title: Ca(2+) /calmodulin- dependent protein kinase II mediates transforming growth factor-β-induced hepatic stellate cells proliferation but not in collagen α1(I) production.
abstract: OBJECTIVE
  Hepatic stellate cells (HSC) are the major players in hepatic fibrosis. As a most potent mitogen, transforming growth factor-β (TGF-β) strongly activates HSC and increases intracellular Ca(2+)  concentration. Here, we assessed the potential role of Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), a main downstream effector of the Ca(2+)  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/CaMKIIα 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 α1(I) expression was detected by immunocytochemistry. The role of CaMKII on TGF-β/Smad-induced collagen α1(I) expression was determined by (CAGA)(12) -MLP luciferase activity assay.
  
  
  RESULTS
  TGF-β dramatically increased CaMKII mRNA, and total and phosphorylated CaMKII expression. KN-93 and CaMKIIα siRNA suppressed TGF-β-mediated HSC proliferation. CaMKII interruption blocked TGF-β-elicited Akt activation. LY294002 arrested HSC proliferation and collagen α1(I) production but had no effect on CaMKII. Furthermore, CaMKII led to increased p21 and p27 expression. KN-93 and CaMKIIα siRNA inhibited TGF-β-induced and basal collagen α1(I) production but had no effect on the activity of (CAGA)(12) -MLP luciferase in response to TGF-β stimulation.
  
  
  CONCLUSIONS
  CaMKII is a pivotal signal in TGF-β-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.
authors.affiliation: Division of Gastroenterology, Renmin Hospital of Wuhan University             Department of Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, China.
authors.forename: Ping
authors.initials: P
authors.lastname: An
authors.forename: Yihao
authors.initials: Y
authors.lastname: Tian
authors.forename: Mingkai
authors.initials: M
authors.lastname: Chen
authors.forename: Hesheng
authors.initials: H
authors.lastname: Luo