Angiotensin II stimulates phosphorylation of high-molecular-mass cytosolic phospholipase A2 in vascular smooth-muscle cells.
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ABSTRACT:
SUBMITTER: Rao GN
PROVIDER: S-EPMC1138041 | biostudies-other | 1994 Apr
REPOSITORIES: biostudies-other
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Project description:BackgroundAngiotensin II (Ang II) induces the phenotypic modulation and hypertrophy of vascular smooth muscle cells (VSMCs), which is implicated in the pathogenesis of hypertension, atherosclerosis, and diabetes. In this study, we tested the hypothesis that histone deacetylases 5 (HDAC5) and its signal pathway play a role in Ang II-induced VSMC hypertrophy.Methods and resultsVSMCs were isolated from the thoracic aortas of male Sprague-Dawley rats and treated with Ang II. We found that Ang II rapidly stimulated phosphorylation of HDAC5 at Serine259/498 residues in a time- and dose- dependent manner. Ang II receptor-1, protein kinase C, and protein kinase D1 (PKD1) mediated HDAC5 phosphorylation. Furthermore, we observed that Ang II stimulated HDAC5 nuclear export, which was dependent on its PKD1-dependent phosphorylation. Consequently, both inhibiting PKD1 and HDAC5 Serine259/498 to Alanine mutant significantly attenuated Ang II-induced myocyte enhancer factor-2 (MEF2) transcriptional activity and protein synthesis in VSMCs.ConclusionThese findings demonstrate for the first time that PKD1-dependent HDAC5 phosphorylation and nuclear export mediates Ang II-induced MEF2 activation and VSMC hypertrophy, and suggest that PKD1 and HDAC5 may emerge as potential targets for the treatment of pathological vascular hypertrophy.
| S-EPMC4259271 | biostudies-literature
Project description:Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle alpha-actin (SM-alpha-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-alpha-actin protein. Arginine vasopressin, which stimulated SM-alpha-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-alpha-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-alpha-actin protein, and stimulation of SM-alpha-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-alpha-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-alpha-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-alpha-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.
| S-EPMC1218847 | biostudies-other
Project description:Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.
| S-EPMC2133140 | biostudies-literature
Project description:We previously showed that angiotensin II (Ang II) and angiotensin-(2-8)-peptide [Ang-(2-8)] activate a phosphoinositide-specific phospholipase C (PLC) and cause calcium mobilization in rat aortic vascular smooth-muscle cells (VSMC), while Ang II and Ang-(1-7) produce prostaglandins. To define further the signal-transduction mechanisms activated by angiotensin peptides in smooth-muscle cells, we measured diacylglycerol (DAG) accumulation in response to different angiotensin peptides and its inhibition by subtype-selective receptor antagonists. Both an initial (10 s) and secondary (10 min) phase of DAG production in response to 100 nM Ang II were inhibited by 1 microM losartan (DuP 753), an AT1 antagonist, while 1 microM PD 123177, an AT2 antagonist, was ineffective. In contrast, the heptapeptide Ang-(1-7) did not produce DAG in VSMC. Ang II also caused the hydrolysis of phosphatidylinositol and phosphatidylcholine, the formation of phosphatidic acid and the formation of phosphatidylethanol (PEt) in the presence of ethanol, through activation of a PLD and a PLD-induced transphosphatidylation reaction. A similar concentration of Ang-(2-8) also activated PLD; in contrast, Ang-(1-7) was ineffective. PEt production by 100 nM Ang II was significantly attenuated by the AT1 antagonists losartan, its metabolite EXP 3174 or L-158,809 (all at 1 microM), whereas a similar concentration of the AT2 antagonists CGP 42112A or PD 123177 was ineffective. The production of PEt by Ang II was also partially attenuated by the removal of extracellular calcium and potentiated by increasing calcium concentrations, indicating that PLD activity is partially dependent on extracellular calcium. Thus VSMC PLD is coupled to an AT1 receptor and occurs in response to Ang II or Ang-(2-8), but not Ang-(1-7). Since AT1 receptors in VSMC are also coupled to activation of PLC, both PLC and PLD may be coupled to the same or a different AT1 receptor. Alternatively, PLD may be sequentially activated in response to Ang II activation of PLC and a subsequent increase in calcium concentration.
| S-EPMC1137526 | biostudies-other
Project description:PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.
