ABSTRACT
Gqα signaling has been implicated in cardiac hypertrophy. In addition, angiotensin II (Ang II) was also shown to induce its hypertrophic effect through Gqα and PKCδ activation. We recently showed the role of enhanced expression of Gqα/PLCß1 proteins in vascular smooth muscle cell (VSMC) hypertrophy, however, the role of PKCδ in VSMC hypertrophy in animal model is still lacking. The present study was therefore undertaken to examine the role of PKCδ and the associated signaling mechanisms in VSMC hypertrophy using 16-week-old spontaneously hypertensive rats (SHR). VSMC from 16-week-old SHR exhibited enhanced phosphorylation of PKCδ-Tyr311 and increased protein synthesis, marker of hypertrophy, as compared to WKY rats which was attenuated by rottlerin, an inhibitor of PKCδ. In addition, knocking down of PKCδ by PKCδ-siRNA also attenuated enhanced protein synthesis in VSMC from SHR. Furthermore, rottlerin attenuated the increased production of superoxide anion, NAD(P)H oxidase activity, increased expression of Gqα, phospholipase C (PLC)ß1, insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) proteins in VSMC from SHR. In addition, the enhanced phosphorylation of c-Src, PKCδ-Tyr311, IGF-1R, EGFR and ERK1/2 exhibited by VSMC from SHR was also attenuated by rottlerin. These results suggest that VSMC from SHR exhibit enhanced activity of PKCδ and that PKCδ is the upstream molecule of reactive oxygen species (ROS) and contributes to the enhanced expression of Gqα and PLCß1 proteins and resultant VSMC hypertrophy involving c-Src, growth factor receptor transactivation and MAP kinase signaling.
Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Myocytes, Smooth Muscle/metabolism , Phospholipase C beta/metabolism , Protein Kinase C-delta/metabolism , Acetophenones/pharmacology , Animals , Benzopyrans/pharmacology , Blotting, Western , Cells, Cultured , Enzyme Inhibitors/pharmacology , Hypertrophy , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , NADPH Oxidases/metabolism , Phosphorylation/drug effects , Protein Kinase C-delta/genetics , Proto-Oncogene Proteins pp60(c-src)/metabolism , RNA Interference , Rats, Inbred SHR , Rats, Inbred WKY , Receptors, Growth Factor/metabolism , Species Specificity , Superoxides/metabolism , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCß1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCß1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCß1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCß1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCß1 proteins and resultant VSMC hypertrophy.
Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Hypertension/enzymology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Oxidative Stress , Phospholipase C beta/metabolism , Receptors, Growth Factor/metabolism , Animals , Antioxidants/pharmacology , Cell Size , Cells, Cultured , Disease Models, Animal , ErbB Receptors/metabolism , Hypertension/genetics , Hypertension/pathology , Hypertrophy , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Oxidative Stress/drug effects , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Proto-Oncogene Proteins pp60(c-src)/metabolism , RNA Interference , Rats, Inbred SHR , Rats, Inbred WKY , Receptor, IGF Type 1/metabolism , Receptors, Growth Factor/antagonists & inhibitors , Receptors, Growth Factor/genetics , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction , Transfection , Up-RegulationABSTRACT
Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-ß1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-ß1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-ß1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-ß1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-ß1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-ß1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-ß1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy.
Subject(s)
Angiotensin II/metabolism , Endothelin-1/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Hypertension/enzymology , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , Phospholipase C beta/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Hypertension/pathology , Hypertrophy , Male , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , NF-kappa B/metabolism , Phospholipase C beta/genetics , Phosphorylation , RNA Interference , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Receptor, Angiotensin, Type 1/metabolism , Receptor, Endothelin A/metabolism , Signal Transduction , Transcription Factor AP-1/metabolism , Transfection , Up-RegulationABSTRACT
The discovery of endothelin (ET) in 1988 has led to considerable effort to unravel its implication in health and disease and the mechanisms evoked by ET. ET-1 and related signaling aberrancies are believed to be implicated in the pathogenesis of diverse cardiovascular diseases, such as hypertension, atherosclerosis, hypertrophy and diabetes. The endothelin system consists of three potent vasoconstrictive isopeptides, ET-1, ET-2 and ET-3, signaling through two G protein coupled receptors, ETA and ETB, which are linked to multiple signaling pathways. Activated signaling transduction pathways include the modulation of the adenylyl cyclase/cAMP pathway through stimulatory (Gs) and inhibitory (Gi) G proteins, activation of the phosphoinositide pathway through the activation of proteins Gq/11, generation of oxidative stress, growth factor receptor-related mitogenic events, such as the activation of phosphatidylinositol-3 kinase pathway, phosphoinositide pathway and activation of the mitogen-activated protein (MAP) kinase cascade. The levels of ETA and ETB receptors as well as the signaling pathways activated by these receptors are altered in several cardiovascular diseases including hypertension, hypertrophy, atherosclerosis, diabetes, etc. In this review, we provide an overview of the signaling events modulated by ET-1 in vascular smooth muscle cells in both physiological and pathological conditions.
