Identification of a competitive binding component in vitamin D-resistant new world primate cells with a low affinity but high capacity for 1,25-dihydroxyvitamin D3

Open Access
Publisher Website
Google Scholar
Abstract
Monkeys in a number of different New World primate genera express a form of compensated target organ resistance to steroid hormones, including 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Characterization of these phenotypes has previously relied upon the study of the 1,25-(OH)2D3-receptor (VDR) interaction in cultured dermal fibroblasts from affected primates. In this report, we show that three of these prototypic phenotypes can be faithfully reproduced in previously established cultured cell lines: B95–8, EBV-transformed B lymphoblasts from the marmoset (Callithrix jacchus), a New World primate with recognized vitamin D resistance; OMK, renal tubular epithelial cells from the owl monkey (Aotus trivergatus), a New World primate with an Old World primate-like VDR phenotype; and MLA144, transformed B lymphoblasts from a gibbon (Hylobates), an Old World primate that expresses the wild-type VDR phenotype. The rank order of specific nuclear uptake and binding of [3H]1,25-(OH)2D3 to the VDR was OMK ≥ ML A144 » B95–8. Despite a 7- to 9-fold difference in cellular VDR content according to ligand binding analyses, there was no discernible difference in the internalization constant Kin for specific cellular uptake of [3H]1,25-(OH)2D3 (0.12–0.26 nM) or in the quantity of VDR detected by immunoblot analysis. We now speculate that the discrepancy in VDR quantitation by binding and immunoblot analysis in the B95–8 New World primate cell line results from the presence of an intracellular, vitamin D metabolite binding moiety in this cell line that competes with the VDR for metabolite binding. Extracts of B95–8 cells, but not wild-type MLA144 cells, exhibited a binding component that had a relatively low affinity (∼ 50 nM) but high capacity for 1,25-(OH)2D3 compared to the VDR. When mixed with extracts of OMK and MLA144 cells (containing the wild-type VDR) of equivalent protein concentration, B95–8 cell extract inhibited specific 1,25-(OH)2D3-VDR binding 84–86%. Sephadex G-100 gel filtration chromatography of the sterol binding component in the B95–8 cell extract detected a single peak of [3H]1,25-(OH)2D3 and [3H]25-OHD3 binding to a protein eluting with an apparent molecular mass of 58–60 kD. This 58–60 kD sterol binding component was also distinguished from the 50 kD VDR by its elution from DEAE-cellulose anion-exchange HPLC at 0.45 M KCl; the mammalian VDR eluted at 0.25 M KCl. These data suggest that the 1,25-(OH)2D3-resistant phenotype of B95–8 cells may be due to the presence of a relatively plentiful, 58–60 kD intracellular protein that binds 1,25-(OH)2D3 with low affinity but high capacity and inhibits the high-affinity nuclear localization of 1,25-(OH)2D3 to the VDR.
Funding Information
  • U.S. PHS (DK37399)