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.
- Lysosomal enzymes
- Tyrosine kinase
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Orthopedics and Sports Medicine