Concentration-dependent stimulation by tissue inhibitor of metalloproteinases(TIMP)-2 of two signaling pathways in human osteosarcoma (MG-63) Cells

We reported previously that tyrosine kinase (TYK) and mitogen-activated protein kinase (MAPK) play a role in TIMP-dependent growth signaling. Here we demonstrate that the activation of MAPK stimulated by TIMP-2 was sharply inhibited in MG-63 cells by PP1, a selective inhibitor of Src-family tyrosine kinases, suggesting that Src activation is possibly required for the MAPK activity in response to 1 ng/mL TIMP-2. A specific cell-permeable inhibitor of MAPK kinase (MEK), PD 98058 had an inhibitory effect on MAPK activity, indicating that MEK is an upstream effector of MAPK. By transfecting MG-63 cells with dominant-negative Ras, RasN17, and furthermore, by using a selective farnesyltransferase inhibitor, FTI-277, we found that MAPK activation stimulated by TIMP-2 was independent of Ras. We also demonstrated that the activation of MAPK in response to 1 ng/mL TIMP-2 was inhibited by a cAMP agonist, 8-bromo-cAMP. On the contrary, [3H]thymidine incorporation and the activation of MAPK, all of which were heavily suppressed by increasing the TIMP-2 concentration up to 100 ng/mL, were recovered by the addition of a cell-permeable specific PKA inhibitor, H-89. These results strongly suggest the presence of a negative crosstalk from the cAMP/PKA pathway to the TYK/MAPK one.