GSK3 beta-dependent inhibition of AMPK potentiates activation of neutrophils and macrophages and enhances severity of acute lung injury

Abstract

Although AMP-activated protein kinase (AMPK) is involved in regulating carbohydrate and lipid metabolism, activated AMPK also plays an antiinflammatory role in many cell populations. However, despite the ability of AMPK activation to diminish the severity of inflammatory responses, previous studies have found that AMPK activity is diminished in LPS-treated neutrophils and also in lungs of mice with LPS-induced acute lung injury (ALI). Since GSK3 beta participates in regulating AMPK activity, we examined potential roles for GSK3 beta in modulating LPS-induced activation of neutrophils and macrophages and in influencing severity of ALI. We found that GSK3 beta-dependent phosphorylation of T479-AMPK was associated with pT172 dephosphorylation and inactivation of AMPK following TLR4 engagement. GSK3 beta inhibitors BIO (6-bromoindirubin-3=-oxime), SB216763, or siRNA knockdown of GSK3 beta, but not the PI3K/AKT inhibitor LY294002, prevented Thr172-AMPK dephosphorylation. Exposure to LPS resulted in rapid binding between IKK beta and AMPK alpha, and phosphorylation of S485-AMPK by IKK beta. These results suggest that IKK beta-dependent phosphorylation of S485-AMPK was an essential step in subsequent phosphorylation and inactivation AMPK by GSK3 beta. Inhibition of GSK3 beta activity delayed I kappa B alpha degradation and diminished expression of the proinflammatory TNF-alpha in LPS-stimulated neutrophils and macrophages. In vivo, inhibition of GSK3 beta decreased the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNF-alpha and MIP-2, and release of the alarmins HMGB1 and histone 3 in the lungs. These results show that inhibition of AMPK by GSK3 beta plays an important contributory role in enhancing LPS-induced inflammatory responses, including worsening the severity of ALI.