Heat shock protein gp96 and NAD(P)H oxidase 4 play key roles in Toll-like receptor 4-activated apoptosis during renal ischemia/reperfusion injury.
Ischemia/reperfusion injury (IRI) causes inflammation and cell injury as a result of activating innate immune signaling. Toll-like receptor 4 (TLR4) has a key role in mediating kidney damages during IRI, but the downstream signaling pathway(s) stimulating apoptosis remains debated. In this study we show that TLR4 mediates MyD88-dependent activation of TNF receptor-associated factor 2, apoptosis signal-regulating kinase 1 (ASK1), and Jun N-terminal kinase (JNK) and p38 MAP kinases in ischemic-reperfused kidneys and posthypoxic renal tubule epithelial cells (RTECs). Hypoxia stimulated the expression of the endoplasmic-resident gp96, which co-immunoprecipitated TLR4, whereas silencing gp96 mRNA expression impaired hypoxia-induced apoptosis in TLR4-expressing RTECs. NAD(P)H oxidase 4 (NOX4) was shown to interact with TLR4 and to be required in lipopolysaccharide-induced production of reactive oxygen species (ROS). IRI stimulated the expression of a 28-kDa NOX4 spliced isoform abundantly expressed in wild-type RTECs, which co-immunoprecipitated with TLR4, but not with gp96 in TLR4-deficient RTECs. Silencing NOX4 mRNA expression impaired hypoxia-induced activation of ASK1 and both JNK and p38, leading to the inhibition of ROS production and apoptosis in posthypoxic TLR4-expressing RTECs. These findings show that, concomitantly to the activation of p38, the gp96/TLR4 interaction is required for activation of ASK1/JNK signaling in posthypoxic mouse RTECs, and that the 28-kDa NOX4 has a key role in TLR4-mediated apoptosis during renal IRI.
Ben Mkaddem
S
S
INSERM, Centre de Recherche Biomédicale Bichat-Beaujon CRB3, Université Paris, France.
Pedruzzi
E
E
Werts
C
C
Coant
N
N
Bens
M
M
Cluzeaud
F
F
Goujon
J M
JM
Ogier-Denis
E
E
Vandewalle
A
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D007527:Isoenzymes / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C494827:Bax protein, mouse; D007527:Isoenzymes; D008562:Membrane Glycoproteins; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D047992:TNF Receptor-Associated Factor 2; D051197:Toll-Like Receptor 4; D051028:bcl-2-Associated X Protein; C053387:endoplasmin; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482859:Map3k5 protein, mouse