Selective expression of a normal action of the 1,25-dihydroxyvitamin D3 receptor in human skin fibroblasts with hereditary severe defects in multiple actions of that receptor.

Abstract

We evaluated three actions of 1,25-dihydroxycholecalciferol 11,25-(OH)2D31 in human skin fibroblasts to test for heterogeneity in hormone-response coupling. In fibroblasts from normal subjects the 1,25-OH)2D3 concentrations for half-maximal effect (EC50) were: for mitogenic effect 0.0001-0.0005 nM, for antimitogenic effect 1 nM, and for induction of 25-OHD3 24-hydroxylase (24-OHase) 5 nM. To evaluate the effects of mutations presumed to be in the gene for the 1,25-(OH)2D3 receptor we examined cell lines representing four kindreds with hereditary resistance to 1,25-(OH)2D3 ("mutant" cell lines). In one mutant cell line all three 1,25-(OH)2D3 actions were severely abnormal. In one mutant cell line 24-OHase induction and mitogenic action were undetectable, but EC50 and maximal effect were normal for antimitogenic action of 1,25-(OH1)D3. In two mutant cell lines 24-OHase induction and antimitogenic actions were undetectable or severely impaired but mitogenic action was normal in EC50 and normal or increased in maximal effect. The mitogenic and antimitogenic actions in normal cells showed a similar profile of potency ratios for 1,25-(OH)2D3 and six analogues. Whenever a mutant cell showed a normal or even an abnormal mitogenic or antimitogenic effect of 1,254-OH)2D3, these effects showed potency ratios similar to wild type, suggesting mediation by a similar 1,2540H)2D3 receptor.We conclude that three 1,25-(OHhD3 actions show important differences in hormone response coupling indicated by differences in EC50 for 1,25-(OH)2D3 and by different consequences of receptor mutations.

Subcellular Distribution of Normal and Mutant Vitamin D Receptors in Living Cells

“…Cell Culture-Normal human skin fibroblasts and skin fibroblasts from patients with hereditary resistance to calcitriol were from biopsies as described previously (16). In fibroblasts from patients with vitamin D-dependent rickets type II, previous studies characterized the homozygous point mutations in the VDR gene and the resulting defects in VDR functions (5,16,17,(27)(28)(29).…”

“…In fibroblasts from patients with vitamin D-dependent rickets type II, previous studies characterized the homozygous point mutations in the VDR gene and the resulting defects in VDR functions (5,16,17,(27)(28)(29). MCF7 human breast cancer cells were a gift from M. Lippman (Georgetown University, Washington, D. C.), LLC-PK1 porcine kidney epithelial cells were from American Type Culture Collection, and ROS 17/2.8 rat osteosarcoma cells were a gift from G. Rodan (Merck Sharp and Dohme).…”

“…Cytoplasmic VDR is another possible candidate to mediate nongenomic actions. We have demonstrated that cytoplasmic VDR mediates a rapid increase in intracellular cGMP (16,17). Recent articles show that VDR, together with other members of the steroid receptor superfamily of proteins, associates with calreticulin, the major calcium-sequestering protein of the endoplasmic reticulum (ER) lumen (18 -20).…”

Effect of vitamin D on growth cartilage cell proliferation in vitro

“…1,25(OH)zD3 inhibits proliferation of several cell lines [18,20] and biphasic effects on growth have been demonstrated in the human breast cancer cell line T47D [17], human bone cells [21] and fibroblasts [22].…”

Rapid accumulation of cyclic GMP near activated vitamin D receptors.

“…Normal dermal fibroblasts were from three subjects. Dermal fibroblasts from seven subjects with hereditary resistance to calcitriol (mutant fibroblasts) had VDR functional defects characterized previously (19,21 …”