Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of the lipid-derived second messenger phosphatidic acid (PA) in mediating this effect and, in particular, to determine its interaction with the extracellular signal-regulated kinase (ERK) cascade. HSCs were isolated from rat livers. PA production was determined by lipid extraction and thin-layer chromatography (TLC) after prelabeling cells with [ 3 H]myristate. ERK activity was measured by an in vitro kinase assay after immunoprecipitation. Mitogenic concentrations of PDGF, but not those of the relatively less potent mitogen, transforming growth factor ␣ (TGF-␣), stimulated the sustained production of PA from HSCs. Exogenous PA stimulated HSC proliferation and a sustained increase in ERK activity, and proliferation was completely blocked by the inhibition of ERK activation with PD98059. The stimulation of ERK by PDGF was of a similar magnitude but more sustained than that caused by TGF-␣. These results suggest that the potent mitogenic effect of PDGF in HSCs may be caused, in part, by the generation of PA and subsequently by a more sustained activation of ERK than occurs with less potent mitogens that do not induce the production of this lipid second messenger. (HEPATOLOGY 2000;31:95-100.)Hepatic stellate cells (HSCs) are regarded as the principal cell type responsible for the development and progression of liver fibrosis. 1 After liver injury, they transform from quiescent cells into myofibroblast-like cells that proliferate in response to platelet-derived growth factor (PDGF), epidermal growth factor, transforming growth factor ␣ (TGF-␣), and basic fibroblast growth factor. As the most potent mitogen, studies aimed at understanding the signaling mechanisms involved in HSC mitogenesis have focused largely on PDGF. The PDGF receptor consists of 2 subunits with intrinsic tyrosine kinase activity. After ligand binding, the subunits autophosphorylate several tyrosine residues, 2 which then act as binding sites for molecules containing src homology 2 domains. 3 These include the adaptor protein Grb2, which recruits the Ras-activating protein, Sos, and triggers a kinase cascade that results in the sequential activation of Raf-1, mitogen-activated protein kinase kinase and the extracellular signal-regulated kinases, ERK1 and ERK2. 4 Recently, studies in HSCs 5 and many other cell types 6 have established an important role for this mitogen-activated protein kinase cascade in the proliferative response.A second protein recruited to the autophosphorylated PDGF receptor by virtue of its src homology 2 domain is phospholipase C ␥ (PLC-␥). 7,8 Once at the plasma membrane, PLC-␥ initiates a distinct signaling cascade beginning with the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol (DAG). DAG stimulates one or more members of a family of kinases known collectively as protein kinase C, which are established as key ...