Nongenotropic, Anti-Apoptotic Signaling of 1α,25(OH)2-Vitamin D3 and Analogs through the Ligand Binding Domain of the Vitamin D Receptor in Osteoblasts and Osteocytes

Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors, we have explored the possibility that the vitamin D nuclear receptor (VDR) might exhibit similar nongenotropic actions. We report that the conformationally flexible full VDR agonist, 1α,25(OH) 2 -vitamin D 3 (1α,25(OH) 2 D 3 ), and the 6-s-cis-locked 1α,25(OH) 2 -lumisterol 3 (JN) analog, also acting through the VDR but with poor transcriptional activity, protected murine osteoblastic or osteocytic cells from apoptosis. This effect was reproduced in HeLa cells transiently transfected with either wild type VDR or a mutant consisting of only the VDR ligand binding domain. The VDR ligand binding domain bound [ 3 H]1α,25(OH) 2 D 3 as effectively as wild type VDR but did not induce vitamin D response element-mediated transcription. The anti-apoptotic effects of 1α,25(OH) 2 D 3 and the 6-s-cis-locked 1α,25(OH) 2 -lumisterol 3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors: Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP1), phosphatidylinositol 3 kinase inhibitor Wortmannin, and the JNK kinase inhibitor SP600125. However, inhibition of p38 with SB203580 or ERK with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions. Further, 1α,25(OH) 2 D 3 induced rapid (5 min) association of VDR with Src kinase in OB-6 cells. Finally, actinomycin D or cycloheximide prevented the anti-apoptotic effect of 1α,25(OH) 2 D 3 , indicating that transcriptional events are also required. These findings suggest that nongenotropic modulation of kinase activity is also a general property of the VDR and that ligands that activate nongenotropic signals, but lack transcriptional activity, display different biological profiles from the steroid hormone 1α,25(OH) 2 D 3 .