Tyrosyl Protein Phosphorylation in Human Bone Cells : Its Potential Role in Mediating Fluoride Induced Osteoblast Proliferation
This dissertation was designed to test the hypothesis that fluoride-stimulated osteoblast proliferation is mediated by specific inhibition of a phosphotyrosyl protein phosphatase, resulting in increased steady state levels of tyrosyl phosphorylation of key cellular proteins. Cellular levels of tyrosyl phosphorylation were determined by an immunoblot detection assay in which cellular proteins were separated by SDS-PAGE, transblotted to nitrocellulose membranes and the phosphotyrosyl proteins were identified by anti-phosphotyrosine monoclonal antibodies. The relative amount of tyrosyl phosphorylation was quantitated by laser densitometric analysis of the autoradiograms. This method showed that fluoride induced dose- and time-dependent changes in the level of tyrosyl phosphorylation of proteins with apparent M.W.’s of 244, 220, 170, 96, 62, 48, 34 and 32kD. These changes in tyrosyl phosphorylation appeared to be determining factors in mediating fluoride induced osteoblast proliferation based on the following: a) Some of the fluoride-dependent changes in tyrosyl phosphorylation appeared to exhibit dose-dependent relationships similar to fluoride induced bone cell mitogenesis (i.e. 220, 34 and 32kD), b) Several other bone cell mitogens stimulated tyrosyl phosphorylation of similar molecular weight proteins as fluoride, suggesting that these proteins are important mediators of bone cell proliferation (e.g. vanadate 244, 220, 96, 48, 34 and 32kD; IGF-I 220, 170 and 96kD; PDGF 244 and 220kD; EGF 244, 220, 170 and 96kD; IGF-II 220 and 96kD; electromagnetic field exposure 220 and 96kD), and c) Cells which did not respond to fluoride mitogenically were shown to have no significant fluoride-dependent changes in their levels of tyrosyl phosphorylation. With regards to the mechanism by which fluoride increased tyrosyl phosphorylation, the time course for fluoride-dependent tyrosyl phosphorylation was not compatible with tyrosyl kinase activation (i.e. several hours were required to observe fluoride-dependent tyrosyl phosphorylation while the tyrosyl kinase activating growth factors elicited immediate increases in steady state levels of tyrosyl phosphorylation). Taken together, these data are consistent with the hypothesis that fluoride induced osteoblast proliferation is mediated by an increase in the steady state level of tyrosyl phosphorylation via phosphotyrosyl protein phosphatase inhibition.
Biochemistry, Fluorides; Phosphorylation; Protein-Tyrosine-Phosphatase; Osteoblasts -- drug effects
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