| S-EPMC1513282 | biostudies-literature
Project description:Bradykinin stimulates cAMP synthesis in cultured airway smooth muscle (ASM) cells. This occurs via a pathway that involves: (1) the protein kinase C (PKC)-dependent activation of mitogen-activated protein kinase (MAPK); (2) the MAPK-dependent phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) and (3) the utilization of cPLA2-derived arachidonate by the cyclo-oxygenase pathway to produce prostaglandin E2 (PGE2). PGE2 is released and binds to cell surface receptors to stimulate intracellular cAMP synthesis. The signalling pathway was confirmed by the use of PD098059 [the inhibitor of MAPK kinase-1 (MEK-1) activation], AACOCF3 (an inhibitor of cPLA2) and indomethacin (an inhibitor of cyclo-oxygenase), which all reduced bradykinin-stimulated cAMP synthesis. Bradykinin also elicits the inhibition of approx. 60% of the total cAMP phosphodiesterase activity in the cell [Stevens, Pyne, Grady and Pyne (1994) Biochem. J. 297, 233-239]. This is likely to decrease the rate of cAMP degradation markedly and therefore to potentiate PGE2-stimulated cAMP synthesis. Acute treatment of ASM cells with PMA (a direct activator of PKC) also stimulated the MAPK-dependent phosphorylation of cPLA2. However, in contrast with bradykinin, PMA did not stimulate arachidonate release, suggesting that additional signals (e.g. Ca2+ ions) are required for phosphorylation by MAPK to activate cPLA2. PMA was also without effect on PGE2 release and cAMP synthesis. Evidence that PKC can also directly regulate adenylate cyclase was obtained by using cells pretreated with cholera toxin. Under these conditions, PMA stimulated cAMP synthesis independently of arachidonate metabolites. Furthermore the combined treatment of cells with PMA (to activate PKC) and PGE2 (to activate Gs) stimulated synergistic cAMP synthesis. This might be due to the presence of the type 2 adenylate cyclase, which is synergistically activated by Gs and PKC.
| S-EPMC1218972 | biostudies-other
Project description:Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs) as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.
| S-EPMC4404349 | biostudies-literature
Project description:Understanding the mechanisms that regulate cell cycle progression in vascular smooth muscle cells (VSMCs) is key to understanding and modulating vascular lesion formation. Results of the present study provide the first evidence that phosphorylation of the helix-loop-helix factor Id3 in VSMCs occurs in vitro and in vivo and provides a regulatory switch controlling Id3-induced regulation of p21Cip1 and VSMC growth.
| S-EPMC2929385 | biostudies-literature
Project description:Increased interferon (IFN)-? signaling in patients with insufficient coronary collateralization and an inhibitory effect of IFN? on collateral artery growth in mice have been reported. The mechanisms of IFN?-induced inhibition of arteriogenesis are unknown. In stimulated monocytes from patients with chronic total coronary artery occlusion and decreased arteriogenic response, whole genome expression analysis showed increased expression of IFN?-regulated genes. Immunohistochemically, the IFN? receptor was localized in the vascular media of murine collateral arteries. Treatment of vascular smooth muscle cells (VSMC) with IFN? resulted in an attenuated proliferation, cell-cycle arrest, and increased expression of cyclin-dependent kinase inhibitor-1A (p21). The growth inhibitory effect of IFN? was attenuated by inhibition of p21 by RNA interference. IFN?-treated THP1 monocytes showed enhanced apoptosis. Subsequently, we tested if collateral artery growth can be stimulated by inhibition of IFN?-signaling. RNA interference of the IFN? receptor-1 (IFNAR1) increased VSMC proliferation, cell cycle progression, and reduced p21 gene expression. IFN? signaling and FAS and TRAIL expression were attenuated in monocytes from IFNAR1(-/-) mice, indicating reduced monocyte apoptosis. Hindlimb perfusion restoration 1 week after femoral artery ligation was improved in IFNAR1(-/-) mice compared with wild-type mice as assessed by infusion of fluorescent microspheres. These results demonstrate that IFN? inhibits collateral artery growth and VSMC proliferation through p21-dependent cell cycle arrest and induction of monocyte apoptosis. Inhibition of IFN? stimulates VSMC proliferation and collateral artery growth.
| S-EPMC2966083 | biostudies-literature
Project description:We have shown previously that activation of protein kinase C-delta (PKC delta) is required for angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC delta phosphorylation at Tyr(311) plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC delta phosphorylation at Tyr(311), and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC delta was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr(311) by Ang II. This phosphorylation was markedly blocked by an Src family kinase inhibitor and dominant-negative Src but not by an epidermal growth factor receptor kinase inhibitor. Ang II-induced Akt phosphorylation and hypertrophic responses were significantly enhanced in VSMCs expressing PKC delta wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC delta Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC delta K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC delta kinase activation but also the Src-dependent Tyr(311) phosphorylation contributes to Akt activation and subsequent VSMC hypertrophy induced by Ang II, thus signifying a novel molecular mechanism for enhancement of cardiovascular diseases induced by Ang II.
| S-EPMC2728048 | biostudies-literature