Roles of RhoA and phospholipase A(2) in the elevation of intracellular H2O2 by transforming growth factor-beta in Swiss 3T3 fibroblasts

We have investigated the mechanisms by which transforming growth factor-bet a(TGF-beta) increased intracellular H2O2 in Swiss 3T3 fibroblasts. Increase of intracellular H2O2 by TGF-beta was maximal at 30 min and blocked by cat alase from Aspergillus niger. Scrape-loading of C3 transferase, which down- regulated RhoA, inhibited the production of H2O2 in response to TGF-beta. T GF-beta stimulated release of arachidonic acid, which was completely inhibi ted by mepacrine, a phospholipase Az inhibitor. Mepacrine also blocked the increase of H2O2 by TGF-beta. In addition, arachidonic acid increased intra cellular H2O2. Furthermore, TGF-beta stimulated stress fibre formation, whi ch was blocked by catalase, without membrane ruffling. Catalase also inhibi ted stimulation of thymidine incorporation by TGF-beta. These results sugge sted that TGF-beta increased intracellular H2O2 through RhoA and phospholip ase A(2), and also suggested that intracellular H2O2 was required for the s timulation of stress fibre formation and DNA synthesis in response to TGF-b eta. (C) 1999 Elsevier Science Inc.