Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin

Abstract

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.

FIGURE 1.

Yap1 expression is increased during VSMC phenotypic switch induced by PDGF-BB and in vessel injury. A, RASMCs grown to 60–70% confluence underwent mitogenic quiescence by serum starvation (DMEM + 0.5% FBS) for 48 h and were subsequently stimulated with 10 ng/ml PDGF-BB for the indicated periods. Time point expression of Yap1 and markers of VSMC proliferation and contractile phenotype were analyzed by quantitative RT-PCR. 18S RNA served as internal control. CCND1, cyclin D1; CCND2, cyclin D2; SMA, SM α-actin; MyoCD, myocardin; SMMHC, smooth muscle myosin heavy chain. n = 3. Data are expressed as mean ± S.D. *, p < 0.05. B, representative Western blot analysis of total protein lysates of cultured RASMCs. RASMCs were treated with 10 ng/ml PDGF-BB for 24 h after serum starvation for 48 h and harvested for Western blot. C, representative Western blot analysis of total protein lysates of carotid arteries. Rat carotid arteries subjected to balloon injury and sham surgery controls were harvested 14 days after surgery and ground for Western blot analysis as indicated in the figure. n = 3.

FIGURE 2.

Knockdown of Yap1 attenuates VSMC proliferation. A, RASMCs infected with lentiviruses encoding Yap1 shRNA (shYap1) or control scrambled shRNA were seeded in 6-well plates, and cell proliferation was measured by counting cell numbers at each time point as described under “Experimental Procedures.” n = 4. Data are expressed as mean ± S.D. (error bars). *, p < 0.05. B, representative Western blot analysis of total protein lysates from RASMCs infected with lentiviruses encoding Yap1 shRNA (shYap1) or control scrambled shRNA actively growing in 10% FBS-containing growth medium using the indicated antibodies. GAPDH served as internal control.

FIGURE 3.

Knockdown of Yap1 enhances contractile phenotype-specific gene expression in VSMCs. A and B, RASMCs infected with shYap1 were cultured in 10% FBS-containing DMEM and harvested for quantitative RT-PCR (A) or Western blotting (B) to detect gene expression as indicated. Cells infected with lentiviral scrambled vector served as a control. C, a luciferase reporter for the SMMHC gene promoter was co-transfected with myocardin (MyoCD) and/or Yap1 expression plasmids in HEK293 cells, and the luciferase activity was measured 48 h after transfection as described under “Experimental Procedures.” Thymidine kinase Renilla served as internal control. The basal activity of pcDNA3.1 empty vector on SMMHC promoter activity was normalized to 1. Data are expressed as mean ± S.D. (error bars). n = 3. *, p < 0.05.

FIGURE 4.

Yap1 binds myocardin. A, HEK 293 cells were transfected with pcDNA-myocardin-FLAG and/or pcDNA4-Yap1-Myc expression plasmids. The specificity of binding between Yap1 and myocardin was confirmed by precipitation with FLAG antibody and further immunoblot with anti-Myc antibody. B, HEK 293 cells were transfected with pcDNA-myocardin-FLAG and SRF while pcDNA4-Yap1-Myc expression plasmid was introduced with increased dosage. Total cell lysates were precipitated with FLAG antibody and further immunoblot with anti-SRF antibodies, FLAG, and Yap1 as indicated. IB, immunoblot.

FIGURE 5.

Yap1 attenuates the SRF-dependent transcriptional activation of CArG box containing contractile VSMC-specific genes. ChIP assay were performed on crossed-linked chromatin from RASMCs with Yap1 knockdown (shYap1) immunoprecipitated with anti-SRF antibody. The precipitated DNA was amplified by real-time PCR with VSMC gene-specific primers spanning the CArG region. Data represent the relative enrichment of IP DNA samples as compared with input DNA and are expressed as mean ± S.D. (error bars). n = 3. *, p < 0.05.

FIGURE 6.

Working hypothesis whereby Yap1 regulates VSMC phenotypic switch via myocardin. Upon stimuli driving VSMC phenotypic switch from contractile to synthetic, like PDGF-BB, Yap1 interferes with myocardin activity, resulting in reduced expression of VSMC contractile genes and, through an as yet undefined mechanism, increased proliferation markers in the VSMCs.