G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells

We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca(2+)](i). GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca(2+)](i) in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.     

Endogenous G protein-coupled receptor kinase 6 triggers homologous beta-adrenergic receptor desensitization in primary uterine smooth muscle cells

We previously reported that G protein-coupled receptor kinase (GRK) may contribute to beta-adrenergic receptor (beta-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous beta-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the beta-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated beta-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants ((K215R)GRK5 and (K215R)GRK6). Expression of (K215R)GRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of (K215R)GRK5 or (K220R)GRK2 had no effect on beta-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous beta-AR desensitization in myometrial cells.     

Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells

Recent studies have demonstrated that upregulated Rho-kinase plays an important role in the pathogenesis of arteriosclerosis and vasospasm in both animals and humans. However, little is known about the molecular mechanism(s) involved in the Rho-kinase upregulation. Since inflammatory mechanisms have been implicated in the pathogenesis of arteriosclerosis and vasospasm, we examined whether inflammatory stimuli upregulate Rho-kinase in vitro and in vivo. In cultured human coronary vascular smooth muscle cells (hcVSMC), inflammatory stimuli, such as angiotensin II and interleukin-1beta, increased Rho-kinase expression (at both mRNA and protein levels) and function (as evaluated by the extent of the phosphorylation of the ERM (the ezrin/radixin/moesin) family, substrates of Rho-kinase) in a time- and concentration-dependent manner. The expression of Rho-kinase was inhibited by blockades of protein kinase C (PKC) (by either GF109253 or prolonged treatment with phorbol myristate acetate for 24 h) and an adenovirus-mediated gene transfer of dominant-active Ikappa-B, suggesting an involvement of PKC and NF-kappaB in the intracellular signal transduction pathway for the Rho-kinase expression. Furthermore, coronary vascular lesion formation (characterized by medial thickening and perivascular fibrosis) induced by a long-term administration of angiotensin II was markedly suppressed in NF-kappaB(-/-) mice with reduced expression and activity of Rho-kinase in vivo. These results indicate that the expression and function of Rho-kinase are upregulated by inflammatory stimuli (e.g. angiotensin II and IL-1beta) in hcVSMC with an involvement of PKC and NF-kappaB both in vitro and in vivo.     

Role of smooth muscle Nox4-based NADPH oxidase in neointimal hyperplasia

Elevated levels of reactive oxygen species (ROS) in the vascular wall play a key role in the development of neointimal hyperplasia. Nox4-based NADPH oxidase is a major ROS generating enzyme in the vasculature, but its roles in neointimal hyperplasia remain unclear.ObjectiveOur purpose was to investigate the role of smooth muscle cell (SMC) Nox4 in neointimal hyperplasia.Approach and resultsMice overexpressing a human Nox4 mutant form, carrying a P437H dominant negative mutation (Nox4DN) and driven by SM22α promoter, to achieve specific expression in SMC, were generated in a FVB/N genetic background. After wire injury-induced endothelial denudation, Nox4DN had significantly decreased neointima formation compared with non-transgenic littermate controls (NTg). ROS production, serum-induced proliferation and migration, were significantly decreased in aortic SMCs isolated from Nox4DN compared with NTg. Both mRNA and protein levels of thrombospondin 1 (TSP1) were significantly downregulated in Nox4DN SMCs. Downregulation of TSP1 by siRNA decreased cell proliferation and migration in SMCs. Similar to Nox4DN, downregulation of Nox4 by siRNA significantly decreased TSP1 expression level, cell proliferation and migration in SMCs.ConclusionsDownregulation of smooth muscle Nox4 inhibits neointimal hyperplasia by suppressing TSP1, which in part can account for inhibition of SMC proliferation and migration.     

