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.