title: Proteolytic Cleavage of AMPKα and Intracellular MMP9 Expression Are Both Required for TLR4-Mediated mTORC1 Activation and HIF-1α Expression in Leukocytes.
abstract: LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPKα is cleaved and its relevance to human health. Our data indicate that AMPKα cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1α expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.
authors.affiliation: Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903;
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903; and.
authors.forename: Louis F
authors.initials: LF
authors.lastname: Amorosa
authors.affiliation: Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903;
authors.forename: Susette M
authors.initials: SM
authors.lastname: Coyle
authors.affiliation: Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903;
authors.forename: Marie A
authors.initials: MA
authors.lastname: Macor
authors.affiliation: Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903; and.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Lubitz
authors.affiliation: Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903; and.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Carson
authors.affiliation: Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
authors.forename: Morris J
authors.initials: MJ
authors.lastname: Birnbaum
authors.affiliation: Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903;
authors.forename: Leonard Y
authors.initials: LY
authors.lastname: Lee
authors.affiliation: Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08903; haimovic@rwjms.rutgers.edu.
authors.forename: Beatrice
authors.identifier: http://orcid.org/0000-0001-9726-8254
authors.initials: B
authors.lastname: Haimovich
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000368:Aged; D000818:Animals; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000378:metabolism; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D015151:Immunoblotting; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008070:Lipopolysaccharides; D046912:Multiprotein Complexes; D051197:Toll-Like Receptor 4; D000076222:Mechanistic Target of Rapamycin Complex 1; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9