Caveolae and caveolin-1 are implicated in 1α,25(OH)2-vitamin D3-dependent modulation of Src, MAPK cascades and VDR localization in skeletal muscle cells

We previously reported that 1 alpha,25(OH)(2)D-3 induces non-transcriptional rapid responses through activation of MAPKs in C2C12 skeletal muscle cells. However, there is little information on the molecular mechanism underlying the initiation of 1 alpha,25(OH)(2)D-3 signaling through this pathway. Plasma membrane components have been involved in some non-genomic effects. In this work, we investigated the role of caveolae and caveolin-1 (cav-1) in 1 alpha,25(OH)(2)D-3-stimulation of c-Src and MAPKs. When proliferating cells were pretreated with methyl beta cyclodextrin (M beta CD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1 alpha,25(OH)(2)D-3-dependent activation of ERK1/2, p38 MAPK and c-Src was suppressed. Similar results were obtained by siRNA technology whereby silencing of cav-1 expression abolished activation of c-Src and MAPKs induced by the hormone. By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment disrupted the colocalization of these proteins and redistributed them into cytoplasm and nucleus. Co-immunoprecipitation assays corroborated these observations. Changes in VDR localization after 1 alpha,25(OH)(2)D-3 exposure were also investigated. Confocal microscopy images showed that the hormone induces VDR translocation to the plasma membrane, and this effect is abolished by M beta CD. Altogether, these data suggest that caveolae is involved upstream in c-Src-MAPKs activation by 1 alpha,25(OH)(2)D-3 and that VDR and cav-1 participate in the rapid signaling elicited by the hormone. (C) 2010 Elsevier Ltd. All rights reserved.