Nupur B. Dey

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.

Cyclic GMP–Dependent Protein Kinase Inhibits Osteopontin and Thrombospondin Production in Rat Aortic Smooth Muscle Cells

Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-Ialpha or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.

Inhibition of cGMP-dependent protein kinase reverses phenotypic modulation of vascular smooth muscle cells.

We have previously shown that type I cGMP-dependent protein kinase (PKG) can alter the phenotype of cultured vascular smooth muscle cells (VSMCs). Although the expression of contractile proteins in VSMCs has been shown to be modulated with the induction of PKG, experiments in which PKG inhibition brings about reduced expression of contractile markers have not been performed. To more thoroughly examine the role of PKG in the expression of contractile proteins, recombinant adenovirus containing the PKG coding sequence (AD-PKG) was used to induce gene expression and morphologic changes in adult rat aortic VSMCs. Cells expressing PKG, but not control adenovirus-infected cells, began to express a specific marker protein for the contractile phenotype, smooth muscle myosin heavy chain (SMMHC), within 48 hours of PKG induction. The morphology of the AD-PKG-infected cells began to change from a fibroblastic phenotype to a spindle-shaped phenotype within 72 hours after PKG induction. The specific cell-permeable PKG inhibitory peptide DT-2, but not control peptides, reversed the biochemical and morphologic changes associated with PKG expression. These results suggest that PKG expression and activity in cultured VSMCs is capable of altering the VSMC phenotype. These data also verify the intracellular action of DT-2 and reveal uptake and dynamic properties of this PKG-inhibiting peptide.

Glc-6-PD and PKG contribute to hypoxia-induced decrease in smooth muscle cell contractile phenotype proteins in pulmonary artery

Persistent hypoxic pulmonary vasoconstriction (HPV) plays a significant role in the pathogenesis of pulmonary hypertension, which is an emerging clinical problem around the world. We recently showed that hypoxia-induced activation of glucose-6-phosphate dehydrogenase (Glc-6-PD) in pulmonary artery smooth muscle links metabolic changes within smooth muscle cells to HPV and that inhibition of Glc-6PD reduces acute HPV. Here, we demonstrate that exposing pulmonary arterial rings to hypoxia (20-30 Torr) for 12 h in vitro significantly (P < 0.05) reduces (by 30-50%) SM22α and smooth muscle myosin heavy chain expression and evokes HPV. Glc-6-PD activity was also elevated in hypoxic pulmonary arteries. Inhibition of Glc-6-PD activity prevented the hypoxia-induced reduction in SM22α expression and inhibited HPV by 80-90% (P < 0.05). Furthermore, Glc-6-PD and protein kinase G (PKG) formed a complex in pulmonary artery, and Glc-6-PD inhibition increased PKG-mediated phosphorylation of VASP (p-VASP). In turn, increasing PKG activity upregulated SM22α expression and attenuated HPV evoked by Glc-6-PD inhibition. Increasing passive tension (from 0.8 to 3.0 g) in hypoxic arteries for 12 h reduced Glc-6-PD, increased p-VASP and SM22α levels, and inhibited HPV. The present findings indicate that increases in Glc-6-PD activity influence PKG activity and smooth muscle cell phenotype proteins, all of which affect pulmonary artery contractility and remodeling.

Genetic modification of smooth muscle cells to control phenotype and function in vascular tissue engineering.

Rat smooth muscle cells (SMCs) stably transfected with the gene for the phenotype regulating protein cyclic guanosine monophosphate-dependent protein kinase (PKG) were used as a cell source in the preparation of three-dimensional (3D) collagen type I vascular constructs. PKG-transfected cells expressed severalfold higher levels of the contractile protein smooth muscle alpha-actin (SMA), relative to untransfected SMCs, both in monolayer culture and in 3D gels. The proliferation rate of PKG-transfected cells was lower than that of untransfected cells in both culture geometries. Three-dimensional collagen constructs made with PKG-transfected cells compacted to a similar degree as those made with untransfected cells, and this compaction could be augmented by biochemical stimulation with platelet-derived growth factor BB (PDGF) or transforming growth factor beta(1) (TGF). Application of cyclic mechanical strain to tubular collagen gels seeded with PKG-transfected cells resulted in a higher degree of gel compaction and circumferential matrix alignment, relative to statically grown controls, but cell proliferation and SMA expression were not affected. These results show that genetic modification of SMCs can be used as a tool to control cell function in vascular tissue engineering, and that the function of such cells can be further modulated by application of biochemical and mechanical stimulation.

UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase.

Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer from Kaufman oculocerebrofacial syndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene UBE3B. However, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein with homologous to the E6-AP C terminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758–1068) results in loss of UBE3Bs ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29–58) results in loss of calmodulin binding and a significant increase in the in vitro ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels in vitro disrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.

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.