F, an effective agent to increase bone formation in humans, at micromolar doses stimulates HBC proliferation in vitro. The mitogenic action of F is closely associated with increases in steady state tyrosyl phosphorylalion (PY) level of cellular proteins in HBCs. Because the ras-Raf-MAPK pathway is the major PY signal transduction pathway, we tested the hypothesis that F acts through this signal transduction pathway to stimulate HBC proliferation. Activation of MAPK & Raf-1 was assessed by increases in the PY level of the corresponding protein, as the activation of these two signaling proteins are closely associated with their PY level. Activation of these proteins was subsequently confirmed by the respective in vitro kinase assays- Because ras is inactivated by the hydrolysis of the bound GTP which is catalyzed by rasGAP, & because PY of rasGAP inactivates its activity, increased PY level of rasGAP would lead to ras activation. Accordingly, the ras activation was assessed by the increase in PY level of rasGAP. Steady state PY level of MAPK, Raf-1, & rasGAP was determined by immunoprecipitation with an PY antibody, followed by Western analysis using anli-MAPK, anli-Raf, & anti-rasGAP antibodies, respectively, and ECL. We found that l h treatment with mitogenic doses of F significantly & reproducibly increased PY of D441 (2-3-fold, p<.001), Raf-1 (5-10-fold, p<.001), rasGAP (5-15-fold, p< 001) in a dose-dependent, biphasic manner with the optimal dose of 50-100 uM F for each, which was similar to that of the HBC mitogenic effect of F Time course studies revealed that the F-mediated increases in PY level of MAPK, Raf-1, & rasGAP was gradual & sustained with maximal increases (10-15 fold for p44p, 12-fold for Raf-1, & 12-fold for rasGAP, p<.001 for each) occurring after 1-3 h of F treatment. The increases were sustained for as long as 12 h. Because we found that vanadate, a PTP inhibitor, also caused sustained increases in PY of these proteins whereas the effect of EOF, a tyrosyl kinase activator, on PY in HBC was acute (within 10 min) & transient (dissipated within 1 h), these findings arc consistent with our hypothesis that the HBC mitogenic action of F is mediated by an inhibition of PTP activity. In conclusion, these findings provide strong circumstantial evidence that F stimulâtes HBC proliferation through a sustained activation of the ras-Raf-MAPK pathway through an inhibition of PTP activity in HBCs.
|出版狀態||Published - 1996 十二月 1|
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