.? ROLE OF CAVEOLAE AND CAVEOLIN-1 IN 1,25(OH)2-VITAMIN D3 ?DEPENDENT MODULATION OF SRC, MAPK CASCADES AND VDR LOCALIZATION IN SKELETAL MUSCLE CELLS?

BUITRAGO C; BOLAND R

Bélgica

Workshop; Workshop of Vitamin D; 2009

1,25(OH)2D3 induces non-transcriptional rapid responses through activation of MAPKs in the skeletal muscle cell line C2C12. However, there is no information on the molecular mechanism underlying the initiation of 1,25(OH)2D3 signaling through this pathway. Plasma membrane components have been involved in some non-genomic effects. In the present work we investigated the role of caveolae and caveolin-1 (cav-1) in 1,25(OH)2D3 stimulation of c-Src and MAPKs. When proliferating C2C12 cells were pre-treated with methyl-beta-cyclodextrin, a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1,25(OH)2D3-dependent activation (phosphorylation) of ERK 1/2, p38 MAPK and c-Src was suppressed. Similar results were obtained by siRNA technology where silencing of cav-1 expression abolished phosphorylation of c-Src and MAPKs induced by 1,25(OH)2D3. By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment redistributed these proteins into cytoplasm and nucleus and disrupted their colocalization. Co-immunoprecipitation assays corroborated these observations. Changes in VDR localization after 1,25(OH)2D3 exposure were also investigated in C2C12 cells. Confocal microscopy showed that the hormone induces VDR translocation to the plasma membrane, and this effect is abolished by metil beta cyclodextrin. Preliminary studies revealed 1,25(OH)2D3 -dependent VDR-cSrc association. Altogether, these data suggest that intact caveolae participate in an early upstream step in 1,25(OH)2D3 signal transduction via c-Src-MAPKs and that the VDR and cav-1 are involved in the rapid events triggered by the hormone.