Chung-Sik Choi

Regulation of vascular smooth muscle cell phenotype by cyclic GMP and cyclic GMP-dependent protein kinase.

This basic science review examines the role of cGMP and cGMP-dependent protein kinase (PKG) in the regulation of vascular smooth muscle cell (VSMC) phenotype. The first such studies suggested a role for nitric oxide (NO) and atrial natriuretic peptides (ANP), and the downstream second messenger cGMP, in the inhibition of VSMC proliferation. Subsequently, many laboratories confirmed the anti-proliferative effects of the cGMP pathway in cultured cells and the anti-atherosclerotic effects of the pathway in in vivo animal models. Other studies suggested that the cGMP target, PKG, mediated the anti-proliferative effects of cGMP although other laboratories have not consistently observed these effects. On the other hand, PKG mediates cGMP-dependent increases in smooth muscle-specific gene expression, and in vivo studies suggest that PKG expression itself reduces vascular lesions. The mechanisms by which PKG regulates gene expression are addressed, but it still unknown how the cGMP-PKG pathway is involved in smooth muscle-specific gene expression and phenotype.

cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation

Although the regulation of smooth muscle cell (SMC) gene expression by cGMP-dependent protein kinase (PKG) is now recognized, the mechanisms underlying these effects are not fully understood. In this study, we report that PKG-I stimulates myocardin/serum response factor (SRF)-dependent gene expression in vascular SMCs. The expression of PKG in PKG-deficient cells enhanced myocardin-induced SM22 promoter activity in a concentration-dependent fashion. However, neither SRF nor myocardin expression was affected. To investigate alternative mechanisms, we examined whether PKG affects the phosphorylation of E26-like protein-1 (Elk-1), a SRF/myocardin transcription antagonist. The activation of PKG caused an increase in a higher molecular mass form of phospho-Elk-1 that was determined to be small ubiquitin-related modifier (sumo)ylated Elk-1. PKG increased Elk-1 sumoylation twofold compared with the PKG-deficient cells, and Elk-1 sumoylation was reduced using dominant-negative sumo-conjugating enzyme, DN-Ubc9, confirming PKG-dependent sumoylation of phospho-Elk-1 in vascular SMCs. In addition, PKG stimulated Elk-1 sumoylation in COS-7 cells overexpressing Elk-1, sumo-1, and PKG-I. The increased expression of PKG in vascular SMCs inhibited Elk-1 binding to SMC-specific promoters, SM22 and smooth muscle myosin heavy chain, as measured by EMSA and chromatin immunoprecipitation assay, and PKG suppressed the Elk-1 inhibition of SM22 reporter gene expression. Taken together, these data suggest that PKG-I decreases Elk-1 activity by sumo modification of Elk-1, thereby increasing myocardin-SRF activity on SMC-specific gene expression.

Changes in Vascular Reactivity and Endothelial Ca2+ Dynamics with Chronic Low Flow

Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling and atherosclerosis. Recent findings suggest that spatial and temporal tuning of local Ca2+ signals along the endothelium is vital to vascular function. In this study, we examined whether chronic flow disruption causes alteration of dynamic endothelial Ca2+ signal patterning associated with changes in vascular structure and function. For these studies, we performed surgical PL of the left carotid arteries of mice to establish chronic low flow for 2 weeks; right carotid arteries remained open and served as controls (C). Histological sections showed substantial remodeling of PL compared to C arteries, including formation of neointima. Isometric force measurements revealed increased PE‐induced contractions and decreased KCl‐induced contractions in PL vs C arteries. Endothelium‐dependent vasorelaxation in response to ACh; 10−8 to 10−5 mol/L) was significantly impaired in PL vs C vessels. Evaluation of endothelial Ca2+ using confocal imaging and custom analysis exposed distinct impairment of Ca2+ dynamics in PL arteries, characterized by reduction in active sites and truncation of events, corresponding to attenuated vasorelaxation. Our findings suggest that endothelial dysfunction in developing vascular disease may be characterized by distinct shifts in the spatial and temporal patterns of localized Ca2+ signals.