Adrenal G protein-coupled receptor kinase-2 in regulation of sympathetic nervous system activity in heart failure

Heart failure (HF), the number one cause of death in the western world, is caused by the insufficient performance of the heart leading to tissue underperfusion in response to an injury or insult. It comprises complex interactions between important neurohormonal mechanisms that try but ultimately fail to sustain cardiac output. The most prominent such mechanism is the sympathetic (adrenergic) nervous system (SNS), whose activity and outflow are greatly elevated in HF. SNS hyperactivity confers significant toxicity to the failing heart and markedly increases HF morbidity and mortality via excessive activation of adrenergic receptors, which are G protein-coupled receptors. Thus, ligand binding induces their coupling to heterotrimeric G proteins that transduce intracellular signals. G protein signaling is turned-off by the agonist-bound receptor phosphorylation courtesy of G protein-coupled receptor kinases (GRKs), followed by βarrestin binding, which prevents the GRK-phosphorylated receptor from further interaction with the G proteins and simultaneously leads it inside the cell (receptor sequestration). Recent evidence indicates that adrenal GRK2 and βarrestins can regulate adrenal catecholamine secretion, thereby modulating SNS activity in HF. The present review gives an account of all these studies on adrenal GRKs and βarrestins in HF and discusses the exciting new therapeutic possibilities for chronic HF offered by targeting these proteins pharmacologically.     

PTEN as an effector in the signaling of antimigratory G protein-coupled receptor

PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) by cell surface receptors has not been described. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P), which acts through the S1P2 receptor (S1P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S1P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S1P was unable to efficiently inhibit the migration of Pten(DeltaloxP/DeltaloxP) mouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S1P2R activation of Rho GTPase is not affected in Pten(DeltaloxP/DeltaloxP) cells, and dominant-negative Rho GTPase reversed S1P inhibition of cell migration in WT cells but not in Pten(DeltaloxP/DeltaloxP) cells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S1P2R receptor stimulated the coimmunoprecipitation of S1P2R and PTEN. Interestingly, S1P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S1P2R signaling. These data suggest that the S1P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.     

前肾小球平滑肌细胞中内皮素介导的钙信号(英文)

<正> Abstract This study was performed to test the hypothesisthat endothelin peptides differentially influence intracellularcalcium concentration([Ca~(2+)]i) in preglomerular microvascu-lar smooth muscle cells (MVSMC),in part through activationof endothelin (ET) A receptors.Experiments were performedin vitro with the use of single MVSMC freshly isolated fromrat preglomerular microvessels.The effect of ET-1,ET-2,and ET-3 on[Ca~(2+)]i was measured with the use of the calci-um-sensitive dye, fura 2, and standard fluorescence mi-croscopy techniques. Baseline [Ca~(2+)]i averaged (84±3)nmol/L (n=141 cells from 23 dispersions). ET-1 concentra-tions of 1,10,and 100 nmol/L evoked peak increases in[Ca~(2+)]i of(48±16),(930±125),and (810±130)nmol/L,respectively.The time course of the [Ca~(2+)]i response wasbiphasic,beginning with a rapid initial increase followed by asustained plateau phase or a period during which [Ca~(2+)]i os-cillated sharply.Similar responses were observed after ET-2administration.In contrast,E     

SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells

Recent evidence infers a contribution of smooth muscle cell (SMC) progenitors and stromal cell-derived factor (SDF)-1alpha to neointima formation after arterial injury. Inhibition of plaque area and SMC content in apolipoprotein E-deficient mice repopulated with LacZ+ or CXCR4-/- BM or lentiviral transfer of an antagonist reveals a crucial involvement of local SDF-1alpha and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1alpha expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1alpha binds to platelets at the site of injury, triggers CXCR4- and P-selectin-dependent arrest of progenitor cells on injured arteries or matrix-adherent platelets, preferentially mobilizes and recruits c-kit-/platelet-derived growth factor receptor (PDGFR)-beta+/lineage-/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1alpha/CXCR4 axis is pivotal for vascular remodeling by recruiting a subset of SMC progenitors in response to apoptosis and in concert with platelets, epitomizing its importance for tissue repair and identifying a prime target to limit lesion development.     

