Bone resorption, mediated by osteoclasts, is essential for bone growth and repair. Past c-src knockout mice studies have indicated that the protein tyrosine kinase (PTK) activity of c-src is essential for mature osteoclastic activity. The c-src PTK activity is regulated predominately by the phosphorylation status of a C-terminal tyr-527 residue: phosphorylation inactivates, and dephosphorylation activates, c-src PTK activity. The phosphorylation is catalyzed by a cytosolic PTK, Csk, but the identity of the phosphotyrosine phosphatase (PTP) activity that mediates the dephosphorylation is unknown. We have recently cloned a structurally unique PTP, termed PTP-oc, from rabbit osteoclast cDNA library. It is expressed preferentially in osteoclasts and precursors. We have a large body of preliminary data to support that PTP-oc may dephosphorylate c-src and may regulate c-src PTK activity in osteoclasts. These findings led us to formulate a working model, in which PTP-oc acts to dephosphorylate tyr-527 of c-src in osteoclasts, resulting in activation of its PTK activity, which in turn leads to activation of osteoclast activity and resorption. We further speculate that some bone resorption activators may stimulate osteoclastic resorption through an increase in the expression and/or the activity of PTP-oc in osteoclasts; whereas some inhibitors may reduce osteoclastic resorption by reducing the PTP-oc expression and/or activity. This application will test three specific hypotheses: 1) suppression of PTP- oc expression in osteoclasts increases c-src tyr-527 phosphorylation, reduces c-src PTK activity, decreases their resorptive activity, and impairs actions of some resorption activators on their c-src PTK and resorptive activities; 2) overexpression of PTP-oc in osteoclasts reduces tyr-527 phosphorylation of c-src, stimulates c-src PTK activity, increases their resorptive activity, and reduces actions of some resorption inhibibitors on their c-src PTK and resorptive activities; 3) overexpression of an inactive PTP-oc mutant in osteoclasts reduces c-src tyr-527 dephosphorylation, leading to a reduction in c-src PTK activity and a decrease in their osteoclast activity; and it also suppresses the actions of some resorption activators on their c-src PTK and resorptive activities. The first hypothesis will be tested in primary rabbit osteoclasts by using specific PTP-oc antisense oligodeoxynucleotides to suppress PTP-oc expression. The other two hypotheses will be evaluated by stably transfecting either a wild type or a """"""""phosphatase-dead"""""""" PTP-oc inducible vector in a human U937/ osteoclast cell model. The stable clones will be induced to overexpress either the wild-type or mutant PTP-oc for evaluation. If our hypotheses are correct, this work would provide insights into a) molecular mechanism of osteoclastic resorption, b) regulation of osteoclast activity, and c) the role of PTP-oc in bone resorption. PTP-oc may also be used as a screening target to identify new agents that alter bone resorption, and as such this study may identify a potential therapeutic target for development of more specific and safer modulators of bone resorption.
