Bone formation is determined largely by osteoblast proliferation, a process which is initiated by multiple signal transduction pathways. There is now evidence that the phosphotyrosyl (P-tyr) phosphorylation of MAPK, (a key mitogenic signaling protein) is a common converging step for all mitogenic signal transduction pathways. There is also evidence that phosphotyrosyl protein phosphatases (PTPPs) are important regulators of cellular P-tyr phosphorylation and cell proliferation. This application deals with investigations of an osteoblastic fluoride(F)-sensitive PTPP, which we discovered during our pursuit of the molecular mechanism of action of F. Over the past several years we have accumulated evidence to support the concept that the effect of F to increase cell proliferation is mediated by inhibition of a PTPP. This evidence includes the findings that F, at concentrations of 12 cellular proteins in osteoblasts, 3 of which have Mr identical to that of MAPK, Raf and rasGAP. [Moreover, recent preliminary data shows that F increases P-tyr phosphorylation of MAPK, and rasGAP in time and dose-dependent manners]. Because P-tyr phosphorylation of these 3 signaling proteins (MAKP, Raf and rasGAP) is closely associated with cell proliferation, we have proposed a molecular model of F action which contains 4 hypotheses: l) inhibition of the F-sensitive PTPP leads to stimulation of bone cell proliferation; 2) stimulation of bone cell proliferation by inhibitors of the F-sensitive PTPP is mediated via in phosphorylation level of key cellular P-tyr signaling proteins; 3) stimulation of bone cell proliferation by F is mediated by inhibition of the F-sensitive PTPP which leads to increased P-tyr phosphorylation of MAPK; 4) F-induced increases in p-tyr phosphorylation of MAPK is mediated through increased P-tyr phosphorylation of Raf and/or rasGAP. These hypotheses will be tested by: a) determine whether inhibition of the expression of F-sensitive PTPP (by antisense oligo) would stimulate osteoblast proliferation and whether the overexpression of this enzyme in F-nonresponsive cells (e.g., skin fibroblasts) would convert these cells into F-responsive cells; and b) determining whether inhibition of this F- sensitive PTPP would increase the steady state P-tyr phosphorylation level of MAPK, Raf, and rasGAP and whether reduction of the level of these key signaling proteins (using specific antisense oligo against specific signaling protein) would inhibit osteoblast proliferation. To perform the molecular biology studies, we need to know the nucleotide sequence of the enzyme; and thus we will also clone the F-sensitive PTPP gene. The proposed work has significance in 2 areas: l) it advances our understanding of the molecular action of F, information which may help to optimally utilize this drug or any future new F-like drugs (i.e., PTPP inhibitors) to treat osteoporosis. Accordingly, this F sensitive enzyme and other PTPPs may have regulatory role in normal bone cell proliferation and could be subverted in certain bone wasting diseases; and 2) it supports the concept that PTPPs constitute a new level of regulation of cell proliferation of cell proliferation which may be applicable to all renewing cell populations.
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