Involvement of transforming growth factor-β1 signaling in hypoxia-induced tolerance to glucose starvation

Because survival and growth of human hepatoma cells are maintained by nutrient, especially glucose, glucose starvation induces acute cell death. The cell death is markedly suppressed by hypoxia, and we have reported involvement of AMP-activated protein kinase-α (AMPK-α), Akt, and ARK5 in hypoxia-induced tolerance. In the current study we investigated the mechanism of hypoxia-induced tolerance in human hepatoma cell line HepG2. ARK5 expression was induced in HepG2 cells when they were subjected to glucose starvation, and we found that glucose starvation transiently induced Akt and AMPK-α phosphorylation and that hypoxia prolonged phosphorylation of both protein kinases. We also found that hypoxia-induced tolerance was partially abrogated by blocking the Akt/ARK5 system or by suppressing AMPK-α expression and that suppression of both completely abolished the tolerance, suggesting that AMPK-α activation signaling and the Akt/ARK5 system play independent essential roles in hypoxia-induced tolerance. By using chemical compounds that specifically inhibit kinase activity of type I-transforming growth factor-β (TGF-β) receptor, we showed an involvement of TGF-β in hypoxia-induced tolerance. TGF-β1 mRNA expression was induced by hypoxia in an hypoxia-inducible factor-1α-independent manner, and addition of recombinant TGF-β suppressed cell death during glucose starvation even under normoxic condition. AMPK-α, Akt, and ARK5 were activated by TGF-β1, and Akt and AMPK-α phosphorylation, which was prolonged by hypoxia, was suppressed by an inhibitor of type I TGF-β receptor. Based on these findings, we propose that hypoxia-induced tumor cell tolerance to glucose starvation is caused by hypoxia-induced TGF-β1 through AMPK-α activation and the Akt/ARK5 system.