Effect of cyclosporin A treatment on the in vivo regulation of type I MHC gene expression.

Abstract

Rat soleus muscle consists predominantly of slow type I fibers. We have shown previously through deletion analysis that the highest level of reporter activity that we measure when injecting type I myosin heavy chain (MHC) promoter (MHC(1))-linked luciferase plasmid into soleus muscles depends on the presence of a 550-bp upstream enhancer (3,450-2,900) region of the promoter. Because the calcineurin-nuclear factor of activated T cells (NFAT) pathway has been implicated in the regulation of the slow muscle gene program, particularly the MHC(1) isoform, and the MHC(1) promoter contains several putative NFAT sites, we examined via deletion and mutation analyses whether this pathway is involved in the regulation of promoter activity in soleus. Nine days of treatment with the calcineurin inhibitor cyclosporin A (CsA) caused a significant decrease in activity of the -3,500- and -3,450-bp promoters compared with vehicle-treated rats. Truncation of the promoter to -2,900 bp or smaller reduced the activity and also eliminated the CsA responsiveness, thus implying that the enhancer region is required for CsA responsiveness. Surprisingly, mutating the two NFAT elements within the enhancer region had no obvious effect on promoter activity. CsA treatment resulted in an increase in the mRNA levels of fast-type IIa and IIx MHC isoforms, but RT-PCR analysis of MHC(1) pre-mRNA and mature mRNA expression in soleus muscles revealed no differences between vehicle- and CsA-treated rats. Although CsA affects the activity of the MHC(1) promoter, it appears that its effect is not through direct binding of NFAT to sites on the promoter.