杉本 大樹

Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in cellular and tissue adaptation to low oxygen conditions. Although inflammatory stimuli such as lipopolysaccharide (LPS) also increase HIF-1α levels under normoxia, its transcriptional activity and regulatory mechanisms in this context remain unclear. To address this, we performed chromatin immunoprecipitation sequencing and transcriptome analyses in murine macrophages stimulated with either LPS or hypoxia. Both stimuli stabilized HIF-1α protein but via distinct mechanisms: hypoxia acted post-translationally, whereas LPS increased Hif-1α mRNA expression. Genome-wide HIF-1α binding was observed under both conditions; however, only hypoxia induced broad transcriptional activation of target genes, whereas LPS upregulated a restricted set, mostly glycolytic genes. Motif enrichment analysis revealed that hypoxia, but not LPS, promoted cooperative transcription factor engagement, including HIF-1β, ETS, and bZIP family members. Hypoxia also increased H3K27 acetylation at HIF-1α target loci, consistent with a transcriptionally permissive chromatin state. In contrast, LPS led to reduced H3K27ac at noninduced loci, suggesting epigenetic repression. Mechanistically, HIF-1α exhibited a phosphorylation-dependent band shift under hypoxia but not LPS. Although both conditions showed comparable overall phosphorylation levels by Phos-tag analysis, only hypoxia triggered a conformational change, suggesting site-specific phosphorylation linked to transcriptional competence. These findings demonstrate that HIF-1α binding alone is insufficient for gene activation and that phosphorylation and chromatin context determine its transcriptional output in a stimulus-dependent manner.

BACKGROUND: Cortical spreading depression is thought to be the underlying mechanism of migraine aura. In 2006, three relatives having the point mutation E700K in ATP1A2 exon 15 were diagnosed with familial hemiplegic migraine 2 characterized by complicated forms of aura. Here, we generated a transgenic mouse model having the human E700K mutation in the Atp1a2 orthologous gene. OBJECTIVE: To investigate the characteristics of cortical spreading depression in a mouse model with E700K mutation in the Atp1a2. METHODS: Cortical spreading depression was induced by applying stepwise increases of KCl concentration or electrical stimulation intensity to C57BL/6J-Tg(Atp1a2*E700K)9151Kwk mice (Tg, both sexes) and corresponding wild-type animals. Under urethane anesthesia, the responsiveness and threshold to cortical spreading depression were examined and the distribution of c-Fos expression, a neuronal activity marker, was immunohistochemically determined. RESULTS: Overall, Tg mice showed significantly faster propagation velocity (p < 0.01) and longer full-width-at-half-maximum (p < 0.01) than wild-type animals, representing a slower recovery from direct current potential deflection. The cortical spreading depression threshold tended to be lower in Tg, especially in females. c-Fos-positive cells were significantly enhanced in the ipsilateral somatosensory cortex, piriform cortex, amygdala and striatum (each p < 0.05 vs. contralateral side). Numbers of c-Fos positive cells were significantly higher in the ipsilateral amygdala of Tg, as compared with wild-type animals (p < 0.01). CONCLUSION: The effect of cortical spreading depression may be greater in E700K transgenic mice than that in wild-type animals, while the threshold for cortical spreading depression shows little change. Higher c-Fos expression in the amygdala may indicate alterations of the limbic system in Tg, suggesting an enhanced linkage between cortical spreading depression and amygdala connectivity in familial hemiplegic migraine 2 patients.

The ATP1A2 coding α2 subunit of Na,K-ATPase, which is predominantly located in astrocytes, is a causative gene of familial hemiplegic migraine type 2 (FHM2). FHM2 model mice (Atp1a2tmCKwk/+ ) are susceptible to cortical spreading depression (CSD), which is profoundly related to migraine aura and headache. However, astrocytic properties during CSD have not been examined in FHM2 model mice. Using Atp1a2tmCKwk/+ crossed with transgenic mice expressing G-CaMP7 in cortical neurons and astrocytes (Atp1a2+/- ), we analyzed the changes in Ca2+ concentrations during CSD. The propagation speed of Ca2+ waves and the percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- were higher than those in wild-type mice. Increased percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- may contribute to FHM2 pathophysiology.