Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)-2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP-2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide-producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP-2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose-dependent manner. The addition of antioxidants as well as MMP-2-specific inhibitors impaired these phenomena. X/XO treatment increased MMP-2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane-type I MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO-induced MMP-2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K) abrogated X/XO-elicited MMP-2 upregulation and completely prevented X/XO-induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP-2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS-mediated MMP-2 expression.