Abstract
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.
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Abbreviations
- ASK1:
-
apoptosis signal-regulating kinase 1
- DAMP:
-
damage-associated molecular pattern
- DPI:
-
diphenyleneiodonium chloride
- HMGB1:
-
high-mobility group box 1
- Hsps:
-
heat shock proteins
- JNK:
-
Jun N-terminal kinase
- LPS:
-
lipopolysaccharide
- MAPK:
-
mitogen-activated protein kinase
- MyD88:
-
myeloid differentiation factor-88
- NOX:
-
NAD(P)H oxidase
- PP5:
-
protein phosphatase 5
- ROS:
-
reactive oxygen species
- RTEC:
-
renal tubule epithelial cell
- siRNA:
-
small-interfering RNA
- TRAF:
-
TNF receptor-associated factor
- UPR:
-
unfolded protein response
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Acknowledgements
We thank S Akira for the use of Tlr4−/− and MyD88−/− mice. We also thank KH Krause for stimulating discussions, and for providing us with the NOX1−/− mice. We thank MW Hornef (Institut of Medical Microbiology and Hygiene, Hannover Medical School, Hannover, Germany) for the gift of the TLR4 antibody. We also thank M Fay and G Pinon (INSERM U773, Paris, France), and B Fernandez and N Quellard (Service d’Anatomie et Cytologie Pathologiques, Hopital Universitaire de Poitiers, France) for their expert technical assistance. This work was funded by INSERM and in part by an Agence Nationale de la Recherche grant (ANR-08-MIEN-030, to AV). SBM was supported by a PhD student grant from the Ligue Nationale contre le Cancer, AV was in receipt of an Interface INSERM-AP-HP fellowship.
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Ben Mkaddem, S., Pedruzzi, E., Werts, C. et al. 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. Cell Death Differ 17, 1474–1485 (2010). https://doi.org/10.1038/cdd.2010.26
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DOI: https://doi.org/10.1038/cdd.2010.26
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