Glucocorticoids play an important role in the therapy of arthritic diseases. We sought, firstly, to identify, characterize and localize glucocorticoid receptors (GR) in normal human chondrocytes and, secondly, to determine whether glucocorticoid suppression of human recombinant interleukin-1 beta (rhIL-1 beta)-stimulated metalloproteases (MPs) synthesis by chondrocytes requires GR occupancy. Radioligand binding studies with cultured chondrocytes revealed the presence of high affinity-low capacity [3H]dexamethasone (DEX) binding sites with the following kinetic parameters: Kd = 12.5 +/- 1.4 nmol/L, Nmax = 57,560 +/- 3,960 sites per cell. Competition studies indicated that the DEX binding site was glucocorticoid specific and the competitive hierarchy established was: DEX greater than RU-26988 greater than RU-486 greater than cortisol greater than progesterone much greater than testosterone greater than estradiol-17 beta. Immunocytochemical studies using a specific anti-human GR antiserum identified immunoreactive material primarily in the cytoplasm with cells cultured in the absence of glucocorticoids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western immunoblotting analysis of chondrocyte cytosol detected the presence of a macromolecular species comigrating with a standard protein possessing a molecular weight of 94 kilodalton. rhIL-1 beta provoked the synthesis and secretion of the MPs stromelysin and collagenase from human chondrocytes in a saturable, coordinate, and dose-dependent fashion. DEX and cortisol inhibited the cytokine-stimulated MP synthesis in similar dose-dependent fashions: DEX, IC50 for stromelysin and collagenase suppression was 1.12 X 10(-8) mol/L and 1.26 X 10(-9) mol/L, respectively and the IC50 for cortisol was 6.3 X 10(-7) mol/L and 4.9 X 10(-8) mol/L, respectively. rhIL-1 beta failed to stimulate metalloprotease synthesis and release from chondrocytes pretreated with 10 nmol/L DEX, even after 20 days of incubation. The antiglucocorticoid, RU-486 completely reversed the DEX induced suppression of MP synthesis at 10(-7) mol/L. RU-486 alone had no effect on MP synthesis. We believe there is a biochemical rationale for the therapeutic efficacy of glucocorticoid administration in the management of arthritic diseases such as osteoarthritis and rheumatoid arthritis, and cytokines such as IL-1 are likely to be involved in the increase in MP synthesis.

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SUMMARY Incubation, with [4-14C]progesterone, of adrenocortical tissue (posterior cardinal vein preparations) from the eel yielded 14C-labelled cortisol and cortisone. These two hormones, containing both 14C and 3H, appeared when similar preparations were incubated simultaneously with [4-14C]progesterone and [16-3H]pregnenolone. In addition, the following intermediaries were isolated: [14C, 3H]progesterone; [14C, 3H]17α-hydroxyprogesterone; [3H]17α-hydroxypregnenolone and [14C, 3H]21-deoxycortisol. Analysis of the isotope content of the end products and isolated intermediaries seemed to indicate that the transformation of pregnenolone to corticosteroids proceeded to a large extent through intermediaries other than progesterone. In none of these experiments could the formation of aldosterone be demonstrated. Similarly, incubation of eel adrenocortical tissue with a mixture of [4-14C]-progesterone and [1,2-3H]corticosterone failed to yield detectable aldosterone. A further search for aldosterone, using a large amount of eel adrenocortical tissue with [4-14C]progesterone and [16-3H]pregnenolone as substrates with added angiotensin, also gave negative results. In similar preparations, [1-14C]sodium acetate was not transformed to any recognizable corticosteroids.