Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptor-positive resistance to 1,25-dihydroxyvitamin D3.
We describe studies of the molecular defect in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action in cultured skin fibroblasts from a patient previously reported to have vitamin D-dependent rickets, type II. Binding of [3H]1,25-(OH)2D3 in fibroblast cytosol was normal with a Bmax (amount of high affinity binding) of 26 fmol/mg protein and a half-maximal saturation of 0.2 nM. Nuclear binding of [3H]1,25-(OH)2D3 following whole cell uptake was 1.5 fmol/micrograms DNA in patient fibroblasts compared with a range of 0.5-2.9 fmol/micrograms DNA in five control strains. The size of the [3H]1,25-(OH)2D3-receptor complex on sucrose density gradients, 3.8 S, was the same as in normal cells. This patient, therefore, appeared to have a receptor-positive form of resistance to 1,25-(OH)2D3. To document resistance to 1,25-(OH)2D3 in the fibroblasts we developed a method for detection of 1,25-(OH)2D3 action in normal skin fibroblasts. Following treatment of normal cell monolayers with 1,25-(OH)2D3 there was more than a 20-fold increase of 25-hydroxy-vitamin D-24-hydroxylase (24-hydroxylase) activity. Treatment of 10 control cell strains with 1,25-(OH)2D3 for 8 h increased the formation of 24,25-dihydroxy-vitamin D3 from 25-hydroxyvitamin D3 in cell sonicates from less than 0.02 to 0.11-0.27 pmol/min per mg protein. When cells from the patient with vitamin D-dependent rickets, type II were treated with 1,25-(OH)2D3 in a similar manner, maximal 24-hydroxylase activity was only 0.02 pmol/min per mg protein, less than a fifth the lower limit of normal. 24-Hydroxylase activity in fibroblasts from the parents of the patient increased normally following treatment with 1,25-(OH)2D3. We conclude that impaired induction of 24-hydroxylase in the presence of normal receptor binding is evidence for postreceptor resistance to the action of 1,25-(OH)2D3.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism*; D000328:Adult; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007015:Hypophosphatemia, Familial / Q000235:genetics / Q000378:metabolism*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000032:analysis*; D013250:Steroid Hydroxylases / Q000378:metabolism*
D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol