The rapid effects of 1,25-dihydroxyvitamin D3 require the vitamin D receptor and influence 24-hydroxylase activity -: Studies in human skin fibroblasts bearing vitamin D receptor mutations

If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D-3 in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D-3 (1 pM-10 nM) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D-3 (profile and magnitude) and 24-hydroxylase responses to low (1 pM-100 pM) 1,25-(OH)(2)D-3 concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca2+ channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D-3 rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D-3 catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex.