ß1-integrins co-localize with Na, K-ATPase, epithelial sodium channels (ENaC) and voltage activated calcium channels (VACC) in mechanoreceptor complexes of mouse limb-bud chondrocytes

Abstract

Interactions between chondrocytes and their extracellular matrix are partly mediated by ß1-integrin receptors. Recent studies have shown that ß1-integrins co-localize with a variety of cytoskeletal complexes, signaling proteins and growth factor receptors. Since mechanosensitive ion channels and integrins have been proposed to participate in skeletal mechanotransduction, in this study, we investigated the possible co-localization of ß1-integrins with two ion channels and a P-type ATPase in mouse limb-bud chondrocytes. The a subunits of Na, K-ATPase, the epithelial sodium channel (ENaC) and the voltage activated calcium channel (VACC) were immunostained in organoid cultures derived from limb-buds of 12-day-old mice using wellcharacterized antibodies . Indirect immunofluorescence revealed abundant expression of ß1-integrins and each of the selected systems in limb-bud chondrocytes. Twofluorochrome immunostaining demonstrated that ß1- integrin, Na, K-ATPase, ENaC and VACC co-localize in chondrocytes. Co-imunoprecipitation experiments revealed co-localization and association of integrins with ENaC, VACC and Na, K-ATPase. Cellular responses and signaling cascades initiated by the influx of calcium or sodium through putative mechanosensitive channels may be regulated more effectively if such channels were organized around integrins with receptors, kinases and cytoskeletal complexes clustered about them. The close proximity of ATPase ion pumps such as Na, K-ATPase to chondrocyte mechanoreceptor complexes could facilitate rapid homeostatic responses to the ionic perturbations brought about by activation of mechanically gated cation channels and efficiently regulate the intracellular milieu of chondrocytes.