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 (Nm.x) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), N., 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. Finally, 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 la-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. Introduction It is well established that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)' is a crucial hormone in Ca2' homeostasis (1). (13) and also is biosynthesized in several ofits peripheral target cells (4-6) in addition to the traditional renal site of formation. Because endothelial cells are a dynamic tissue with spontaneous or injury-dependent cell renewal and expression of specific cell functions at the blood/vessel-wall interface, these cells were examined to determine whether they are potential targets for 1,25(OH)2D3. Initially, the possible presence of specific binding sites for 1,25(OH)2D3 was probed using cultured bovine aortic endothelial cells (BAEC) as a model. When receptors for 1,25(OH)2D3 were observed, the following hypotheses were tested: (a) that the growth state of BAEC may be associated with changes in VDR activity; (b) that BAEC differentiation induced by activators of protein kinase C (PKC) (14-17) may be associated with VDR regulation; (c) that growth parameters of BAEC may be altered in response to 1,25(OH)2D3; (d) that the receptor may be expressed in vivo in endothelial cells in venules and capillaries of human skin; and (e) that BAEC may possess la-hydroxylase activity to form the sterol hormone ligand for the receptor. These studies describe the ...

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Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)2D3 receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [3H]-1,25(OH)2D3 is noted in both the logarithmic growth phase and at confluence (Nmax 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. KD was 10(-11) M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)2D3 receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest Nmax in the proliferating cartilage. 1,25(OH)2D3 in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10(-12) M and dose-dependent inhibition at 10(-10) M and below. Inhibition was specific and not seen with 24,25(OH)2D3 or dexamethasone. Growth phase-dependent 1,25(OH)2D3 receptor expression and effects of 1,25(OH)2D3 on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.

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