Ca2+/calmodulin- dependent protein kinase II mediates transforming growth factor-β-induced hepatic stellate cells proliferation but not in collagen α1(I) production

Aim: Hepatic stellate cells (HSC) are the major players in hepatic fibrosis. As a most potent mitogen, transforming growth factor-beta (TGF-beta) strongly activates HSC and increases intracellular Ca2+ concentration. Here, we assessed the potential role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a main downstream effector of the Ca2+ signal in liver fibrogenesis cascade. Methods: A human immortal HSC cell line, LX-2, and primary rat hepatic stellate cells were used in current study. CaMKII blockage and Akt inhibition were performed by KN-93/CaMKIIa siRNA and LY294002, respectively. HSC proliferation was detected by 5-bromodeoxyuridine incorporation assay. Real-time polymerase chain reaction, western blot and enzyme-linked immunosorbent assay were used to measure mRNA, cellular protein and protein in medium, respectively. Procollagen a1(I) expression was detected by immunocytochemistry. The role of CaMKII on TGF-beta/Smad-induced collagen a1(I) expression was determined by (CAGA)12-MLP luciferase activity assay. Results: TGF-beta dramatically increased CaMKII mRNA, and total and phosphorylated CaMKII expression. KN-93 and CaMKIIa siRNA suppressed TGF-beta-mediated HSC proliferation. CaMKII interruption blocked TGF-beta-elicited Akt activation. LY294002 arrested HSC proliferation and collagen a1(I) production but had no effect on CaMKII. Furthermore, CaMKII led to increased p21 and p27 expression. KN-93 and CaMKIIa siRNA inhibited TGF-beta-induced and basal collagen a1(I) production but had no effect on the activity of (CAGA)12-MLP luciferase in response to TGF-beta stimulation. Conclusion: CaMKII is a pivotal signal in TGF-beta-induced fibrogenic cascades by means of stimulating HSC proliferation, and involved in a basal collagen production. Therefore, CaMKII will be a potentially effective target in the development of therapeutic intervention strategies to attenuate hepatic fibrosis.