Smooth Muscle Specific Overexpression of p22phox Potentiates Carotid Artery Wall Thickening in Response to Injury

We hypothesized that transgenic mice overexpressing the p22phox subunit of the NADPH oxidase selectively in smooth muscle (Tgp22smc) would exhibit an exacerbated response to transluminal carotid injury compared to wild-type mice. To examine the role of reactive oxygen species (ROS) as a mediator of vascular injury, the injury response was quantified by measuring wall thickness (WT) and cross-sectional wall area (CSWA) of the injured and noninjured arteries in both Tgp22smc and wild-type animals at days 3, 7, and 14 after injury. Akt, p38 MAPK, and Src activation were evaluated at the same time points using Western blotting. WT and CSWA following injury were significantly greater in Tgp22smc mice at both 7 and 14 days after injury while noninjured contralateral carotids were similar between groups. Apocynin treatment attenuated the injury response in both groups and rendered the response similar between Tgp22smc mice and wild-type mice. Following injury, carotid arteries from Tgp22smc mice demonstrated elevated activation of Akt at day 3, while p38 MAPK and Src activation was elevated at day 7 compared to wild-type mice. Both increased activation and temporal regulation of these signaling pathways may contribute to enhanced vascular growth in response to injury in this transgenic model of elevated vascular ROS.

Cyclic GMP-Dependent Protein Kinase

Publisher Summary This chapter addresses three aspects of PKG function that include the role of PKG-I targeting to subcellular proteins especially in smooth muscle cells (SMC), the role of PKG-I in regulating vascular SMC (VSMC) gene expression, and the regulation of the expression of PKG-I. The serum response factor (SRF) interacts with numerous co-transcriptional regulators, including myocardin, a smooth muscle specific co-transcription factor and member of the larger myocardin-related factor (MRF) family of proteins. PKG-I enhances myocardin-stimulated SRF activity in part through the phosphorylation of a myocardin regulatory protein, cysteine-rich protein-2 (CRP-2) in VSMC. The enhanced myocardin-dependent SRF activity in a non-smooth muscle cell line is observed, when it is transfected with myocardin and PKG-I cDNA. PKG-I also increases SRF binding to smooth muscle-specific promoter regions of SMMHC and SM22. The Kruppel-like factor (KLF-4) binds to the Sp1 sites on the PKG-I proximal promoter and regulates gene transcription. The small molecular weight G proteins, RhoA and rac, regulate KLF-4 binding and PKG-I gene expression. The inflammatory, atherogenic cytokines such as IL-1β and TNFα decrease PKG-I mRNA and protein levels in bovine aortic VSMC. One mechanism responsible for this event is a cytokine-dependent increase in iNOS expression, NO biosynthesis, and a decrease in Sp1 binding to the PKG-I promoter. Suppression of iNOS activity or sGC activity inhibits the downregulation of PKG-I mRNA induced by the cytokines. PKA inhibition also suppresses the effects of cytokines on PKG-I mRNA expression, suggesting that high elevations in cGMP in response to iNOS expression cross-activate PKA and lead to decreased Sp1 protein binding to the PKG-I promoter.

Regulation of smooth muscle specific gene expression by PKG and mechanisms regulating PKG expression in smooth muscle cells

Cyclic GMP-dependent protein kinase I (PKG) is highly expressed in smooth muscle cells (SMC) and mediates the effects of nitric oxide (NO) on smooth muscle relaxation and SMC-specific gene expression. To understand the mechanisms by which PKG stimulates SMC-specific gene expression, we examined the effects of PKG over-expression in passaged rat aortic SMC that express low levels of PKG and SMC-specific genes. PKG enhances serumresponse factor (SRF) and myocardin (MY)induced reporter gene expression in SMC. These effects were not dependent on induction of either SRF or MY. The Ternary Complex Element transcription factor, Elk-1, is known to inhibit SRF-MY induced SMC gene expression when phosphorylated in response to platelet derived growth factor (PDGF). PKG inhibited Elk-1 repression of SRF-MY gene transcription by stimulating post-translational modification of phospho-Elk-1 via the small ubiquitin-like modifier (SUMO). The mechanism of PKG-induced sumoylation of Elk-1 may be dependent on the phosphorylation of SENP-1 (sumo-specific protease 1) at ser-125. These results suggest that PKG regulates SRF-MY gene expression through de-repression of Elk-1 on SMC-specific promoters.

