Lysophosphatidic acid increases intracellular H2O2 by phospholipase D and RhoA in rat-2 fibroblasts

We have investigated the possible roles of phospholipase D (PLD) and RhoA i n the production of intracellular H2O2 and actin polymerization in response to lysophosphatidic acid (LPA) in Rat-2 fibroblasts. LPA increased intrace llular H2O2, with a maximal increase at 30 min, which was blocked by the ca talase from Aspergillus niger, The LPA-stimulated production of H2O2 was in hibited by 1-butanol or PKC-downregulation, but not by 2-butanol, Purified phosphatidic acid (PA) also increased intracellular H2O2 and the increase w as inhibited by the catalase, The role of RhoA was studied by the scrape-lo ading of C3 transferase into the cells. The C3 toxin, which inhibited stres s fiber formation stimulated by LPA, blocked the H2O2 production in respons e to LPA or PA, but had no inhibitory effect on the activation of PLD by LP A, Exogenous H2O2 increased F-actin content by stress fiber formation. In a ddition, catalase inhibited actin polymerization activated by LPA, PA, or H 2O2, indicated the role of H2O2 in actin polymerization, These results sugg est that LPA increased intracellular H2O2 by the activation of PLD and RhoA , and that intracellular H2O2 was required for the LPA-stimulated stress fi ber formation.