CONICET | Buscador de Institutos y Recursos Humanos

We previously reported that the classical receptor of vitamin D (VDR) participates in non-transcriptional events triggered by the steroid hormone 1alpha,25-dihydroxyvitamin D3 [1,25D] in skeletal muscle cells. Thus, the hormone induces rapid activation of Src, ERK 1/2, p38 MAPK and Akt in a VDR-dependent manner in C2C12 myoblasts cell line. In separate studies we have demonstrated that 1,25D promotes muscle cell proliferation and differentiation, however we did not go in deep into the hormone actions on these processes. In this work we present data indicating that VDR also plays a role in the mechanism by which the 1,25D mediates these effects. To investigate VDR contribution on hormone regulation of cell cycle, we significantly reduced VDR expression by infection of C2C12 murine myoblasts with lentiviral particles containing the pLKO.1 plasmid with information to express a shRNA against mouse VDR. By this way, the vitamin D receptor was knocked down (80%) in these cells, called now C2C12 (-VDR). Cell cycle studies, performed by flow cytometry using propidium iodide staining, evidenced that hormone stimulation prompts a peak of S phase followed by an arrest in G0/G1 phase, events which were dependent on VDR expression. When we investigate changes in cellular cycle regulating proteins, it was evidenced that 1,25D induces an augment of cyclin D3 expression, while protein levels of cyclin D1 do not change significantly during G0/G1 arrest. The up-regulation of cyclin D3 by 1,25D was abolished in C2C12 (-VDR). At same time, we observed the 1,25D ?dependent acute increase in myogenin expression, indicating that G0/G1 arrest of cells was a pro-differentiate event. Accordingly with a differentiate stimulus, the hormone promotes an increase of cyclins inhibitors p21Waf1/Cip1 and p27Kip1 levels, only in C2C12 wild type cells. The results indicate that VDR is involved in the control of cellular cycle by 1,25D in skeletal muscle cells, providing relevant information on the VDR ?dependent mechanism of myogenesis modulation.