title: Integrin-linked kinase regulates endothelial cell nitric oxide synthase expression in hepatic sinusoidal endothelial cells.
abstract: OBJECTIVE
  Portal hypertension results from endothelial dysfunction after liver injury caused in part by abnormal production of endothelial cell derived nitric oxide synthase (eNOS). Here, we have postulated that endothelial mechanosensing pathways involving integrin-linked kinase (ILK) may play a critical role in portal hypertension, eNOS expression and function. In this study, we investigated the role of ILK and the small GTP-binding protein, Rho, in sinusoidal endothelial cell (SEC) eNOS regulation and function.
  
  
  METHODS
  Primary liver SECs were isolated using standard techniques. Liver injury was induced by performing bile duct ligation (BDL). To examine the expression of Rho and ILK in vivo during wound healing, SECs were infected with constitutively active Rho (V14), a dominant negative Rho (N19) and constructs encoding ILK and a short hairpin-inhibiting ILK.
  
  
  RESULTS
  Integrin-linked kinase expression was increased in SECs after liver injury; endothelin-1, vascular endothelial growth factor, and transforming growth factor beta-1 stimulated ILK expression in SECs. ILK expression in turn led to eNOS upregulation and to enhance eNOS phosphorylation and NO production. ILK knockdown or ILK (kinase) inhibition reduced eNOS mRNA expression, promoter activity, eNOS expression and ultimately NO production. In contrast, ILK overexpression had the opposite effect. Inhibition of ILK activity also disrupted the actin cytoskeleton in isolated SECs. Rho overexpression suppressed phosphorylation of the serine-threonine kinase, Akt and inhibited eNOS phosphorylation. Finally, inhibition of Rho function with the RGS domain of the p115-Rho-specific GEF (p115-RGS) significantly increased eNOS phosphorylation.
  
  
  CONCLUSIONS
  Our data suggest a potential role for ILK, the cytoskeleton and ILK signalling partners including Rho in regulating intrahepatic SEC eNOS expression and function.
authors.affiliation: Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Mahnoush S
authors.initials: MS
authors.lastname: Shafiei
authors.forename: Songling
authors.initials: S
authors.lastname: Lui
authors.forename: Don C
authors.initials: DC
authors.lastname: Rockey
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000201:enzymology* / Q000235:genetics; D002779:Cholestasis / Q000201:enzymology* / Q000235:genetics; D003599:Cytoskeleton / Q000201:enzymology; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver / Q000201:enzymology*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014162:Transfection; D014945:Wound Healing; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099200:Scaffold Protein ILK
chemical_list: D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020741:rho GTP-Binding Proteins; D000099200:Scaffold Protein ILK; C496316:Nos3 protein, rat
keywords: cirrhosis; liver; portal hypertension; rho; rho kinase; signalling