SARAF represses the mild hypothermia response through the regulation of JUN

Abstract

The mild hypothermia response (MHR) is a conserved mammalian cytoprotective program activated upon exposure to mild hypothermia (32 degrees C) that contributes to the neuroprotective effects of therapeutic hypothermia following hypoxic injury. Although rapid changes in intracellular calcium occur upon cooling, the mechanisms linking calcium dynamics to the activation of core MHR factors such as SP1 and RBM3 remain incompletely defined. In this study, we used siRNA-mediated knockdown (KD) of candidate regulators in conjunction with novel mild hypothermia indicator (MHI) reporters to identify upstream modulators of MHR-associated transcription. We identify SARAF, a negative regulator of store-operated calcium entry (SOCE), as a repressor of both SP1 and RBM3 under normothermic conditions. SARAF depletion is associated with increased intracellular calcium release and enhanced SP1- and RBM3-linked transcriptional outputs. We identify JUN as an important downstream factor mediating SARAF depletion-dependent de-repression of the MHR and demonstrate that it undergoes activation rapidly upon cooling. Finally, SARAF depletion conferred significant cytoprotection against hypoxia-induced early apoptosis. Collectively, these findings establish SARAF as an upstream regulator of MHR-associated transcription and provide a functional link between cold-induced intracellular calcium dynamics and the induction of core MHR effectors.