Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

Lin, Guiting; Chow, Samson A.; Lin, Jackie Jin; Wang, Guifang; Lue, Tom F.; Lin, Chen

doi:10.1002/jcb.20043

Cited by 22 publications

(19 citation statements)

References 23 publications

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“…cGMP was found to increase nuclear PKGI␥ abundance in BHK and RFL-6 cells expressing PKGI␤-FLAG ( Figure 3A). Moreover, because PKGI expression is increased in postconfluent SMCs, 19 the influence of SMC density on PKGI␥ levels in purified nuclei was examined. cGMP increased nuclear PKGI␥ levels in sparsely plated A7r5 cells, but as SMC density increased, so did the level of nuclear PKGI␥ immunoreactivity in the absence of cGMP stimulation (Figure 3B).…”

Section: Cgmp Increases Pkgi␥ Nuclear Localizationmentioning

confidence: 99%

Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells

Sugiura

Nakanishi²,

Roberts³

2008

Circulation Research

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Abstract-Cyclic GMP modulates gene expression in vascular smooth muscle cells (SMCs) in part by stimulating cGMP-dependent protein kinase I (PKGI) and the phosphorylation of transcription factors. In some cells, cGMP increases nuclear translocation of PKGI and PKGI-dependent phosphorylation of transcription regulators; however, these observations have been variable, and the mechanisms mediating nuclear PKGI translocation are incompletely understood. We tested the hypothesis that proteolytic cleavage of PKGI is required for cGMP-stimulated nuclear compartmentation of PKGI and phosphorylation of transcription factors. We detected an NH 2 -terminal PKGI fragment with leucine zipper domain immunoreactivity in the cytosol and endoplasmic reticulum of SMCs, but only a COOH-terminal PKGI fragment containing the catalytic region (now termed PKGI␥) was observed in the Golgi apparatus (GA) and nucleoplasm. Posttranslational PKGI processing in the GA was critical for nuclear compartmentation of PKGI␥ because GA disruption with nocodazol or brefeldin A inhibited PKGI␥ nuclear localization. PKGI␥ immunoreactivity was particularly abundant in the nucleolus of interphase SMCs where its colocalization with the nucleolar dense fibrillar component protein fibrillarin closely matched the level of nucleolar assembly. Purified nucleolar PKGI␥ enzyme activity was insensitive to cGMP stimulation, which is consistent with its lack of the NH 2 -terminal autoinhibitory domain. Mutation of a putative proteolytic cleavage region in PKGI inhibited cGMPmediated phosphorylation of cAMP response element-binding protein, cAMP response element-dependent transcription, and nuclear localization of PKGI␥. These observations suggest that posttranslational modification of PKGI critically influences the nuclear translocation of PKGI and activities of cGMP in SMCs. Key Words: cGKI Ⅲ guanylate cyclase Ⅲ gene expression regulation Ⅲ signal transduction Ⅲ vascular disease C yclic GMP is a key regulator of vascular smooth muscle cell (SMC) cytoskeletal kinetics, proliferation, and differentiation, and abnormalities in cGMP signaling have been associated with pulmonary and peripheral vascular disease. 1 cGMP is synthesized by guanylyl cyclases, which are activated by nitric oxide, carbon monoxide, and natriuretic peptides, and is metabolized by phosphodiesterases. Through the interaction with cytoplasmic proteins, cGMP influences SMC shape and migration and decreases vascular tone. Recently, cGMP has also been observed to modulate the expression of genes that influence SMC phenotype and proliferation. 2 For example, cGMP has been noted to regulate SMC gene expression by increasing the phosphorylation of transcription factors, such as cAMP response element-binding protein (CREB) [3][4][5] and activating transcription factor-1 (ATF-1), 5 altering the expression of transcription regulators such as activator protein-1 (AP-1) 3 and the growth arrest-specific homeobox transcription factor (GAX), 6 and regulating the activity of other nuclear factors, such as se...

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Section: Cgmp Increases Pkgi␥ Nuclear Localizationmentioning

confidence: 99%

Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells

Sugiura

Nakanishi²,

Roberts³

2008

Circulation Research

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“…However, all gene expression is not increased across cell passages. Research indicates that protein kinase G expression in vascular smooth muscle cells is decreased across cell passages [14]. In addition, membrane estrogen receptor-alpha (mERalpha) and intracellular ERalpha expression have been shown to decline across cell passages [15].…”

Section: Discussionmentioning

confidence: 99%

Increased expression of IL-1 receptors in response to IL-1β may produce more IL-6, IL-8, VEGF, and PGE2 in senescent synovial cells induced in vitro than in presenescent cells

et al. 2011

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“…However, others found little differences in PKG expression at different cell densities (9). We examined PKG I expression at two different cell densities in the rat pulmonary artery smooth muscle cell line CS54, in primary bovine aortic smooth muscle cells (BASMCs), and in REF52 fibroblasts; cells were plated at 0.1-0.2 ϫ 10 6 /10-cm dish or at a 10-fold higher density and cultured for 48 h to generate subconfluent and postconfluent cultures, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Zeng

Zhuang

Gloddek

et al. 2006

Journal of Biological Chemistry

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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...

show abstract

Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

Cited by 22 publications

References 23 publications

Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells

Proteolytic Processing of cGMP-Dependent Protein Kinase I Mediates Nuclear cGMP Signaling in Vascular Smooth Muscle Cells

Increased expression of IL-1 receptors in response to IL-1β may produce more IL-6, IL-8, VEGF, and PGE2 in senescent synovial cells induced in vitro than in presenescent cells

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

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