ABSTRACT
Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation.
Subject(s)
14-3-3 Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , 14-3-3 Proteins/antagonists & inhibitors , 3-Phosphoinositide-Dependent Protein Kinases/antagonists & inhibitors , 3-Phosphoinositide-Dependent Protein Kinases/metabolism , Animals , Apoptosis/drug effects , Autophagy/drug effects , Benzamides/pharmacology , Cell Line , Colitis/chemically induced , Colitis/metabolism , Colitis/pathology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Interferon-gamma/pharmacology , Intestinal Mucosa/cytology , Male , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrazoles/pharmacology , Signal Transduction/drug effects , Threonine/metabolismABSTRACT
Neutrophil transepithelial migration (TEM) during acute inflammation is associated with mucosal injury. Using models of acute mucosal injury in vitro and in vivo, we describe a new mechanism by which neutrophils infiltrating the intestinal mucosa disrupt epithelial homeostasis. We report that junctional adhesion molecule-like protein (JAML) is cleaved from neutrophil surface by zinc metalloproteases during TEM. Neutrophil-derived soluble JAML binds to the epithelial tight junction protein coxsackie-adenovirus receptor (CAR) resulting in compromised barrier and inhibition of wound repair, through decreased epithelial proliferation. The deleterious effects of JAML on barrier and wound repair are reversed with an anti-JAML monoclonal antibody that inhibits JAML-CAR binding. JAML released from transmigrating neutrophils across inflamed epithelia may thus promote recruitment of leukocytes and aid in clearance of invading microorganisms. However, sustained release of JAML under pathologic conditions associated with persistence of large numbers of infiltrated neutrophils would compromise intestinal barrier and inhibit mucosal healing. Thus, targeting JAML-CAR interactions may improve mucosal healing responses under conditions of dysregulated neutrophil recruitment.
