Role of growth arrest and DNA damage-inducible α in Akt phosphorylation and ubiquitination after mechanical stress-induced vascular injury.
Am J Respir Crit Care Med
; 184(9): 1030-40, 2011 Nov 01.
Article
en En
| MEDLINE
| ID: mdl-21816939
ABSTRACT
RATIONALE 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. OBJECTIVES:
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. MEASUREMENTS AND MAINRESULTS:
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 receptorassociated 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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Nucleares
/
Proteínas de Ciclo Celular
/
Células Endoteliales
/
Proteínas Proto-Oncogénicas c-akt
/
Lesión Pulmonar Inducida por Ventilación Mecánica
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Am J Respir Crit Care Med
Asunto de la revista:
TERAPIA INTENSIVA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Estados Unidos