Fluoride at Mitogenic Doses Induces a Sustained Activation of p44, but Not p42, in Human TE85 Osteosarcoma Cells*

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 mmol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44, but not p42, with the maximal increase (4to 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 was dose dependent, with the optimal dose being 100 mmol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44, but not that of p42, in a time-dependent manner similar to the effect on the p44 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 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, but not p42, 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. (J Clin Endocrinol Metab 82: 1126–1135, 1997) F IS an effective agent to increase spinal bone density in osteoporotic patients (1, 2). Bone morphometric studies showed that the osteogenic action of fluoride is mediated by an increase in the number of osteoblasts (3, 4). Farley and co-workers (5) have shown that fluoride in vitro stimulated the proliferation and differentiation of bone cells in monolayer cultures, indicating a direct anabolic action of fluoride on bone cells. The mitogenic effect of fluoride was subsequently confirmed by several laboratories in bone cells of various species, including humans (6–11). The optimal dose of fluoride to stimulate bone cell proliferation and differentiation in vitro is low, between 10–100 mmol/L (depending on the type of culture medium used) (5, 6), which corresponds to the serum fluoride concentration in fluoridetreated patients (12). The mitogenic action of fluoride appears to be bone cell specific (5, 6, 13). The molecular mechanism by which fluoride stimulates bone cell proliferation is unknown. Although fluoride at millimolar concentrations is a potent activator of adenylyl cyclase through stimulation of Gs, fluoride at the mitogenic, micromolar concentrations did not have an effect on cellular cAMP production in bone cells (13). Recent evidence suggests that the mitogenic action of fluoride could be mediated through a signal transduction pathway involving tyrosyl phosphorylation (13, 14). Our past work suggests that the osteogenic action of fluoride could be mediated at least in part by increasing the steady state tyrosyl phosphorylation of key signaling proteins through inhibition of an osteoblastic fluoride-sensitive phosphotyrosyl phosphatase (PTP) (13). Our hypothesis is supported by several pieces of circumstantial evidence: 1) fluoride at micromolar concentrations specifically inhibited an osteoblastic PTP, and the apparent Ki for fluoride inhibition was within the mitogenic fluoride dose range for bone cells (5, 6, 15); 2) fluoride treatment increased the net protein tyrosyl phosphorylation level in intact bone cells and isolated cell membrane (13); 3) other PTP inhibitors of this fluoride-sensitive PTP, i.e. vanadate and molybdate, also stimulated bone cell proliferation at doses that inhibited this fluoride-sensitive PTP activity (13, 16); 4) treatment with human TE85 osteosarcoma cells with mitogenic doses of fluoride for 24 h significantly increased the steady state tyrosyl phosphorylation level of at least 13 cellular proteins in human bone cells (17); and 5) the apparent molecular masses of some of these proteins were similar to Received October 23, 1996. Revision received December 27, 1996. Accepted January 7, 1997. 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@LLVAMC.VA. GOV. * This work was supported in part by research grants from the NIH (DE-08681 to K.-H.W.L.) and from the V.A. (to D.J.B.). 0021-972X/97/$03.00/0 Vol. 82, No. 4 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright © 1997 by The Endocrine Society