Interaction of 1,25-dihydroxyvitamin-D3 with keratinocytes and fibroblasts from skin of normal subjects and a subject with vitamin-D-dependent rickets, type II: a model for study of the mode of action of 1,25-dihydroxyvitamin D3.

Abstract

The binding of [3H]l,25-dihydroxyvitamin D3 (1,25-(OH)2-[3H]D3) was examined in the cytosol of epidermal keratinocytes and dermal fibroblasts grown from normal human skin. Both cell types contained macromolecules with high affinity for 1,25-(OH)2D3, as demonstrated by sucrose density gradient centrifugation, saturation binding analysis, and DNA-cellulose chromatography. Scatchard analysis of cytosol binding of the hormone yielded affinity constants of 1.0 × 10−10 and 0.8 × 10−10 M and binding capacities of 6.4 and 8.7 fmol/mg protein for fibroblasts and keratinocytes, respectively. In parallel studies, binding of 1,25-(OH)2-[3H]D3 was evaluated in the cytosol from keratinocytes and fibroblasts cultured from a skin biopsy of a patient with vitamin D-dependent rickets, type II (DDRII), an inheritable disorder characterized by extreme end-organ resistance to the action of 1,25-(OH)2D3. Multiple analyses by sucrose density gradient or saturation binding assays failed to reveal specific saturable binding of ho...