Nuclear uptake of 1,25-dihydroxy[3H]cholecalciferol in dispersed fibroblasts cultured from normal human skin.

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Abstract
Because of the relative inaccessiblity of known calciferol target tissues (i.e., intestine and bone), fibroblasts derived from normal human skin and grown in tissue culture were examined as a means of evaluating the interaction of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its effector system. When dispersed, intact cells were used, nuclear uptake of 1,25-dihydroxy[23,24(n)-3H[cholecalciferol [1,25(OH)2[3H]D3] was temperature-dependent, optimal at 45 min at 37.degree. C and saturable. In competition experiments with other calciferols, the 1,25(OH)2[3H]D3 uptake showed specificity indistinguishable from that reported for 1,25(OH)2D3 receptors from calciferol target tissues. Analysis of 1,25(OH)2[3H]D3 nuclear uptake in fibroblast strains from 6 normal adults (4 male, 2 female) yielded an average binding capacity of 10,600 .+-. 2000 (SEM [standard error of the mean] nuclear sites per cell and an apparent Kd of 0.50 .+-. 0.07 (SEM) .times. 10-9 M. Donor sex, donor age or anatomic site of origin of the cell line did not affect the characteristics of uptake. Similar nuclear uptake was demonstrable with cultured MCF-7 cells (derived from human breast cancer) when assayed in the same fashion. When hypertonic extracts of nuclei obtained from skin fibroblasts incubated with 1,25(OH)2[3H]D3 were subjected to centrifugation on sucrose gradients, a single peak of radioactivity sedimented at .apprxeq. 3 S; when excess 1,25 (OH)2D3 was coincubated during the cellular uptake phase, this 3S peak was not observed. Molybdate was an essential buffer component for receptor stabilization during cell fractionation and sedimentation analysis. By using fibroblasts culturated from normal human skin, a process of nuclear uptake of 1,25(OH)2[3H]D3 with the affinity, saturability and specificity characteristics of a steroid hormone-receptor interaction was identified. This method should be useful in studying 1,25(OH)2D3 receptor physiology in cells from normal persons and in cells from patients who have disorders in the responsiveness of calciferol target tissues.