Inducible mouse model of skeletal muscle specific deletion of the Vitamin D Receptor (VDR)

Abstract

Vitamin D3 has beneficial effects on skeletal muscle and can prevent falls leading to reduced bone fracture risk. Excess of glucocorticoids (GC), either endogenous as in aging or due to glucocorticoid administration as immunosuppressants, leads to muscle loss mass and increases the risk of bone fractures. Earlier findings showed that 1,25(OH)2 vitamin D3 (1,25D3) prevents GC-induced skeletal muscle atrophy in vivo, in muscle organ cultures ex vivo, and in C2C12 myoblasts/ myotubes in vitro. Based on these findings, we formulated the hypothesis that the beneficial actions of Vitamin D3 are mediated by direct hormonal effects on skeletal muscle cells. The purpose of this work was to generate mice lacking the receptor for Vitamin D (VDR) in skeletal muscle and test their response to Vitamin D3 signaling. Towards this end, we crossed transgenic mice expressing a tamoxifen-inducible Mer-Cre-Mer driven by Human skeletal muscle alfa actin promoter with mice whose Receptor for Vitamin D, VDR, was flanked with LoxP sites at the exon 3 locus of the gene. So, tamoxifen-induced specific activation of Cre, produces a new genomic structure of VDR. Males and females 3 months old mice VDRf/f;human skeletal muscle α-actin (HSA)-Cre+/- and their littermate control VDRf/f;HSA-Cre-/- mice (C) were injected with tamoxifen for 5 days (2mg/d 1x/d for 5d). In some experiments, vehicle was injected to check the effects of tamoxifen. Fifteen days after the last tamoxifen injection, at 4months old mice, animals were implanted with slow-release pellets of 2.1mg/kg/d prednisolone or placebo and were treated with 50ng/kg/d 1,25D3 or vehicle 5x/wk for 4wks. Mice were fed a regular Vitamin D3-containing diet and maintained in a 12h light/dark cycle. First, we confirmed tissue-specific VDR deletion. HSA-CRE was present in gDNA of CRE positive mice. Also, we confirmed the deletion of VDR only in induced by tamoxifen in CRE-positive mice. The excised form of the VDR is only detected in skeletal muscle (plantaris and tibialis anterior), but not in kidney, intestine, or bone, of Cre positive mice (VDR f/f;HSA-Cre +/-) treated with tamoxifen. VDR deletion induced by tamoxifen is only detected in CRE positive mice (VDR f/f;HSA-Cre +/-), but not in any tissues from control littermate mice (VDR fl/fl;HSA-Cre -/-). In conclusion this model achieves adult-onset deletion of the VDR in skeletal muscle (Cre and tamoxifen dependent) and thus it will allow its use to determine the direct effects of vitamin D3 signaling in this tissue.