The Src signaling pathway regulates osteoclast lysosomal enzyme secretion and is rapidly modulated by estrogen

To investigate the role of the pp60src signaling pathway in osteoclast activity, we have used dominant negative pp60src, c-ras, and c-raf expression vectors to individually disrupt their functions in osteoclasts. Osteoclasts were transiently transfected and secretions of cathepsin B/K and tartrate-resistant acid phosphatase (TRAP) were monitored. Expression of these constructs increased secretion of lysosomal enzymes. In contrast, constitutively active pp60src expression caused decreased lysosomal enzyme secretion. Osteoclasts respond to 17-aβ estradiol (171βE₂) treatment with decreased lysosomal enzyme secretion. Therefore, we investigated the effects of E₂ on pp60src kinase activity and observed an E₂ time- and dose-dependent decrease in cytoskeletal membrane-associated pp60src tyrosine kinase activity. We have shown that estrogen decreases lysosomal enzyme gene expression and secretion; so we have examined the effects of the expression constructs on estrogen regulation of enzyme secretion. Constitutively active pp60src blocked E₂ effects on secretion whereas expression of dominant negative pp60src, c-Ras, or c-Raf enhanced E₂ effects. These data support that the kinase domain of cytoskeletal-associated pp60src is likely to be involved in the regulation of lysosomal enzyme secretion.