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. 2010 Sep;17(9):1474-85. doi: 10.1038/cdd.2010.26. Epub 2010 Mar 12.

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.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Cell Hypoxia / physiology
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Female
  • Isoenzymes / metabolism
  • JNK Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • JNK Mitogen-Activated Protein Kinases / metabolism
  • Kidney / cytology
  • Kidney / metabolism*
  • Kidney Tubules / cytology
  • Kidney Tubules / metabolism
  • MAP Kinase Kinase Kinase 5 / metabolism
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Myeloid Differentiation Factor 88 / genetics
  • Myeloid Differentiation Factor 88 / metabolism
  • NADPH Oxidase 4
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Protein Binding / physiology
  • Protein Kinase Inhibitors / pharmacology
  • RNA, Small Interfering / genetics
  • Reactive Oxygen Species / metabolism
  • Reperfusion Injury / genetics
  • Reperfusion Injury / metabolism*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • TNF Receptor-Associated Factor 2 / genetics
  • TNF Receptor-Associated Factor 2 / metabolism
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • bcl-2-Associated X Protein / metabolism
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Bax protein, mouse
  • Isoenzymes
  • Membrane Glycoproteins
  • Myd88 protein, mouse
  • Myeloid Differentiation Factor 88
  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • Reactive Oxygen Species
  • TNF Receptor-Associated Factor 2
  • Toll-Like Receptor 4
  • bcl-2-Associated X Protein
  • endoplasmin
  • NADPH Oxidase 4
  • NADPH Oxidases
  • Nox4 protein, mouse
  • JNK Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase Kinase 5
  • Map3k5 protein, mouse