Role of growth arrest and DNA damage-inducible α in Akt phosphorylation and ubiquitination after mechanical stress-induced vascular injury

Sumegha Mitra, Saad Sammani, Ting Wang, David L. Boone, Nuala J. Meyer, Steven M. Dudek, Liliana Moreno-Vinasco, Joe G N Garcia, Jeffrey R. Jacobson

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 methyl-specific polymerase chain reaction assay. Measurements and Main 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.

Original languageEnglish (US)
Pages (from-to)1030-1040
Number of pages11
JournalAmerican Journal of Respiratory and Critical Care Medicine
Volume184
Issue number9
DOIs
StatePublished - Nov 1 2011
Externally publishedYes

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Ventilator-Induced Lung Injury
Mechanical Stress
Ubiquitination
Vascular System Injuries
DNA Damage
Phosphorylation
Ubiquitin
Hydrolases
Endothelial Cells
Growth
Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
Polyubiquitin
Ubiquitin-Protein Ligases
Capillary Permeability
Lung Injury
Bronchoalveolar Lavage
DNA Methylation
Transgenes
Immunoprecipitation
Knockout Mice

Keywords

  • AKT
  • GADD45a
  • Mechanical stress
  • Ubiquitin
  • UCHL1

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Critical Care and Intensive Care Medicine

Cite this

Role of growth arrest and DNA damage-inducible α in Akt phosphorylation and ubiquitination after mechanical stress-induced vascular injury. / Mitra, Sumegha; Sammani, Saad; Wang, Ting; Boone, David L.; Meyer, Nuala J.; Dudek, Steven M.; Moreno-Vinasco, Liliana; Garcia, Joe G N; Jacobson, Jeffrey R.

In: American Journal of Respiratory and Critical Care Medicine, Vol. 184, No. 9, 01.11.2011, p. 1030-1040.

Research output: Contribution to journalArticle

Mitra, Sumegha ; Sammani, Saad ; Wang, Ting ; Boone, David L. ; Meyer, Nuala J. ; Dudek, Steven M. ; Moreno-Vinasco, Liliana ; Garcia, Joe G N ; Jacobson, Jeffrey R. / Role of growth arrest and DNA damage-inducible α in Akt phosphorylation and ubiquitination after mechanical stress-induced vascular injury. In: American Journal of Respiratory and Critical Care Medicine. 2011 ; Vol. 184, No. 9. pp. 1030-1040.
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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 methyl-specific polymerase chain reaction assay. Measurements and Main 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.",
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AU - Sammani, Saad

AU - Wang, Ting

AU - Boone, David L.

AU - Meyer, Nuala J.

AU - Dudek, Steven M.

AU - Moreno-Vinasco, Liliana

AU - Garcia, Joe G N

AU - Jacobson, Jeffrey R.

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N2 - 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 methyl-specific polymerase chain reaction assay. Measurements and Main 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.

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