Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.     

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, alpha-smooth muscle actin (alphaSMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on alphaSMA and TSP. Treatment with 30 microM Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, alphaSMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.     

环鸟苷酸依赖的蛋白激酶对心血管功能的调节作用

环鸟苷酸(cGMP)依赖的蛋白激酶(PKG)是一氧化氮-cGMP的主要细胞内受体,在哺乳动物细胞中分为PKG-I和PKG-II两型。在PKG介导的血管平滑肌舒张作用中,其主要通过活化细胞膜上的钙活化的钾通道(BK通道),磷酸化肌质网上的受磷蛋白(phospholamban,PLB)和三磷酸肌醇受体相关的PKG-I底物(IP3receptor-associated PKG-I substrate,IRAG),降低细胞内Ca2 浓度。PKG还可通过活化肌球蛋白轻链磷酸酶及抑制Rho激酶降低肌球蛋白对Ca2 敏感性。PKG调节血管平滑肌细胞的基因表达和表型调变,调节细胞增生。PKG活化以后还具有抑制血小板聚集,抑制心肌细胞肥大等功能。最近的研究证明,PKG的表达水平和活性改变与动脉粥样硬化和再狭窄、高血压、糖尿病心血管病变以及硝酸盐耐受等的发病机制有密切关系。     

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca²⁺-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.     

Atorvastatin Calcium Inhibits Phenotypic Modulation of PDGF-BB-Induced VSMCs via Down-Regulation the Akt Signaling Pathway

Plasticity of vascular smooth muscle cells (VSMCs) plays a central role in the onset and progression of proliferative vascular diseases. In adult tissue, VSMCs exist in a physiological contractile-quiescent phenotype, which is defined by lack of the ability of proliferation and migration, while high expression of contractile marker proteins. After injury to the vessel, VSMC shifts from a contractile phenotype to a pathological synthetic phenotype, associated with increased proliferation, migration and matrix secretion. It has been demonstrated that PDGF-BB is a critical mediator of VSMCs phenotypic switch. Atorvastatin calcium, a selective inhibitor of 3-hydroxy-3-methyl-glutaryl l coenzyme A (HMG-CoA) reductase, exhibits various protective effects against VSMCs. In this study, we investigated the effects of atorvastatin calcium on phenotype modulation of PDGF-BB-induced VSMCs and the related intracellular signal transduction pathways. Treatment of VSMCs with atorvastatin calcium showed dose-dependent inhibition of PDGF-BB-induced proliferation. Atorvastatin calcium co-treatment inhibited the phenotype modulation and cytoskeleton rearrangements and improved the expression of contractile phenotype marker proteins such as α-SM actin, SM22α and calponin in comparison with PDGF-BB alone stimulated VSMCs. Although Akt phosphorylation was strongly elicited by PDGF-BB, Akt activation was attenuated when PDGF-BB was co-administrated with atorvastatin calcium. In conclusion, atorvastatin calcium inhibits phenotype modulation of PDGF-BB-induced VSMCs and activation of the Akt signaling pathway, indicating that Akt might play a vital role in the modulation of phenotype.     

Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.     

Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

培养的血管平滑肌细胞ERα和ERβ的表达与细胞表型转变及增殖时相有关

目的探讨雌激素对血管平滑肌细胞（VSMC）增殖的双重效应机制。方法采用Westernblot、电镜形态定量及细胞计数的方法,动态检测原代培养大鼠VSMC在有或无10^-8mol/L17β-雌二醇（E2）存在下,雌激素受体（ER）α和β表达变化与细胞表型转变及增殖时相的关系。结果无E2存在时,VSMC在从收缩型向合成型转变（原代培养第0到5天）及活跃增殖（第5到12天）过程中,ERβ表达无明显变化,但ERα表达明显上升,导致ERα/ERβ比值升高。这种变化并不随VSMC表型的恢复及增殖停止而逆转。有E2存在时,ERα/ERβ比值在第5天时低于对照组,而第9天后各时点均高于对照组;这种影响与E2对不同状态VSMC的不同作用基本对应,即延长原代收缩型SMC的增殖潜伏期,但促进已发生表型转变的VSMC增殖。结论雌激素对不同表型VSMC的双重效应与表型转变前后ERα/ERβ比值变化有关。     

Effect of NO donors on protein phosphorylation in intact vascular and nonvascular smooth muscles

It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.     

Human-derived vascular smooth muscle cells produce angiotensin II by changing to the synthetic phenotype

