Hassan Sellak

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.

α1G T-type calcium channel selectively regulates P-selectin surface expression in pulmonary capillary endothelium

Regulated P-selectin surface expression provides a rapid measure for endothelial transition to a proinflammatory phenotype. In general, P-selectin surface expression results from Weibel-Palade body (WPb) exocytosis. Yet, it is unclear whether pulmonary capillary endothelium possesses WPbs or regulated P-selectin surface expression and, if so, how inflammatory stimuli initiate exocytosis. We used immunohistochemistry, immunofluorescence labeling, ultrastructural assessment, and an isolated perfused lung model to demonstrate that capillary endothelium lacks WPbs but possesses P-selectin. Thrombin stimulated P-selectin surface expression in both extra-alveolar vessel and alveolar capillary endothelium. Only in capillaries was the thrombin-stimulated P-selectin surface expression considerably mitigated by pharmacologic blockade of the T-type channel or genetic knockout of the T-type channel alpha(1G)-subunit. Depolarization of endothelial plasma membrane via high K(+) perfusion capable of eliciting cytosolic Ca(2+) transients also provoked P-selectin surface expression in alveolar capillaries that was abolished by T-type channel blockade or alpha(1G) knockout. Our findings reveal an intracellular WPb-independent P-selectin pool in pulmonary capillary endothelium, where the regulated P-selectin surface expression is triggered by Ca(2+) transients evoked through activation of the alpha(1G) T-type channel.

KLF4 and SOX9 Transcription Factors Antagonize β-Catenin and Inhibit TCF-Activity In Cancer Cells

The transcriptional activator β-catenin is a key mediator of the canonical Wnt signaling pathway. β-catenin itself does not bind DNA but functions via interaction with T-cell factor (TCF)/lymphoid-enhancing factor (LEF) transcription factors. Thus, in the case of active Wnt signaling, β-catenin, in cooperation with TCF/LEF proteins family, activates the expression of a wide variety of genes. To date, the list of established β-catenin interacting targets is far from complete. In this study, we aimed to establish the interaction between β-catenin and transcription factors that might affect TCF activity. We took advantage of EMSA, using TCF as a probe, to screen oligonucleotides known to bind specific transcription factors that might dislodge or antagonize β-catenin/TCF binding. We found that Sox9 and KLF4 antagonize β-catenin/TCF binding in HEK293, A549, SW480, and T47D cells. This inhibition of TCF binding was concentration-dependent and correlated to the in vitro TCF-luciferase functional assays. Overexpression of Sox9 and KLF4 transcription factors in cancer cells shows a concentration-dependent reduction of TCF-luciferase as well as the TCF-binding activities. In addition, we demonstrated that both Sox9 and KLF4 interact with β-catenin in an immunoprecipitation assay and reduce its binding to TCF4. Together, these results demonstrate that Sox9 and KLF4 transcription factors antagonize β-catenin/TCF in cancer cells.

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.

Transcriptional and post-transcriptional regulation of cGMP-dependent protein kinase (PKG-I): pathophysiological significance

The ability of the endothelium to produce nitric oxide, which induces generation of cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG-I), in vascular smooth muscle cells (VSMCs), is essential for the maintenance of vascular homeostasis. Yet, disturbance of this nitric oxide/cGMP/PKG-I pathway has been shown to play an important role in many cardiovascular diseases. In the last two decades, in vitro and in vivo models of vascular injury have shown that PKG-I is suppressed following nitric oxide, cGMP, cytokine, and growth factor stimulation. The molecular basis for these changes in PKG-I expression is still poorly understood, and they are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stability, protein synthesis, or protein degradation. Emerging studies have begun to define mechanisms responsible for changes in PKG-I expression and have identified cis- and trans-acting regulatory elements, with a plausible role being attributed to post-translational control of PKG-I protein levels. This review will focus mainly on recent advances in understanding of the regulation of PKG-I expression in VSMCs, with an emphasis on the physiological and pathological significance of PKG-I down-regulation in VSMCs in certain circumstances.

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.