Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor beta (TGF-beta) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-beta on CTGF expression. We found that myoblasts treated with LPA and TGF-beta 1 produced an additive effect on CTGF expression. In the absence of TGF-beta, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-beta receptor type II (TGF-beta RII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-beta RI, suggesting that CTGF induction is dependent on LPA and requires active TGF-beta Rs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-beta RI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-beta RI-mediated activity. These novel results for the mechanism of action of LPA and TGF-beta are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases. (C) 2010 Elsevier Inc. All rights reserved.