Rho and PKN regulate ANF gene expression in cardiac myocytes via the SRE

Stimulation of the α₁-adrenergic receptor in neonatal rat ventricular myocytes elicits a hypertrophic response which includes increased expression of the atrial natriuretic factor (ANF) gene. We have previously shown that this response is mediated by the small GTPase Rho. Rho has been shown to regulate transcriptional activation of the c-fos serum response element (SRE) by a ternary complex factor (TCF)-independent mechanism. The ANF promoter contains several SREs which lack the capacity to bind TCF. We tested the hypothesis that Rho regulates ANF gene expression through these SREs. Using truncated and mutant ANF-luciferase reporter gene constructs, we demonstrate that a dominant negative Rho inhibits expression of all constructs containing an intact SRE. Protein kinase N (PKN) is a putative downstream effector of Rho. A constitutively activated mutant of PKN (PKN*), which lacks its regulatory (Rho-binding) domain, markedly stimulates reporter gene expression regulated by the ANF promoter or the ANF SRE sequences in cardiomyocytes. Mutation of the SRE attenuates this stimulatory response. Since the catalytic domain of PKN is highly homologous to that of protein kinase C (PKC), we compared their effects. In preliminary studies, PKN* activated the ANF SRE better than it did the c-fos SRE, whereas an activated PKCε showed the opposite preference. Thus PKN maybe the effector of SRF mediated, Rho dependent transcriptional activation of the ANF gene.