ESTROGEN MODULATION OF AVIAN OSTEOCLAST LYSOSOMAL GENE-EXPRESSION

We have previously demonstrated that avian osteoclasts contain high levels of 17beta-estradiol (17betaE2) receptors and respond to 17betaE2 treatment with a dose-dependent decrease in in vitro resorption of [H-3] proline-labeled bone particles. To more accurately assess the influence of 17betaE2 on osteoclastic activity, the specificity of estrogen modulation of resorption levels was determined using a quantitative pit resorption assay. Treatment with 17betaE2 significantly decreased the number of osteoclast resorption pits formed compared with that after either vehicle or 17alphaE2 treatment. Cotreatment with 17betaE2 and hydroxytamoxifen (a complete 17betaE2 antagonist in birds) abrogated the influence of 17betaE2 on resorption activity. To elucidate the mechanism by which 17betaE2 inhibits osteoclast activity, the effects of 17betaE2 on the steady state mRNA levels of two avian osteoclast lysosomal proteins, lysozyme and a lysosomal membrane protein (LEP-100), were examined. Using highly purified avian osteoclasts, 17betaE2 was shown to decrease lysosomal protein mRNA levels in a dose-dependent manner within 8 h of treatment in a process that required de novo protein synthesis. This response was specific for 17betaE2, since the inactive stereoisomer 17alphaE2 had no effect. Furthermore, coincubation of 17betaE2 with hydroxytamoxifen eliminated the 17betaE2 influence. After removal of 10(-8) m 17betaE2, lysosomal gene mRNA levels returned to near-normal levels within 24 h. This is consistent with the previously reported ability of avian osteoclast-mediated resorption activity to recover from 17betaE2 treatment. Lysozyme protein levels similarly decreased after 17betaE2 treatment. These data suggest that avian osteoclasts are target cells for 17betaE2 in vitro, that osteoclast activity in vivo is likely to be modulated by circulating levels of 17betaE2, and that the 17betaE2 inhibition of osteoclast resorption activity may be mediated at least in part via regulation of osteoclast lysosomal gene expression.