Rho kinase–dependent CCL20 induced by dynamic compression of human chondrocytes

Abstract

Mechanical stimulation of cartilage affects tissue homeostasis and chondrocyte function. The chondrocyte phenotype is dependent on cell shape, which is largely determined by the actin cytoskeleton. Reorganization of the actin cytoskeleton results from Rho GTPase activation. The purpose of this study was to examine the roles of both actin and Rho in mechanotransduction in chondrocytes. We embedded human articular chondrocytes in 2 x 6-mm agarose discs at 5 x 10(6) cells/ml and subjected the discs to unconfined dynamic compression at 0.5 Hz. By comparing samples with and without dynamic compression, we identified Rho activation according to the GTP-bound active RhoA measured in cell lysates. We identified rearrangements in filamentous actin structures using fluorescence-labeled phalloidin and confocal microscopy of fixed samples. We identified altered gene expression using TaqMan quantitative reverse transcription-polymerase chain reaction analysis. We tested for a requirement for Rho signaling by performing the dynamic compression in the presence of Rho kinase inhibitors. RhoA activation occurred within 5-10 minutes of dynamic compression. Rho kinase-dependent actin reorganization occurred within 20 minutes after application of dynamic compression and was apparent as "punctate" F-actin structures that were visible under confocal microscopy. We identified early-phase mechanoresponsive genes (CCL20 and inducible nitric oxide synthase) that were highly up-regulated within 1 hour of dynamic compression in a Rho kinase-dependent and actin-dependent manner. Together, these results are the first demonstration that the Rho-Rho kinase pathway and actin cytoskeletal reorganization are required for changes in the expression of genes involved in human chondrocyte mechanotransduction.