AMPK activation stabilizes HIF-2α by disrupting VHL binding and promotes erythroid differentiation under anemic stress via the mTOR pathway

Anemia affects over two billion people globally, and ESA resistance poses major clinical hurdles. HIF-2α governs erythropoiesis, yet how AMPK/mTOR energy sensors regulate HIF-2α in erythroid cells remains unclear. This study explored whether AMPK activation stabilizes HIF-2α via impaired VHL binding to boost erythroid differentiation under anemic stress through mTOR signaling.

UT-7/EPO cells were cultured under 1% O₂ with low EPO to mimic anemic stress, treated with AMPK/mTOR pharmacological modulators. CHX chase and Co-IP detected HIF-2α stability and VHL interaction; Ser672 phosphorylation, erythroid and iron-related genes, hepcidin and intracellular iron were measured.

Anemic stress suppressed cell viability, stabilized HIF-2α, activated AMPK and inhibited mTOR, upregulated erythroid markers and triggered apoptosis. AICAR extended HIF-2α half-life, disrupted HIF-2α-VHL binding and elevated Ser672 phosphorylation, while mTOR activator MHY1485 reversed these effects. AMPK activation elevated EPOR, DMT1, FPN1, lowered hepcidin and ferritin, and restored intracellular iron; these effects vanished upon HIF-2α knockdown or mTOR overactivation.

AMPK stabilizes HIF-2α by blocking VHL binding to facilitate erythroid maturation and iron homeostasis under anemic stress via mTOR. The AMPK/mTOR/HIF-2α cascade controls stress erythropoiesis, and targeting this axis may treat ESA-insensitive anemia.