Protein kinase Cµ plays an essential role in hypertonicity-induced heat shock protein 70 expression

Abstract

Abbreviations: HSF1, heat shock transcription factor 1; HSP70, heat shock protein 70; TonEBP, the tonicity-responsive enhancer binding protein AbstractHeat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Gö6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKCμ, we then evaluated the role of PKCμ in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKCμ with siRNA or the inhibition of PKCμ activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKCμ evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKCμ performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification.

How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5?

“…at 2 μmol/L failed to inhibit high NaCl-induced increase of protein abundance of HSP70, a transcriptional target of NFAT5 [82,83] , in NIH3T3 cells [84] . This is probably due to that the concentration of H89 is too low.…”

“…PKD acts upstream of ERK1/2 under certain contexts [95] . However, it remains unclear whether PKD1 involves in PKCα-ERK1/2 signaling activation of NFAT5, since elimination of high NaCl-induced phosphorylation of ERK1/2 by PD98059 (20 micromol/L) does not reduce tonicitydependent increase of HSP70 protein abundance [84] . (3) The AKT protein kinase family comprises three highly related isoforms encoded by different genes.…”

“…PKD is highly mobile and functions as a "communicator"between different subcellular compartments [95] . General PKC inhibitors, Go6976 and GF109203X, and siRNA-mediated knockdown of PKD1 reduce high NaCl-induced increase of protein abundance of HSP70 [84] . The general inhibitors reduce high NaCl-induced NFAT5 mobility shift and have no significant effect on NFAT5 nuclear localization [84] .…”

“…General PKC inhibitors, Go6976 and GF109203X, and siRNA-mediated knockdown of PKD1 reduce high NaCl-induced increase of protein abundance of HSP70 [84] . The general inhibitors reduce high NaCl-induced NFAT5 mobility shift and have no significant effect on NFAT5 nuclear localization [84] . The latter effect is consistent with the lack of effect of PKCα and ERK1/2 on NFAT5 nuclear accumulation.…”

“…High NaCl increases PLCγ1 activity [24] , diacylglycerol and total PKC activity [90] . PKC inhibitors reduce NFAT5 transcriptional activity in mIMCD3 [91] and NIH3T3 cells [84] . We recently identified PKCα involved in regulation of NFAT5 activity [29] .…”