title: Role of growth arrest and DNA damage-inducible α in Akt phosphorylation and ubiquitination after mechanical stress-induced vascular injury.
abstract: BACKGROUND
  The stress-induced growth arrest and DNA damage-inducible a (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a(-/-)) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown.
  
  
  OBJECTIVE
  We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress.
  
  
  METHODS
  VILI-challenged GADD45a(-/-) mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methylspecific polymerase chain reaction assay.
  
  
  RESULTS
  Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI challenged GADD45a(-/-) mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a silenced endothelial cells and GADD45a(-/-) mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1(UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a significantly reduces UCHL1 expression via UCHL1 promoter methylation resulting in increased Akt K48 ubiquitination and reduced Akt levels.
  
  
  CONCLUSIONS
  These studies highlight a novel role for GADD45a in the regulation of site-specific Akt ubiquitination and activation and implicate a significant functional role for GADD45a in the clinical predisposition to VILI.
authors.affiliation: Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago, 60612, USA.
authors.forename: Sumegha
authors.initials: S
authors.lastname: Mitra
authors.forename: Saad
authors.initials: S
authors.lastname: Sammani
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.forename: David L
authors.initials: DL
authors.lastname: Boone
authors.forename: Nuala J
authors.initials: NJ
authors.lastname: Meyer
authors.forename: Steven M
authors.initials: SM
authors.lastname: Dudek
authors.forename: Liliana
authors.initials: L
authors.lastname: Moreno-Vinasco
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Jacobson
mesh_terms: D000818:Animals; D018893:Bronchoalveolar Lavage; D002452:Cell Count; D018797:Cell Cycle Proteins / Q000235:genetics*; D004249:DNA Damage; D019175:DNA Methylation; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000473:pathology; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics*; D010766:Phosphorylation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D011651:Pulmonary Artery; D015398:Signal Transduction / Q000235:genetics; D054875:Ubiquitination / Q000235:genetics; D055397:Ventilator-Induced Lung Injury / Q000235:genetics* / Q000503:physiopathology; D000097642:GADD45 Proteins
chemical_list: D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D011506:Proteins; D051057:Proto-Oncogene Proteins c-akt; C499072:Gadd45a protein, mouse; D000097642:GADD45 Proteins