Expressions of hypoxic stress sensor proteins after transient cerebral ischemia in mice

The role of hypoxia sensor proteins is important in responding and protecting cells against hypoxic/ischemic injury in brain. Seven in absentia homolog 1 (Siah1) regulates primarily the downstream sensor proteins factor inhibiting alpha subunit of hypoxia-inducible factor-1 (FIH) under normoxic conditions and prolyl hydroxylases domain 3 (PHD3) under hypoxic conditions. In the present study, we investigated the temporal and spatial changes of these hypoxia sensor proteins, Siah1, FIH, and PHD3, after 60 min of transient middle cerebral artery occlusion (tMCAO) up to 72 hr after reperfusion in ICR mice. Immunohistochemistry and Western blot analyses showed that Siah1 was quickly and strongly induced in neuronal cells of the ischemic penumbra, with a peak at 2 hr, and gradually returned toward the sham control (SC) level until 72 hr. In contrast, the expressions of FIH and PHD3 were strongly visualized in the SC brains, and significantly reduced in a time-dependent manner with reperfusion until 72 hr. In the ischemic core region, Siah1, FIH, and PHD3 showed a similar change of strong and progressive decrease until 72 hr. Double-immunofluorescence analyses showed a cytoplasmic localization of Siah1 and both cytoplasmic and nuclear localizations of FIH and PHD3 and that Siah1 plus FIH or PHD3 were well colocalized in same neuron at 2 hr after tMCAO. The present study suggests that hypoxia sensor proteins (Siah1, FIH, and PHD3) showed temporally and spatially different expressions after tMCAO, which could provide an effective neuroprotective reaction through their further downstream proteins after cerebral ischemia.