Upon liver injury, hepatic stellate cells (HSC) show increased proliferation, motility, and extracellular matrix (ECM) production. The extracellular signal-regulated kinases (ERK) control different functions in a cell-specific manner. In this study, we evaluated the role of ERK activation in cultured HSC stimulated with platelet-derived growth factor (PDGF) and after induction of liver injury in vivo. HSC were isolated from normal human liver tissue, cultured on plastic, and used in their myofibroblast-like phenotype. In in vivo experiments, HSC were isolated from normal rats or at different time points after a single intragastric administration of CCl 4 . Nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, reduced PDGF-induced activation of ERK in a dosedependent fashion. Suppression of ERK activation was associated with complete inhibition of HSC proliferation and with a 57% reduction in chemotaxis. In the presence of the ERK inhibitor, binding of the AP-1 complex and of STAT1 to the related regulatory elements was inhibited. The inhibition of the DNA binding activity of STAT1 was mediated by a reduction in PDGF-induced tyrosine phosphorylation. Expression of c-fos in response to PDGF was also reduced, but not suppressed, by treatment with PD98059. In HSC isolated from CCl 4 -treated rats, ERK activity increased as early as 6 hours following liver damage, and declined thereafter. The results of this study indicate that ERK activation regulates proliferation and chemotaxis of HSC, and modulates nuclear signaling. Acute liver damage in vivo leads to activation of ERK in HSC. (HEPATOLOGY 1999;30:951-958.)
Hepatic stellate cells (HSC) play a key role in the hepatic wound-healing process. Following acute or chronic liver injury, HSC undergo a process of activation toward a phenotype characterized by increased proliferation, motility, contractility, and synthesis of extracellular matrix (ECM) components. 1 In case of chronic liver damage, HSC activation persists, and progressive accumulation of ECM eventually leads to liver fibrosis, and finally to cirrhosis. The increase in ECM synthesis is sustained by increased secretion of ECM components per cell as well as by an increase in the number of HSC. In addition, in vitro and in vivo data indicate that migration of HSC toward the areas of tissue remodeling may be an additional factor contributing to wound healing. [2][3][4] Therefore, cell proliferation and migration should be regarded as relevant actions for the process of tissue healing and fibrosis.
The extracellular signal-regulated protein kinases (ERK) belong to a group of protein-serine/threonine kinases known as mitogen-activated protein kinases (MAPK). 5 This group of molecules may be activated in virtually all cell types in response to different stimuli, including growth factors, neurotransmitters, differentiating agents, or heat shock. 6 The MAPK signaling pathway is a multistep phosphorylation cascade that transmits signals from the cell' s surface to cytosolic and nuclear targets. 5 In cells exposed to plateletderived growth factor (PDGF), this cascade is initiated by receptor binding, dimerization, and auto-(cross-) phosphorylation on tyrosine residues. 7 Phosphotyrosines serve as docking sites for adapter molecules such as Grb2, Shc, or SHPTP-2 (Syp), which physically interact with the activated receptor through src-homology-2 domains. 7 These interactions result in the recruitment of mSos, a guanine nucleotide exchange factor, and the consequent activation of the small GTP-binding protein, Ras. Active Ras interacts with and activates Raf-1, and in turn Raf-1 activates MEK, a dual specificity kinase, which phosphorylates ERK on threonine and tyrosine residues. 8 Phosphorylation of ERK results in activation of its kinase activity and is associated with translocation to the nucleus. 9 The downstream targets of ERK include cytosolic phospholipase A 2 , the kinases p90 rsk and MAPKAPK2, and a number of transcription factors, such as Elk-1. 5 The control of multiple cellular functions by the ERK cascade occurs in a cell-specific fashion. In vascular smooth muscle cells, ERK may activate both proliferative and growthinhibitory pathways, 10 and in hepatocytes, acute versus chronic activation of ERK results in stimulation or inhibition of DNA synthesis. 11 Furthermore, the role of ERK in the activation of different nuclear signaling pathways has not Abbreviations: HSC, hepatic stellate cells; ECM, extracellular matrix; ERK, extracellular signal-regulated kinases; MAPK, mitogen-activated protein kinase; PDGF, plateletderived growth factor; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.