Demonstration and characterization of 1,25-dihydroxyvitamin D3 receptors in basal cells of epidermis of neonatal and adult mice

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Abstract
Nuclear and cytosolic receptors for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were demonstrated in the epidermis of neonatal and adult mice. The macromolecular binding protein sedimented at 3.5 S (sucrose density gradient) and was distinct from the 6.0 S binding protein for 25-hydroxycholecalciferol [25(OH)D3]. Analysis at different ionic strengths suggested the presence of unoccupied nuclear receptors. Digestion with proteases or nucleases, respectively, and inactivation with alkylating agents demonstrated that the binding macromolecule is a protein with SH groups at the active site. Binding of 1,25(OH)2D3 was specific and reversible. In neonatal mice KD was 1.6 × 10−10 M for both cytosolic and nuclear fractions, binding capacity was 54 fmol/mg protein in the cytosolic and 108 in the nuclear fractions, respectively. The phenotypic expression of the 1,25(OH)2D3 receptor (dissociation constant, binding capacity) was identical in neonatal and adult epidermis. Half maximal displacement of 1,25(OH)2D3 was achieved with an 80-fold and 200-fold molar excess of 25(OH)D3 and 1-alpha-hydroxycholecalciferol [1(OH)D3], respectively. Using Percoll density gradient centrifugation, 1,25(OH)2D3 receptors could be localized in the basal cell fraction. DNA cellulose chromatography with 1.25(OH)2D3 receptor elution from DNA at 0.25 M KCl (linear gradient) points to a possible role in gene transcription. In mouse primary epidermal cell cultures, 1,25(OH)2D3, but not 25(OH)D3, 24,25(OH)2D3, and 1(OH)D3 influenced [3H]thymidine incorporation (at physiological concentrations); the magnitude of change depending on the concentration of 1,25(OH)2D3 and the time of incubation. These data demonstrate that skin is a target organ for the active vitamin D secosterol.

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