DIFFERENTIAL-EFFECTS OF 2 PROTEIN-TYROSINE KINASE INHIBITORS, TYRPHOSTIN AND GENISTEIN, ON HUMAN BONE CELL-PROLIFERATION AS COMPARED WITH DIFFERENTIATION

Protein tyrosyl phosphorylation is a key determinant of cell prolifera tion and differentiation. The aim of this study was to test the hypoth esis that the signal transduction pathway(s) responsible for human bon e cell proliferation may involve different groups of protein tyrosine kinase (PTKs) as compared with that for differentiation. To achieve th is, we investigated the effects of two structurally different PTK inhi bitors viz, tyrphostin A51 and genistein, on the proliferation ([H-3]t hymidine incorporation) and differentiation [alkaline phosphatase (ALP ) specific activity and collagen synthesis] of two normal human bone c ell types: mandible-derived and vertebra-derived bone cells. Tyrphosti n A51 and genistein each markedly reduced cellular tyrosyl phosphoryla tion level (assessed by Western analysis using a commercial anti-phosp hotyrosine antibody and the enhanced chemiluminescence detection assay ), confirming that these two effecters are potent PTK inhibitors in hu man bone cells. Regarding bone cell proliferation, tyrphostin A51 (5-3 0 mu M) caused, a dose-dependent inhibition of basal [3H]thymidine inc orporation of both human bone cell types. In contrast, genistein (5-20 mu M), not only did not inhibit, but significantly stimulated [H-3]th ymidine incorporation of these same cell types in a dose-dependent, bi phasic manner, with the optimal stimulatory dose between 10 and 20 mu M. These effects on cell proliferation were confirmed by cell number c ounting. In addition, whereas the mitogenic activity of 10 ng/ml epide rmal growth factor (EGF) on human mandible-derived bone cells was comp letely abolished by 5-30 mu M tyrphostin A51, genistein at 5-30 mu M e nhanced the EGF-induced bone cell proliferation in an additive manner. With respect to bone cell differentiation, tyrphostin A51 and geniste in each significantly increased basal ALP specific activity and collag en synthesis in human bone cells. In summary, (1) PTKs are involved in human bone cell proliferation and differentiation; (2) tyrphostin A51 inhibited both basal and EGF-induced cell proliferation, thus tyrphos tin-sensitive PTKs are involved in basal and EGF-induced human bone ce ll proliferation; (3) genistein stimulated basal proliferation and enh anced EGF-mediated cell proliferation, suggesting that genistein-sensi tive PTKs may play an inhibitory role in human bone cell proliferation ; and (4) these differential effects of PTK inhibitors on human bone c ell proliferation and differentiation are independent of basal differe ntiation status of the cells.