ACTIVATION OF ERK5, A MEMBER OF THE MAPK FAMILY, DIFFERENTIALLY REGULATES PROLIFERATION AND MIGRATION OF HEPATIC STELLATE CELLS

Background and Aims: ERK5 (also known as big MAPK1-BMK1) a member of the mitogen-activated protein kinase (MAPK) family, is a 98-kDa molecule sharing homology with ERKI/2. ERK5 is activated by a kinase cascade involving the upstream kinases MEK5, MEKK2/3 and Tpl-2. ERK5 plays a role as a redox-sensitive protein kinase, and may be activated in response to growth factor stimulation. Activation of signaling pathways in hepatic stellate cells (HSC) by different soluble mediators plays a critical role in the pathogenesis of fibrosis. Nevertheless, no information is currently available on the possible role of ERK5 in this setting. Aim of this study was to investigate the role of ERK5 in the biologic responses triggered by platelet-derived growth factor (PDGF) in HSC. Methods: Activation ofintracellular signalling pathways was studied using western blot analysis. HSC proliferation and migration was evaluated using tritiated thymidine incorporation and modified Boyden chambers, respectively. Gene silencing with ERK5-specific siRNA or control siRNA was performed using Amaxa | nucleofactor technology. Results: Exposure to PDGF resulted in a rapid induction of ERK5 phosphorylation in activation-specific residues. Induction of ERK5 activation was also observed in response to other soluble mediators, including epidermal growth factor, that signals through a receptor tyrosine kinase, and IP-10, which acts via heterotrimeric G-protein coupled receptors. Upon exposure to PDGF, ERK5 showed a clear and transient translocation from the cytosol to the nucleus, as indicated by immunofluorescence and cell fractionation. To assess the contribution of pathways downstream of the PDGF receptor, activation of ERK was measured in the presence of different pharmacologic inhibitors. While inhibition of phosphatidyl/ inositol 3-1dnase activation did not have any effects on ERK5 activation, several molecules interfering with Src activation inhibited PDGFdependent ERK5 phosphorylation. To establish the biologic significance of ERK5 activation, HSC were incubated with siRNA targeting ERK5 or control siRNA. This strategy resulted in a 50 60% downregulation of ERK5 expression. In these conditions, PDGF-induced cell proliferation was significantly inhibited, while cell migration was increased. Conclusions: Src-dependent activation of ERK5 modulates PDGFdependent biologic activities in human HSC. While ERK5 generates positive signals for cell proliferation, it downregulates the ability of the cells to migrate.