Identification and regulation of 1,25-dihydroxyvitamin D₃ receptor activity and biosynthesis of 1,25-dihydroxyvitamin D₃. Studies in cultured bovine aortic endothelial cells and human dermal capillaries

Because 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)₂D₃ receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (N(max)) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), N(max) increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finaly, additional experiments indicated that BAEC formed the 1,25(OH)₂D₃ hormonal metabolite from 25(OH)D₃ substrate, in vitro, and growth curves of BAEC maintained in the presence of 10^-8 M 1,25(OH)₂D₃ showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1α-hydroxylase enzyme capable of producing 1,25(OH)₂D₃. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)₂D₃ hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.