LPA Activation of a RhoA/CPKCα-Mediated Signaling Pathway Regulates Outer Hair Cell Motility by Phosphorylating the Cytoskeletal Protein Adducin

Abstract

In prior studies on cochlear outer hair cells (OHC) we demonstrated that LPA was able to induce RhoA/ROCK-mediated adducin phosphorylation at Thr445, and RhoA/PKC-mediated adducin phosphorylation at Ser726 (Zhang et al, J Biol Chem 278:35644-50, 2003). Adducin, together with actin and spectrin, is a major component of the OHC's cortical cytoskeleton. Thus, activation of RhoA/PKC pathway could induce changes in OHC length and motility associated with structural modifications in this cytoskeletal structure. In this study, we aimed at determining if PKC inhibitors were able to regulate RhoA/PKC-adducin mediated effects on OHC motility. Actin-dependent changes in cell length (slow motility) and the amplitude of electrically induced changes in cell length (fast motility) were measured in isolated guinea pig OHC, patch clamped in whole-cell mode and internally perfused through the patch pipette with inhibitor of PKC while being externally stimulated with LPA. OHC motility was monitored by high-speed video microscopy, whereas confocal microscopy was used to investigate effects of PKC inhibitor on RhoA/PKC-Adducin signaling pathway. We found that LPA induced expression of cPKCα and nPKCζ, with cPKCα but not nPKCζ phosphorylating adducin both of Ser725 and Thr445. However, treatment with specific pharmacological inhibitors of PKCα reduced adducin phosphorylation only at Ser726. Also, we determined that activation of the RhoA/PKCα-mediated signaling pathway by LPA induced OHC shortening and a simultaneous increase in the amplitude of fast motility. Our results support a positive role for cPKCα in OHC motility and strongly suggest a link between cPKCα, adducin phosphorylation and the regulation of OHC motility.This work was supported by NIDCD/NIH grant R01 DC010146. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.