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 hormone, suggesting that in this disease the lack of an effective cytosolic receptor protein may account for end-organ insensitivity to the hormone. To evaluate the responsiveness of fibroblasts to 1, 25-(OH)2D3, we studied the effect of 1, 25-(OH)2D3 on cell growth. 1, 25-(OH)2D3 (10-10-10-6 M) caused a dose-dependent inhibition of cell growth in receptor-positive normal fibroblasts, whereas these doses of hormone did not affect the growth of receptor-defective DDR-II cells. Hence, the presence of an effective cytosolic receptor in normal skin fibroblasts may be necessary for expression of the growth inhibitory effect of 1, 25-(OH)2D3.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry
- Biochemistry, medical