FIGURE 3.

NFATc3 transcriptionally targets myocardin. A and B, the constitutively active form of NFATc3 (ΔNFATc3) stimulates myocardin expression at protein and mRNA levels. Cardiomyocytes were infected with adenoviral β-gal orΔNFATc3 at a moi of 50. The cells were harvested at the indicated time for the analysis of myocardin protein levels by immunoblot (A) or myocardin mRNA levels by real time PCR (B). *, p < 0.05 versus control. C, myocardin promoter contains a potential NFATc3-binding site. The NFATc3 site is shown between −1734 and −1717 bp. The promoter of myocardin was synthesized and linked to luciferase (Luc) reporter gene. The mutations were introduced to the binding site (BS). D, chromatin immunoprecipitation (ChIP) analysis of in vivo NFATc3 binding to the promoter. Chromatin immunoprecipitation assay was performed using cardiomyocytes treated with or without 10 μm Iso and 1 μm Aldo. The anti-myocardin antibody was used as a negative control. E, NFATc3 activates myocardin promoter activity. Cardiomyocytes were infected with adenoviruses harboring β-gal or ΔNFATc3. 24 h after infection cells were transfected with the constructs of the empty vector (pGL-4.17), the wild type promoter (wt) or the promoter with mutations in the binding site (mBS), respectively. Firefly luciferase activities were normalized to Renilla luciferase activities. F and G, knockdown of endogenous NFATc3 inhibits the elevation of myocardin promoter activity induced by Iso or Aldo. Cardiomyocytes were infected with adenovirus harboring NFATc3 RNAi or its scramble form (NFATc3-S-RNAi) at a moi of 100. 24 h after infection cells were transfected with the construct of wild type promoter. Cardiomyocytes were treated with 10 μm Iso (F) or 1 μm Aldo (G). Firefly luciferase activities were normalized to Renilla luciferase activities. The data are expressed as the means ± S.E. of three independent experiments.