Resveratrol induces p53 and suppresses myocardin-mediated vascular smooth muscle cell differentiation

Abstract

Resveratrol (RSVL), a polyphenolic antioxidant present in red wine, has been shown to provide cardiovascular protection by improving endothelial function and reducing myocardial ischemia. However, little is known about how RSVL affects vascular smooth muscle cells (VSMCs) differentiation. RSVL blocks VSMC proliferation in vitro and neointimal formation following artery injury in vivo. Thus, one might expect that RSVL will promote VSMC differentiation. Unexpectedly, our results in this study show that RSVL induces VSMCs phenotypic modulation; this is characterized by suppressed transcription of SMC-specific marker genes Tagln, Acta2, Myh11, and Smtn in a dose-dependent and time-dependent manner in cultured VSMCs. Consistent with previous studies, RSVL induces the nuclear translocation of p53 and the expression of p53-responsive genes such as Cdkn1a, Gadd45a, Gadd45, and Fas. In an effort to identify the molecular mechanisms whereby RSVL represses VSMC differentiation, we found that RSVL inhibits the transcription of Myocardin (myocd) and Srf, the key VSMC transcriptional regulators. However, knockingdown and overexpressing p53 did not affect RSVL-induced VSMCs phenotypic modulation: this suggests that RSVL may induce VSMC dedifferentiation via p53-independent mechanisms. This study provides the first evidence showing that RSVL induces VSMC dedifferentiation by regulating Myocardin and SRF-mediated VSMC gene transcription.

Fig. 1

RSVL suppresses the transcription and promoter activities of VSMC marker genes in cultured VSMCs (PAC1). (A) Effects of RSVL on VSMCs morphology. (B and C) RSVL significantly downregulated mRNA levels of SMC marker genes in dose- and time-dependent manners by q-RT-PCR assay. (D) RSVL (at 50 µM for 24 hrs) markedly inhibited promoter activities of CArG box-containing promoter activities. * p<0.05 and ** p<0.01 vs vehicle; ^## p<0.01 vs Control.

Fig. 2

RSVL activates p53 signaling in VSMCs. (A) Western blot showed the increased p53 protein expression and nucleus translocation induced by RSVL at 50 µM (left panel). NE and WC indicate nuclear extract and whole cell lysate, respectively. Quantification of p53 protein levels in WC is shown in the right panel. ** p<0.01 vs Vehicle. (B) PAC1 cells were treated with 50 µM RSVL for 24 hours. RSVL did not affect Tp53 mRNA expression but markedly enhanced mRNA expression of p53-responsive genes including Cdkn1a, Fas, Gadd45a and Gadd45b by qRT-PCR. * p<0.05 and ** p<0.01 vs U6.

Fig. 3

RSVL suppresses the transcription of SMC master regulators myocardin/SRF. (A and B) mRNA levels of Myocd and Srf in response to RSVL in dose- and time-dependent manners were measured by the qRT-PCR assay. ** p<0.01 vs Control; ^## p<0.01 vs Vehicle. (C) Effect of actinomycin D (ActD) on RSVL-induced Myocd and Srf mRNA downregulation was determined by the qRT-PCR assay. The mRNA expression levels in VSMCs before adding ActD were set as 100 %. n.s: not significant. (D) The effects of RSVL dosages on Myocd enhancer (MyoE8) driven promoter activities were measured by the luciderase assays at 24 hours in both 10T1/2 cells and PAC1 cells. * p<0.05 and ** p<0.01 vs Vehicle.

Fig. 4

RSVL induces VSMCs phenotypic modulation through p53-independent mechanisms in PAC1 cells. (A) the effect of p53 silencing on nuclear p53 protein level in the presence or absence of RSVL (50 µM for 24 hours) by Western blot (left panel) with the quantification of nuclear p53 protein level change after being normalized by the internal control β-actin (right panel). (B) The effect of knockingdown p53 by p53 siRNA on RSVL-induced VSMC dedifferentiation. (C) The expression level of the nuclear p53 protein level after pCEP4-p53 transfection was measured by Western blot (left panel) with the quantification of the nuclear p53 protein level (right panel). (D) The effect of pCEP4-p53 transfection on Myocd enhancer driven promoter activities in the presence and absence of RSVL. V and R indicate the vehicle and resveratrol respectively. Mock and p53 refer to pCEP4 and pCEP4-p53 plasmids respectively.