A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression

Abstract

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.

FIGURE 1.

Depolarization-induced expression of MEF2-target genes in VSMCs. A, primary VSMCs were depolarized with 60 mm KCl, following pretreatment with 1 μm of nifedipine (Nif), as indicated. Myocardin expression was evaluated by qPCR, corrected for GAPDH using the ΔΔCT method. B, A10 cells were transfected with a myocardin-enhancer reporter gene (MyE-luc) or with a reporter gene with the MEF2 cis element mutated (MyE-mut). Following recovery, cell were depolarized with 60 mm KCl and subjected to luciferase assay. C, quiescent A10 cells were treated with 60 mm KCl following a 15 min treatment of 5 μm Nifedipine (L-type calcium channel blocker). Immunoblotting was performed on protein extracts using c-Jun, c-Fos, and actin antibodies, and RT-PCR was performed on total RNA for c-Jun and SM22 and GAPDH. D, A10 cells were transfected with the c-jun promoter (c-Jun-luc) or with a reporter gene with the MEF2 cis element mutated (c-Jun mut). Following recovery, cells were depolarized with 60 mm KCl and subjected to luciferase assay. E, A10 cells were transfected with the myocardin enhancer and smooth muscle α-actin promoter, as indicated. Cells were treated overnight with endothelin-1 (10 nm), and extracts were subjected to luciferase assay. F, A10 cells were transfected with MEF2C (as indicated). Myocardin expression was evaluated by qPCR and corrected for GAPDH using the ΔΔCT method. Error bars indicate S.E.

FIGURE 2.

Distinct calcium-mediated signaling pathways regulate myocardin and c-Jun expression in VSMCs. A, COS7 cells were transfected with Myocardin-856 and subjected to immunoblotting with myocardin antibody (SC-33766, Santa Cruz Biotechnology). B, A10 cells were treated with 60 mm KCl for 2 h following 15 min pretreatment with 5 μm KN-62 (CaM kinase inhibitor) or DMSO as a vehicle control. Protein extracts were immunoblotted with c-Jun, myocardin, and actin antibodies. C, A10 cells were transfected with a c-jun reporter-gene (c-Jun-luc), MEF2A, HDAC4, and activated CaMKI, CaMKII or CaMKIV, as indicated. D, A10 cells were pretreated with either Y27632 (Y27, 5 μm) or SB203580 (SB, 5 μm), or DMSO as a vehicle control for 15 min, then depolarized for two hours. Extracts were subjected to immunoblotting as indicated. E, A10 cells were pretreated with nifedipine, depolarized, and subjected to immunoblotting. F, model of the distinct signaling pathways that regulate MEF2-dependent myocardin and c-jun expression in VSMCs. G, A10 cells were transfected with MyE or the enhancer with the MEF2 site mutated (MyE mut) along with a MEF2C, and/or an active RhoA (RhoA L63) expression vectors. Extracts were subjected to luciferase assays. H, A10 cells were transfected as described in G, treated with Y27632 (Y27, 5 μm), and harvested for luciferase assay. I, cells were transfected with a plasmid encoding a short-hairpin RNA targeting MEF2C (shMEF2C), and expression vectors for human MEF2C or myocardin, as indicated. Cultures were enriched for expression of the shRNA by puromycin selection, and extracts were subjected to immunoblotting. Error bars indicate S.E.

FIGURE 3.

