Heat Shock Protein gp96 Interacts with Protein Phosphatase 5 and Controls Toll-like Receptor 2 (TLR2)-mediated Activation of Extracellular Signal-regulated Kinase (ERK) 1/2 in Post-hypoxic Kidney Cells

Abstract

Ischemia/reperfusion injury (IRI) induces an innate immune response, leading to an inflammatory reaction and tissue damage that have been attributed to engagement of the Toll-like receptor (TLR) 2 and 4. However, the respective roles of TLR2 and/or TLR4 in mediating downstream activation of mitogenactivated protein kinase (MAPK) pathways during IRI have not been fully elucidated. Here we show that extracellular signalregulated kinase (ERK)1/2 is activated in both intact kidneys and cultured renal tubule epithelial cells (RTECs) from wildtype and Tlr4 knockout mice, but not those from Tlr2 knockout mice subjected to transient ischemia. Geldanamycin (GA), an inhibitor of heat shock protein 90 and reticulum endoplasmicresident gp96, and gp96 mRNA silencing (siRNA), did not affect ERK1/2 activation in either post-hypoxic wild-type or Tlr4-deficient RTECs, but did restore its activation in post-hypoxic Tlr2-deficient RTECs. Immunoprecipitation studies revealed that gp96 co-immunoprecipitates with the serine-threonine protein phosphatase 5 (PP5), identified as a negative modulator of the mitogen extracellular kinase (MEK)-ERK pathway, in unstressed wild-type and post-hypoxic Tlr2-deficient RTECs. In contrast, PP5 co-immunoprecipitation with gp96 was strikingly reduced in post-hypoxic wild-type RTECs, suggesting that the inactivation of PP5 resulting from the dissociation of PP5 from gp96 allows the activation of ERK1/2 to occur. Inhibition of PP5 by okadaic acid, and Pp5 siRNA also restored TLR2-mediated phosphorylation of ERK1/2, and apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK)-mediated apoptosis in post-hypoxic Tlr2-deficient RTECs. These findings indicate that gp96 interacts with PP5 and controls TLR2-mediated induction of ERK1/2 in post-hypoxic renal tubule cells.

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

“…The proapoptotic Bcl-2 homologue BAX, but not the anti-apoptotic Bcl-2, 25 also significantly increased in wild-type, but not in Tlr4 À/À postischemic kidneys ( Figure 1a). These findings indicate that TLR4 mediates predominant activation of p-ASK1 and p-JNK, two key signal events responsible for apoptosis during IRI, 13 in postischemic kidneys.…”

“…7,13 The expression levels of TRAF2 over b-actin, p-ASK1, p-p38, and p-JNK, and BAX, but not of Bcl-2, were also greater in posthypoxic than in untreated wild-type RTECs (Figure 1b). In contrast, the amounts of p-ASK1, p-p38, p-JNK, and BAX remained barely detectable in posthypoxic MyD88 À/À and remained Figure S1a and b).…”

“…Renal IRI caused by bilateral clamping of the renal pedicles from wild-type mice induces marked renal dysfunction and tubule cell damages, which peaked during the first 2 days after the reperfusion of the kidneys. 9,13 The fact that renal dysfunction and tubule cells apoptosis were significantly less marked in Tlr4 À/À mice than in wild-type kidneys 9,13 led us to analyze the activation of MAPK pathways in postischemic, reperfused wild-type and Tlr4 À/À kidneys. IRI increased the expression level of TRAF2, and phosphorylated (p-) ASK1 and p-JNK, and to a lesser extent p-p38, in day-1 and day-2 postischemic wild-type kidneys, but not in postischemic Tlr4 À/À kidneys ( Figure 1a).…”

“…12 We have shown that gp96 interacts with the protein phosphatase 5 (PP5) to control selective TLR2-mediated activation of the mitogen-activated protein kinase (MAPK) ERK 1/2 , but not of JNK. 13 The question therefore arises as to whether gp96, shown to interact with TLR4 in intestinal epithelial cells, 14 is required for TLR4-activated apoptosis during renal IRI.…”

Protein phosphatase 5 modulates SMAD3 function in the transforming growth factor‐β pathway

“…It could be that other SMAD2/3 interacting partners could be dephosphorylated by PP5. Thirdly, PP5 has been implicated in the regulation of multiple pathways, including adipogenesis by controlling the GRα and PPARγ phosphorylation (34), DNA-damage repair by controlling the ATM/ATR/Chk1 and p53 pathway components (21,31,34-37), MAPK-mediated growth and differentiation (38)(39)(40)(41)(42)(43)(44), and cell cycle arrest (45). Interestingly, GRα has also been implicated in the repression of the TGFβ pathway by its association with SMAD3 (46).…”