信号转导子和激活子3通路调控大鼠血管平滑肌细胞增殖迁移机制研究

目的研究转录信号转导子和激活子3（stat3）通路与大鼠血管平滑肌细胞（VSMCs）增殖迁移的关系,明确VSMCs增殖的信号转导过程。方法应用脂质体转染Stat3反义寡核苷酸作用于大鼠VSMCs,应用酶联免疫法（ELISA）检测Stat3水平变化及MTF法检测细胞增殖状态,蛋白免疫印迹法（Western blot）检测stat3、磷酸化stat3及其靶基因产物Cyclin D1、Bcl—XL的表达。结果转染Stat3反义寡核苷酸后,大鼠VSMCs中Stat3水平明显下降（P〈0．01）,同时其增殖水平降低,而相应空白对照组、脂质体组、转染正义寡核苷酸组变化不明显。转染Stat3反义寡核苷酸的VSMCs中stat3、p-Stat 3、Cyclin D1蛋白表达水平随作用时间延长而下降（P〈0．01）,而Bcl—xL水平无明显变化。结论癌基因stat3信号通路与大鼠VSMCs增殖高度相关,可能通过其下游靶基因Cyclin D1影响其增殖,而阻断此通路则可抑制VSMCs的增殖。     

对肺动脉平滑肌细胞趋化反应和DNA合成的研究

探讨肺血管平滑肌细胞（ＰＶＳＭＣ）迁移在缺氧性肺血管结构重组中的作用以及缺氧本身对ＰＶＳＭＣ增殖和迁移的影响。方法利用细胞趋化研究方法和３Ｈ－胸腺嘧啶掺入法研究了血小板衍生生长因子（ＰＤＧＦ）对培养的新生小牛肺动脉平滑肌细胞（ＰＡＳＭＣ）趋化反应和ＤＮＡ合成的作用，以及缺氧和心钠素（ＡＮＰ）对ＰＤＧＦ这种作用的影响。结果表明缺氧可促进ＰＤＧＦ诱导的ＰＡＳＭＣ的趋化反应和ＤＮＡ合成，ＡＮＰ可抑制ＰＡＳＭＣ对ＰＤＧＦ的趋化作用，并抑制ＰＡＳＭＣ的ＤＮＡ合成，鸟苷酸环化酶抑制剂美蓝（ＭＢ）可抑制ＡＮＰ的这种抑制作用。结论研究提示ＰＤＧＦ、ＡＮＰ和缺氧本身对ＰＡＳＭＣ的增殖和迁移有重要作用，这可能对缺氧性肺血管结构重组具有重要意义     

Selective activation of nuclear phospholipase D-1 by g protein-coupled receptor agonists in vascular smooth muscle cells

Recent studies highlight the existence of an autonomous nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PCs as a source of second messengers and, particularly, nuclear phospholipase D (PLD) identification in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). Using immunoblot experiment, in vitro PLD assay with fluorescent substrate and confocal microscopy analysis, we demonstrated that only PLD1 is expressed in VSMC nucleus, whereas PLD1 and PLD2 are present in VSMC. Inhibition of RhoA and protein kinase Czeta (PKCzeta) by C3-exoenzyme and PKCzeta pseudosubstrate inhibitor, respectively, conducted a decrease of phosphatidylethanol production. On the other hand, treatment of intact VSMCs, but not nuclei, with phosphoinositide 3-kinase (PI3K) inhibitors prevented partially nuclear PLD1 activity, indicating for the first time that PI3K may have a role in nuclear PLD regulation. In addition, lysophosphatidic acid (LPA) or angiotensin II treatment of VSMCs resulted in an increase of intranuclear PLD activity, whereas platelet-derived growth factor and epidermal growth factor have no significant effect. Moreover, pertussis toxin induced a decrease of LPA-stimulated nuclear PLD1 activity, suggesting that heterotrimeric G(i)/G(0) protein involvement in intranuclear PLD1 regulation. We also show that LPA-induced nuclear PLD1 activation implied PI3K/PKCzeta pathway activation and PKCzeta nuclear translocation as well as nuclear RhoA activation. Thus, the characterization of an endogenous PLD1 that could regulate PC metabolism inside VSMC nucleus provides a new role for this enzyme in control of vascular fibroproliferative disorders.