Human skin fibroblasts were grown in minimal essential medium with Earle's salts and 15% fetal calf serum. Confluent monolayer subcultures were incubated at 37°C, for 30 min with tritiated glucocorticoid dissolved in medium without fetal calf serum. Free and bound labelled steroids in cell sonicates were separated by chromatography using Sephadex G-25 columns. The specifically bound [3H]-cortisol and [3H]-trimacinolone acetonide represented 75% of the total bound radioactivity. There was no significant displacement of [3H]-cortisol by progesterone, 17-hydroxyprogesterone, testosterone, dihydrotestosterone or estradiol-17β, but 5α-dihydrocortisol, cortisone, corticosterone and asdosterone produced partial displacement. Scatchard analysis of data showed KD and Bmax values of 0.4-8.6 M × 10-9 and 1.5-11.4 fmol/μg DNA for triamcinolone acetonide. Using exclusion chromatography on Sephadex G-150 and sucrose gradient, the molecular weight of the glucocorticoid-receptor complex was estimated to be 40,000. In a cell-free system the glucocorticoid-receptor in cytosol was translocated to nuclei while the glucocorticoid alone was not. Thus subcultured human skin fibroblasts contain a cytosol protein which specifically binds glucocorticoid and which appears to be similar to glucocorticoid receptors described in other cell systems.
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