Involvement of Protein Kinase C and RhoA in Protease-Activated Receptor 1–Mediated F-Actin Reorganization and Cell Growth in Rat Cardiomyocytes

Abstract

Abstract. Protease-activated receptor 1 (PAR1) that can be activated by serine proteinases such as thrombin has been demonstrated to contribute to the development of cardiac remodeling and hypertrophy after myocardial injury. Here, we investigated the mechanisms by which PAR1 leads to hypertrophic cardiomyocyte growth using cultured rat neonatal ventricular myocytes. PAR1 stimulation with thrombin (1 U/ml) or a synthetic agonist peptide (TFLLR-NH 2 , 50 μM) for 48 h induced an increase in cell size and myofibril formation associated with BNP (brain natriuretic peptide) production. This actin reorganization assessed by fluorescein isothiocyanate (FITC)-conjugated phalloidin staining appeared at 1 h after PAR1 stimulation, and this response was reduced by a protein kinase C (PKC) inhibitor, chelerythrine, inhibitors of Rho (simvastatin) and Rho-associated kinase (ROCK) (Y-27632), but not by pertussis toxin (PTX). By Western blot analysis, translocation of PKCα or PKCε from the cytosol to membrane fractions was observed in cells stimulated with thrombin or TFLLR-NH 2 for 2 -5 min. In addition, PAR1 stimulation for 3 -5 min increased the level of active RhoA. Furthermore, inhibitors of PKC and ROCK and Rho abrogated PAR1-mediated increase in cell size. Depletion of PKCα or PKCε by specific small interfering RNA also suppressed both actin reorganization and cell growth. These results suggest that PAR1 stimulation of cardiomyocytes induces cell hypertrophy with actin cytoskeletal reorganization through activation of PKCα and PKCε isoforms and RhoA via PTX-insensitive G proteins.

Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells

“…The family of small Rho GTPases and their downstream target ROCK are implicated in cytoskeletal signaling induced by thrombin (26). Having provided evidence for cytoskeletal involvement in Alk5 transactivation, we sought to determine a possible role of ROCK signaling.…”

Urinary Plasmin Inhibits TRPV5 in Nephrotic-Range Proteinuria

“…The specific subtype of PKC involved in this process has not yet been characterized. However, PKC-a and PKC-« have been reported to mediate PAR-1 signaling, 26 and we would like to suggest that the Ca 2+ -independent PKC-« isoform is more likely to be involved in this pathway because plasmin-mediated PAR-1 activation is sufficient to inhibit TRPV5 in the presence of intracellular Ca 2+ chelator BAPTA. The atypical PKC isoforms were probably not involved because the effect of plasmin is DAG dependent (Supplemental Figure 4B).…”

Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

“…Thus AnxA6-induced Thr654 EGF receptor phosphorylation may link actin and PKCd to EGF receptor recycling. Interestingly, both PKCa and p120GAP interact with members of the actin cytoskeleton and participate in the regulation of actin polymerization at the cell surface and endosomal compartments (98)(99)(100). Together with the ability of AnxA6 to interact with actin and the fact that AnxA6 and its interaction partners including p120GAP, PKCa, and EGF receptor are transported from the plasma membrane to endosomal compartments, it is tempting to speculate that AnxA6-actin interactions provide a scaffold function that coordinates the involvement of negative regulators (p120GAP and PKCa) in EGF receptor/Ras/MAPK pathway signalling along the endocytic pathway.…”