were stimulated with TGF-β1 (3 ng/mL) for 48 h with or without a 1-h pretreatment with simvastatin (10 μM) and addition of geranylgeranyl pyrophosphate (GGPP) (10 μM), farnesyl pyrophosphate (FPP) (10 μM), or mevalonate (200 μM). The protein levels of RhoA, ROCK1, and α-SMA were determined using western blot analysis. (B–D) The densities of RhoA (B), ROCK1 (C), and α-SMA (D) protein bands were quantified and normalized to GAPDH. (E) GO orbital fibroblasts were stimulated with TGF-β1 (3 ng/mL) for 48 h with or without a 1-h pretreatment with geranylgeranyl transferase inhibitor (GGTI-298) (10 μM) or farnesyl transferase inhibitor (FTI-227) (10 μM). The α-SMA protein production was determined using western blot analysis. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05.

Figure 4

Effects of simvastatin and ROCK inhibitor Y-27632 on transforming growth factor-β (TGF-β)-induced signaling. (A) Primary cultured Graves’ ophthalmopathy (GO) orbital fibroblasts were stimulated with TGF-β1 (3 ng/mL) for 48 h with or without a 1-h pretreatment with simvastatin (10 μM) or ROCK inhibitor Y-27632 (30 μM). The expression and phosphorylation levels of Smad2/3, ERK1/2, p38, and JNK were determined using western blot analysis. (B, C) The densities of ERK1/2 (B) and p38 (C) protein bands were quantified and normalized to β-actin. (D) GO orbital fibroblasts were stimulated with TGF-β1 (3 ng/mL) for 30, 60, 120 min with or without a 1-h pretreatment with simvastatin (10 μM) or ROCK inhibitor Y-27632 (30 μM). The expression and phosphorylation levels of Smad2/3, ERK1/2, and p38 were determined using western blot analysis. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05.

Figure 5

Effects of ERK and p38 inhibitors on TGF-β-induced α-SMA expression in Graves’s ophthalmopathy (GO) orbital fibroblasts. Primary cultured GO orbital fibroblasts were stimulated with transforming growth factor-β (TGF-β1) (3 ng/mL) for 48 h with or without a 1-h pretreatment with 10 μM of PD98059 (ERK inhibitor) (A, C) and 10 μM of SB203580 (p38 inhibitor) (B, D). The protein levels of α-SMA were determined using western blot analysis. The densities of α-SMA protein bands were quantified and normalized to β-actin. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05.

Figure 6

Schematic diagram of the possible signaling mechanisms underlying simvastatin- and Y-27632-mediated inhibition of transforming growth factor-β (TGF-β1)-induced orbital tissue fibrosis in Graves’ ophthalmopathy.