Cell growth-promoting activity of TIMPs - Mechanism of signal transduction in human osteoblast-like cells

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We reported earlier that human TIMP-1 and TIMP-2 have potent growth-promoting activity for a wide range of cells (Hayakawa et al. 1992 FEBS Lett 298 : 29-32 & 1994 J Cell Sci 107 : 2373-2379). The cell growth-promoting activity of both TIMP-1 and -2 is suggested to be a direct cellular effect mediated by cell surface receptors. It has also been demonstrated that the binding of TIMP-2 to the cell surface is not competed by TIMP-1, suggesting that both TIMPs have their own specific receptors.In this project, we tried to elucidate the intracellular signal transduction pathways in human osteosarcoma cell line MG-63 cells induced by both TIMP-1 and TIMP-2 as cell growth factors.We first examined the time course of TIMP-dependent [ィイD13ィエD1H]TdR incorporation by MG-63 cells. [ィイD13ィエD1H]TdR incorporation was significantly stimulated at 3 hours after the addition of either TIMP-1 or TIMP-2, and the incorporation remained linear up to 15-20 hours. The effects of TIMPs on the [ィイD13ィエD1H]TdR in … More corporation were dose dependent, showing maximum stimulation at the concentration of either 20 ng/ml TIMP-1 or 1.0 ng/ml TIMP-2. The extent of the stimulation with either TIMP was comparable to that obtained with 10% FCS. We also demonstrated that [ィイD13ィエD1H]TdR incorporation into MG-63 cells by 10% FCS was almost fully dependent on TIMPs in FCS.To get a clue as to the nature of the intracellular signal transduction, next we examined the effects of some protein kinase inhibitors on [ィイD13ィエD1H]TdR incorporation into the cells. Genistein, erbstain and herbimycin A almost completely inhibited the incorporation of [ィイD13ィエD1H]TdR stimulated by either TIMP. However, essentially no effect was observed with H-89, H-7, bisindolymaleimide or K-252a. These inhibition studies thus suggest the possible participation of tyrosine kinase in the signal transduction of TIMPs. To confirm this possibility, we examined the tyrosine-specific phosphorylation stimulated by either TIMP. Tyrosine-specific phosphrylation in MG-63 cells was suppressed by serum depletion and was significantly increased in response to the addition of either TIMP. Next we found that both TIMPs significantly stimulated the activation of one of the MAP kinases, ERK2, and the activity of this kinase in MG-63 cells suggesting the involvement of MAP kinases in the downstream signal transduction pathway.When we increased TIMP-1 and TIMP-2 concentrations up to 500 and 100 ng/ml, respectively, [3H]TdR incorporation was almost completely blocked in either case. Under such conditions, however, all three PKA inhibitions increased [3H]TdR incorporation up to the level obtained in the presence of a low concentration of either TIMP. These results reminded us of the negative crosstalk between cAMP/PKA and Ras/Raf-1 demonstrated by several research groups. By using Raji cells, which express neither TIMP-1 nor TIMP-2, we demonstrated the presence of both high (Kd=0.15 nM) - and low (35 nm) - affinity binding sites on the cell surface. The presence of negative crosstalk explains well why both TIMP-1 and -2 show typical bell-shaped dose response curves when examined for their effect on the proliferation of cells.In conclusion, we, for the first time, have demonstrated that either TIMP-1 or TIMP-2 at the low concentration expressing growth factor activity induces tyrosine phosphorylation, activated MAPKs, and stimulates DNA synthesis in the absence of other exogenous growth factors. At the high concentration, either TIMP seems to bind to a so-called G0-protein-coupled receptor and stimulate cAMP/PKA system which finally inhibits the cell growth-stimulating signal through negative crosstalk to PTK/MAPK system via Ras/Raf-1. Less