The increased incidence and severity of arthritis in women suggest that female sex steroids have an effect on articular integrity. The potential mechanisms by which sex hormones might contribute to chronic joint disease are unknown. A major component of articular degeneration in both osteoarthritis and inflammatory joint disease involves the activity of extracellular matrix proteases. These proteases have been detected in synovial fluid and their expression appears to increase in inflammatory and degenerative joint disease. Further, a variety of inflammatory cytokines, also found in synovial fluid in chronic arthritis or degenerative disease, stimulate production of certain proteases by synovial cells. The effects of gonadal steroids on joint proteolytic activity have not been determined. Previously, the Principal Investigator has observed that estradiol enhances endothelial cell, steady state expression of mRNA and production of protein for certain of these proteases, including gelatinase A and the plasminogen activators urokinase and tissue-type plasminogen activator. At the same time, mRNA for most matrix protease inhibitors remains stable or decreases. This could alter the net protease activity present in the fluid surrounding these cells so that increased connective tissue degradation occurs. Here, we propose to use a comprehensive approach to define the activity of matrix proteases and their inhibitors by cultured human synovial fibroblasts. Our hypothesis is that estradiol will stimulate production of matrix proteases at the level of mRNA and protein, and that net protease activity in the culture supernates will increase as well. To test this hypothesis, three specific aims are proposed. First, we will examine the effect of 17-beta estradiol on cultured human synovial cells, evaluating expression of mRNA, protein and activity for matrix metalloproteinases and plasminogen activators, and for their inhibitors, TIMPs and PAIs, respectively. The net activity resulting from alterations in the balance between proteases and inhibitors will be tested by determining the ability of culture supernatants to release radioactivity or chromogen in solid-phase assays. Second, we will test the effect of inflammatory cytokines on protease production and activity in synovial cell cultures. Estradiol will be added to cultures in the presence and absences of cytokines to determine whether, as is the case with endothelial cells, estradiol enhances cytokine stimulation. Finally, in assays that are demonstrated to be estrogen-sensitive, we will use various estrogen receptor antagonists to determine whether the observed effects are mediated by estrogen receptor binding. The results of these studies will provide data indicating whether a direct link between estrogen and protease activity is likely to contribute to articular degeneration; and they may indicate approaches to therapeutic intervention in chronic joint disease.
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