title: RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response.
abstract: BACKGROUND
  The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.
  
  
  METHODS
  Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT(558A) and pcDNA3/HA-moesinT(558D) were applied in endothelial cells.
  
  
  RESULTS
  The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.
  
  
  CONCLUSIONS
  These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.
authors.affiliation: Department of Pathophysiology, Key Laboratory for Shock and Microcirculation Research, Southern Medical University, Guangzhou, P R China.
authors.forename: Jiping
authors.initials: J
authors.lastname: Wang
authors.forename: Hongxia
authors.initials: H
authors.lastname: Liu
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Xuliang
authors.initials: X
authors.lastname: Huang
authors.forename: Liqun
authors.initials: L
authors.lastname: Wang
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Guo
authors.forename: Qiaobing
authors.initials: Q
authors.lastname: Huang
mesh_terms: D000199:Actins / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D055785:Gene Knockdown Techniques; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology; D006801:Humans; D047468:Immunoprecipitation; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010539:Permeability; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012709:Serum Albumin / Q000378:metabolism*; D000075462:Serum Albumin, Human; D022502:Stress Fibers / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D017127:Glycation End Products, Advanced; D008840:Microfilament Proteins; D047428:Protein Kinase Inhibitors; D012709:Serum Albumin; C109748:advanced glycation end products-human serum albumin; C066737:RHOA protein, human; C071058:moesin; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000075462:Serum Albumin, Human