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

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.Cyclic GMP is produced by soluble and receptor guanylate cyclases in response to nitric oxide (NO) 3 and natriuretic peptides, respectively (1). Cellular targets of cGMP include cGMP-dependent protein kinases (PKGs), cGMP-regulated phosphodiesterases, and cGMP-gated ion channels (1). Type I and II PKG are products of different genes and differ in tissue distribution and function (2). PKG I mediates many cGMP effects on cell proliferation, differentiation, and apoptosis, and PKG I knock-out mice have impaired smooth muscle relaxation, increased platelet aggregation, specific neuronal defects, and a decreased life span (1, 3-5). 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). PKG I␣ is expressed highly in vascular smooth muscle cells (VSMCs), platelets, and cerebellum, whereas PKG I␤ is prevalent in other parts of the central nervous system, the adrenal gland, and uterus (1, 6).Within a given cell type or tissue, PKG I expression varies greatly, depending on growth conditions. 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 (1...

MicroRNA-1 Regulates Smooth Muscle Cell Differentiation by Repressing Kruppel-Like Factor 4

“…KLFs are a subclass of evolutionarily conserved transcription factors [28] and KLF4 expression is associated with growth arrest and the inhibitory effect of SMC marker expression [29]. Previous studies have demonstrated that KLF4 potently represses expression of multiple SMC genes by repressing MyoCD levels during phenotypic switching of SMC in response to vascular injury, transforming growth factor-b, or platelet-derived growth factor-BB [14][15][16][30][31][32][33][34].…”

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

“…For PKG, prolonged activation fosters autophosphorylation, which has been shown to promote ubiquitination and protein breakdown (Dey et al, 2009). PKG expression is also down-regulated by RhoA and up-regulated by Rac1, but whether these processes are in play with prolonged elevation of cGMP is not known (Zeng et al, 2006). These changes in PDEs and PKG in response to sustained increases in the NO/ cGMP/PKG signaling pathway suggest a classic feedback response to blunt or terminate a signal.…”

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

“…Quantitative Reverse Transcription-PCR-One microgram of total cytoplasmic RNA was subjected to reverse transcription with random hexamer primers, and quantitative PCR was performed using an Mx3000 real time PCR detection system (Stratagene) with IQ TM SYBR Green Supermix (Bio-Rad) (22). The following primers were used: 5Ј-GACCAGCCTTTTCA-CAGCTC-3Ј (AAF-44, sense) and 5Ј-GCAAGGGACAG-CAAAGACTC-3Ј (AAF-44, antisense) and 5Ј-AACGGAT-TTGGTCGTATTGGG-3Ј (glyceraldehyde-3-phosphate dehydrogenase, sense) and 5Ј-TGGAGGATGGTGATGGGAT-TTC-3Ј (glyceraldehyde-3-phosphate dehydrogenase, antisense).…”