rottlerin and Hypertrophy

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/PLCβ1 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)β1, 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 PLCβ1 proteins and resultant VSMC hypertrophy involving c-Src, growth factor receptor transactivation and MAP kinase signaling.

Topics: Acetophenones; Animals; Benzopyrans; Blotting, Western; Cells, Cultured; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gq-G11; Hypertrophy; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Phospholipase C beta; Phosphorylation; Protein Kinase C-delta; Proto-Oncogene Proteins pp60(c-src); Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Growth Factor; RNA Interference; Species Specificity; Superoxides; Tyrosine

NADPH oxidase is the major source of superoxide production in cardiovascular tissues. We reported previously that PG (prostaglandin) F2alpha caused hypertrophy of vascular smooth muscle cells by induction of NOX1, a catalytic subunit of NADPH oxidase. PGF2alpha-induced NOX1 expression was mediated by transactivation of the EGF (epidermal growth factor) receptor and subsequent activation of ERK (extracellular-signal-regulated kinase) 1/2, PI3K (phosphoinositide 3-kinase) and ATF-1 (activating transcription factor-1), a member of the CREB (cAMP-response-element-binding protein)/ATF family. As the receptor for PGF2alpha is known to activate PKC (protein kinase C), involvement of PKC in up-regulation of NOX1 expression was investigated in A7r5 cells. GF109203x, a non-selective inhibitor of PKC, dose-dependently suppressed the induction of NOX1 mRNA by PGF2alpha. Whereas an inhibitor of the conventional PKC, Gö 6976, and a PKCeta translocation-inhibitor peptide had no effect, an inhibitor of PKCdelta, rottlerin, significantly attenuated the PGF2alpha-induced increase in NOX1 mRNA. Gene silencing of PKCdelta by RNA interference significantly suppressed the PGF2alpha-induced increase in NOX1 mRNA, as well as phosphorylation of the EGF receptor, ERK1/2 and ATF-1. Silencing of the PKCdelta gene also attenuated the PDGF (platelet-derived growth factor)- induced increase in NOX1 mRNA and transactivation of the EGF receptor. Moreover, the augmented synthesis of the protein induced by PGF2alpha or PDGF was abolished by gene silencing of PKCdelta. These results suggest that PKCdelta-mediated transactivation of the EGF receptor is elicited not only by PGF2alpha, but also by PDGF, and that the subsequent activation of ERK1/2 and ATF-1 leads to up-regulation of NOX1 gene expression and ensuing hypertrophy in the vascular cell lineage.

Topics: Acetophenones; Animals; Benzopyrans; Carbazoles; Catalysis; Cell Line; Enzyme Induction; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Silencing; Hypertrophy; Indoles; NADPH Oxidases; Platelet-Derived Growth Factor; Protein Kinase C-delta; Protein Transport; Rats; Transcriptional Activation