1,25-Dihydroxyvitamin D receptors in an established bone cell line. Correlation with biochemical responses

A stable cell line derived from mouse bone (cell line MMB-1) has been used for studies of the cellular receptor for 1,25-dihydroxyvitamin D3 in osteoblasts. Previous studies have demonstrated that collagen synthesis in the MMB-1 cell line is specifically inhibited by 1,25-dihydroxyvitamin D3 as well as by other bone-regulating hormones. Incubation of cell homogenates with [3H]1,25-dihydroxyvitamin D3 indicated the presence of a specific receptor which was located primarily in the chromatin fraction. Optimum conditions for the receptor assay required the inclusion of 500 kallikrein-inactivating units of Trasylol/ml and 10 mM NaMoO4. Under these conditions the receptors were stable for 2 h at 23 degrees C and for 24 h at 4 degrees C. Cellular content of receptors was dependent upon the state of confluency of the cells: fully confluent cells contained minimal concentrations of receptors. In cultures of 70-80% confluency, the 1,25-dihydroxyvitamin D3 receptors demonstrated linear Scatchard plots with Kd = 0.4 nM. Peak receptor activity was found at 3.7 S in linear sucrose gradient fractions of cell homogenates. The synthesis of collagen by MMB-1 cells was inhibited by 1,25-dihydroxyvitamin D3 in direct proportion to the concentration of cellular receptors at varying levels of culture confluence. The data indicate that MMB-1 cells contain cytoplasmic/nuclear receptors for 1,25-dihydroxyvitamin D3 which are similar to the receptors found in other target tissues for this hormone and suggest that these receptors are mediators of the effects of 1,25-dihydroxyvitamin D3 on collagen synthesis.