Chapter 180 – Cyclic GMP-Dependent Protein Kinase: Targeting and Control of Expression

Publisher Summary This chapter addresses three aspects of PKG function that include the role of PKG-I targeting to subcellular proteins especially in smooth muscle cells (SMC), the role of PKG-I in regulating vascular SMC (VSMC) gene expression, and the regulation of the expression of PKG-I. The serum response factor (SRF) interacts with numerous co-transcriptional regulators, including myocardin, a smooth muscle specific co-transcription factor and member of the larger myocardin-related factor (MRF) family of proteins. PKG-I enhances myocardin-stimulated SRF activity in part through the phosphorylation of a myocardin regulatory protein, cysteine-rich protein-2 (CRP-2) in VSMC. The enhanced myocardin-dependent SRF activity in a non-smooth muscle cell line is observed, when it is transfected with myocardin and PKG-I cDNA. PKG-I also increases SRF binding to smooth muscle-specific promoter regions of SMMHC and SM22. The Kruppel-like factor (KLF-4) binds to the Sp1 sites on the PKG-I proximal promoter and regulates gene transcription. The small molecular weight G proteins, RhoA and rac, regulate KLF-4 binding and PKG-I gene expression. The inflammatory, atherogenic cytokines such as IL-1β and TNFα decrease PKG-I mRNA and protein levels in bovine aortic VSMC. One mechanism responsible for this event is a cytokine-dependent increase in iNOS expression, NO biosynthesis, and a decrease in Sp1 binding to the PKG-I promoter. Suppression of iNOS activity or sGC activity inhibits the downregulation of PKG-I mRNA induced by the cytokines. PKA inhibition also suppresses the effects of cytokines on PKG-I mRNA expression, suggesting that high elevations in cGMP in response to iNOS expression cross-activate PKA and lead to decreased Sp1 protein binding to the PKG-I promoter.

Cyclic GMP-Dependent Protein Kinase: Targeting and Control of Expression

Publisher Summary This chapter addresses three aspects of PKG function that include the role of PKG-I targeting to subcellular proteins especially in smooth muscle cells (SMC), the role of PKG-I in regulating vascular SMC (VSMC) gene expression, and the regulation of the expression of PKG-I. The serum response factor (SRF) interacts with numerous co-transcriptional regulators, including myocardin, a smooth muscle specific co-transcription factor and member of the larger myocardin-related factor (MRF) family of proteins. PKG-I enhances myocardin-stimulated SRF activity in part through the phosphorylation of a myocardin regulatory protein, cysteine-rich protein-2 (CRP-2) in VSMC. The enhanced myocardin-dependent SRF activity in a non-smooth muscle cell line is observed, when it is transfected with myocardin and PKG-I cDNA. PKG-I also increases SRF binding to smooth muscle-specific promoter regions of SMMHC and SM22. The Kruppel-like factor (KLF-4) binds to the Sp1 sites on the PKG-I proximal promoter and regulates gene transcription. The small molecular weight G proteins, RhoA and rac, regulate KLF-4 binding and PKG-I gene expression. The inflammatory, atherogenic cytokines such as IL-1β and TNFα decrease PKG-I mRNA and protein levels in bovine aortic VSMC. One mechanism responsible for this event is a cytokine-dependent increase in iNOS expression, NO biosynthesis, and a decrease in Sp1 binding to the PKG-I promoter. Suppression of iNOS activity or sGC activity inhibits the downregulation of PKG-I mRNA induced by the cytokines. PKA inhibition also suppresses the effects of cytokines on PKG-I mRNA expression, suggesting that high elevations in cGMP in response to iNOS expression cross-activate PKA and lead to decreased Sp1 protein binding to the PKG-I promoter.