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Fluoride at Mitogenic Doses Induces a Sustained Activation of p44^mapk, but Not p42^mapk, in Human TE85 Osteosarcoma Cells¹

Departments of Medicine and Biochemistry, Loma Linda University, and the Mineral Metabolism Unit, Jerry L. Pettis Memorial Veterans Administration Medical Center, Loma Linda, California 92357; and the Department of Pharmacology, University of North Carolina (L.M.G.), Chapel Hill, North Carolina 27514

Address all correspondence and requests for reprints to: Dr. K.-H. William Lau, Mineral Metabolism (151), Jerry L. Pettis Memorial Veterans Administration Medical Center, 11201 Benton Street, Loma Linda, California 92357. E-mail: LAUB{at}LLVAMC.VA.GOV

Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.e. 100 µmol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44^mapk, but not p42^mapk, with the maximal increase (4- to 13-fold) after 1–3 h fluoride treatment. The effect was sustained in that a 9-fold increase was seen after 12 h of the fluoride treatment. The sustained nature of the effect is consistent with an inhibition of dephosphorylation rather than a direct stimulation of phosphorylation. The fluoride effect on the tyrosyl phosphorylation level of p44^mapk was dose dependent, with the optimal dose being 100 µmol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44^mapk, but not that of p42^mapk, in a time-dependent manner similar to the effect on the p44^mapk tyrosyl phosphorylation level. Fluoride at the same micromolar doses did not increase cell proliferation, tyrosyl phosphorylation, or specific activity of any MAPK in human skin foreskin fibroblasts, which are fluoride-nonresponsive cells. Consistent with the interpretation that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44^mapk is a consequence of an inhibition of a phosphotyrosyl phosphatase (PTP), mitogenic doses of orthovanadate, a bone cell mitogen and a PTP inhibitor, also increased the steady state tyrosyl phosphorylation level of p44^mapk, but not p42^mapk, in a time-dependent sustained manner similar to that observed with fluoride. Together, these findings support the concept that inhibition of a PTP activity in bone cells could lead to an activation of MAPK activity.

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