Myocardin expression is opposed by PP1α. A, A10 cells were transfected with HA-PP1α (PP1) and activated myc-RhoA (Myc-RhoA L63) using Lipofectamine reagent and puromycin-selected overnight. Myocardin expression was evaluated by qPCR and corrected for GAPDH using the ΔΔCT method. B, A10 cells were transfected as described in A. Protein extracts were subjected to immunoblotting as indicated. C, A10 cells were treated with Calyculin A (0.5 ng/ml) or DMSO as a vehicle control for 2 h. Protein extracts were immunoblotted as indicated. D, A10 cells were transfected with HA-PP1α and MEF2C as described in A. Protein extracts were subjected to immunoblotting as indicated. E, COS7 cells were transfected with the myocardin enhancer, MEF2C, dHand, and/or PP1α (PP1) as indicated. Luciferase assays were performed on the cells extracts. F and G, A10 cells were transfected with the SM22 promoter, myocardin, an activated type I TGF-β receptor (TBRI), Smad3, or PP1α, as indicated. Extracts were harvested for luciferase. H, A10s were treated with increasing amounts of calyculin A (Cal A; 0.25 ng/ml, 0.5 ng/ml, 1 ng/ml, 2 ng/ml), and I, A10 cells were treated with 0.5 ng/ml of calyculin A and 5 μm of SP600125 for 2 h and harvested for immunoblotting. Error bars indicate S.E.

FIGURE 4.

PP1α-induced repression of myocardin is attenuated by CPI-17. A, COS7 cells were transfected with the myocardin enhancer (MyE), MEF2C, PP1α (PP1), activated RhoA (RhoA L63), or activated MKK6 and p38 (MKK6EE/p38), as indicated. Extracts were subjected to lucifierase assay. B, cells were transfected with the myocardin enhancer, and MEF2C, PP1, or CPI-17 as indicated, followed by luciferase assay. C, COS7 cells were transfected with HA-CPI-17 and HA-PP1α. Protein extracts were immunoprecipitated with PP1α antibody and immunoblotted, as indicated. D, COS7 cells were transfected with MEF2C, HA-PP1, or HA-CPI-17 as indicated. Extracts were immunoprecipitated with MEF2C antibody and immunoblotted for antibodies to HA. Protein extracts were immunoblotted, as indicated, to demonstrate equal loading and transfection efficiency. E, primary VSMCs were fixed, permeabilized, and subjected to immunofluorescence for CPI-17, smooth muscle actin (SMA), and the DAPI nuclear stain. Cells were visualized by standard fluorescence techniques. Relative fluorescence of a representative cell was graphed with ImageJ. F, A10 cells were transfected with HA-CPI-17 and subjected to nuclear/cytosolic fractionation. Lysates were immunoblotted as indicated. Error bars indicate S.E.

FIGURE 5.

Phosphorylation of CPI-17 at threonine 38 regulates MEF2-dependent VSMC differentiation. A, A10 cells were transfected with activated RhoA, ROCKII, or PKN using Lipofectamine reagent and puromycin-selected overnight. Protein extracts were subjected to immunoblotting as indicated. B, COS7 cells were transfected, as described in 4D) with the addition of Thr-38 mutants of CPI-17. Protein extracts were immunoprecipitated and immunoblotted as previously described. C, A10 cells were transfected as described in A with a plasmid encoding a short hairpin RNA targeting CPI-17 (shCPI-17) and an expression vector for active RhoA. Following puromycin selection, myocardin expression was evaluated by qPCR, corrected for GAPDH using the ΔΔCT method. D, A10 were transfected as described in C. Following puromycin selection, extracts were subjected to immunoblotting. E, rat primary VSMCs were transfected by Lipofectamine reagent with the myocardin reporter gene (MyE-luc), increasing amounts of shCPI-17, and an expression plasmid for human CPI-17. Extracts were subjected to luciferase assay. F, 10T1/2 mouse embryonic fibroblast cells were transfected with MEF2C or HA-CPI-17 as indicate. Cells were placed in low serum conditions (5% horse serum) for 96 h and subjected to immunoblotting as indicated. Error bars indicate S.E.

FIGURE 6.

Model of calcium-mediated induction of myocardin expression in VSMCs. This is based on the work presented in this report and previously published observations. MEF2-dependent myocardin expression is regulated by p38 MAPK and RhoA-induced derepression of PP1α by CPI-17. Myocardin activates SRF-dependent VSMC gene expression directly and by dimerizing with myocardin-related transcription factors (MRTFs) that translocate to the nucleus when G- actin polymerizes to form F-actin.