We investigated whether vascular smooth muscle cells (VSMC)-derived from human produce angiotensin (Ang) II upon change from the contractile phenotype to the synthetic phenotype by incubation with fibronectin (FN). Expression of alpha-smooth muscle (SM) actin, apparent in the contractile phenotype, was decreased by FN. Expressions of matrix Gla and osteopontin, apparent in the synthetic phenotype, were increased by FN. Ang II measured by radioimmunoassay (RIA) was significantly increased in human VSMC by FN. Expression of mRNAs for Ang II-generating proteases cathepsin D, cathepsin G, ACE, and chymase was increased by FN. Expressions of cathepsin D and cathepsin G proteins were also increased by FN. Ang I-generating activity, which was inhibited by an aspartyl protease inhibitor pepstatin A, was readily detected in the conditioned medium from human VSMC. Antisense oligodeoxynucleotides (ODNs) that hybridize with cathepsin D and cathepsin G significantly inhibited FN-increased Ang II in conditioned medium and cell extracts. In VSMC conditioned medium, FN-induced elevation of Ang II was significantly inhibited by temocapril but not by chymostatin. Ang II type 1 receptor antagonist CV11974 completely, and antisense cathepsin D and cathepsin G ODNs partially inhibited the FN-stimulated growth of human VSMC. These results indicate that the change of homogeneous cultures of human VSMC from the contractile to the synthetic phenotype sequentially increases expression of proteases cathepsin D, cathepsin G, and ACE, production of Ang II and productions of growth factors, culminating in VSMC proliferation. These findings implicate a new mechanism for the pathogenesis of human vascular proliferative diseases.     

Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.     

Mechanical Stretch Suppresses microRNA-145 Expression by Activating Extracellular Signal-Regulated Kinase 1/2 and Upregulating Angiotensin-Converting Enzyme to Alter Vascular Smooth Muscle Cell Phenotype

Phenotype modulation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of various vascular diseases, including hypertension and atherosclerosis. Several microRNAs (miRNAs) were found involved in regulating the VSMC phenotype with platelet-derived growth factor (PDGF) treatment, but the role of miRNAs in the mechanical stretch-altered VSMC phenotype is not clear. Here, we identified miR-145 as a major miRNA contributing to stretch-altered VSMC phenotype by miRNA array, quantitative RT-PCR and gain- and loss-of-function methods. Our data demonstrated that 16% stretch suppressed miR-145 expression, with reduced expression of contractile markers of VSMCs cultured on collagenI; overexpression of miR-145 could partially recover the expression in stretched cells. Serum response factor (SRF), myocardin, and Kruppel-like factor 4 (KLF4) are major regulators of the VSMC phenotype. The effect of stretch on myocardin and KLF4 protein expression was altered by miR-145 mimics, but SRF expression was not affected. In addition, stretch-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and up-regulated angiotensin-converting enzyme (ACE) were confirmed to be responsible for the inhibition of miR-145 expression. Mechanical stretch inhibits miR-145 expression by activating the ERK1/2 signaling pathway and promoting ACE expression, thus modulating the VSMC phenotype.     

Functional expression of NOS 1 in vascular smooth muscle

Substances that increase intracellular calcium concentration ([Ca(2+)](i)), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca(2+)](i) also activate Ca(2+)/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca(2+)-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeled L-arginine to L-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.     

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type‐Iα (PKG‐Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG‐Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti‐apoptotic Bcl‐2‐related proteins in NCI‐H460 and A549 non‐small cell lung cancer (NSCLC) cells. 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (ODQ), which blocks endogenous NO‐induced activation of cGMP/PKG‐Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c‐IAP1, livin, and survivin. DT‐2 (inhibitor of PKG‐Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c‐IAP1, livin, and survivin expression. Gene knockdown of PKG‐Iα expression using small‐interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c‐IAP1, livin, survivin, and Mcl‐1 expression. Inhibition of PKG‐Iα kinase activity with DT‐2 dramatically enhanced pro‐apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT‐2 and cisplatin increased apoptosis compared with cisplatin or DT‐2 alone, showing a synergistic effect. The data suggest that the PKG‐Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c‐IAP1, livin, survivin, and Mcl‐1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin. J. Cell. Biochem. 113: 3587–3598, 2012. © 2012 Wiley Periodicals, Inc.     

Hydrogen peroxide induces dimerization of protein kinase G type Iα subunits and increases albumin permeability in cultured rat podocytes

Podocytes help regulate filtration barrier permeability in the kidneys. They express contractile proteins that are characteristic of smooth muscle cells as well as receptors for vasoactive factors such as angiotensin II and atrial natriuretic peptide (ANP). The later one generates intracellular cGMP, with subsequent activation of cGMP-dependent protein kinase; PKG (isoform PKGIα and PKGIβ). In this study, we asked whether hydrogen peroxide (H(2)O(2)), a physiological vasorelaxing factor, affected podocyte permeability and the podoctye PKGIα signaling pathway. Expression of PKGIα was confirmed in cultured rat podocytes using RT-PCR, immunofluorescence, and Western blotting. Exposure of podocytes to exogenous H(2)O(2) (100 μM) in non-reducing conditions increased the formation of PKGIα interprotein disulfide bonds, affected the phosphorylation of PKG target proteins, namely MYPT1 (maximal increase of about 57% at 30 min) and MLC (maximal decrease of about 62% at 10 min). Furthermore, H(2)O(2) increased the permeability of a layer of podocytes to albumin: Transmembrane flux for albumin increased five-fold (106.6 ± 5.2 μg/ml vs. 20.2 ± 2.5 μg/ml, P < 0.05, n = 5), and the PKG inhibitor Rp-8-Br-cGMPS (100 μM) prevented the flux increase. These data suggest that oxidative modulation of PKGIα in podocytes plays an important