Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4

Chi-Chuan Tseng,Ying Zeng,Shunhui Zhuang,Jutta Gloddek,Renate B. Pilz,Gerry R. Boss

doi:10.1074/jbc.m602099200

Chi-Chuan Tseng, Ying Zeng + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m602099200

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Abstract

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

Highlights

Defects, and a decreased life span [1, 3,4,5]
Similar results were obtained in bovine aortic smooth muscle cells (BASMCs), and an even greater relative increase in PKG I expression was observed in REF52 cells
We previously showed that all three RhoA effectors can trans-activate an serum response factor (SRF)-dependent promoter, with PKN and PRK2 being most active in vascular smooth muscle cells (VSMCs) but ROK having little effect [33]

Summary

Introduction

Defects, and a decreased life span [1, 3,4,5]. Two isoforms of PKG I, ␣ and ␤, differ in their N-terminal 100 amino acids and are splice variants of the two most 5Ј exons of the PKG I gene, which appear to be flanked by separate GC-rich promoters [6]. A transient decrease in PKG I mRNA occurs in VSMCs exposed to mitogens [7], and some but not all investigators have observed lower PKG I␣ expression in actively proliferating, subconfluent VSMC cultures compared with postconfluent cultures [8, 9]. In early passage VSMCs and cardiomyocytes and in intact blood vessels, NO or cGMP or cAMP analogs decrease PKG I␣ mRNA and protein expression by decreasing transcription; inflammatory cytokines down-regulate PKG I␣ in VSMCs by inducing NO synthase and increasing cGMP production (10 –13). Active RhoA induces actin polymerization and stress fiber formation; it increases the expression of smooth musclespecific genes through actin-regulated cooperation between serum response factor (SRF) and transcription factors of the myocardin family [22, 23]. We found that PKG I expression is controlled by RhoA and Rac activity and that RhoA regulation of the PKG I␣ promoter is mediated, at least in part, through binding of the Kruppel-like transcription factor KLF4 to Sp1 consensus sites in the proximal promoter

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