DNA-dependent protein kinase is involved in heat shock protein-mediated accumulation of hypoxia-inducible factor-1alpha in hypoxic preconditioned HepG2 cells

Hypoxic preconditioning may afford protection against subsequent lethal hypoxia. As hypoxic tolerance induces changes in the expression of genes involved in DNA damage and repair response pathways, we investigated whether DNA-dependent protein kinase (DNA-PK), one of the DNA double-strand break repair proteins, could be involved in hypoxic preconditioning-induced protective signaling cascades. We showed that induction of hypoxia-inducible factor-1alpha expression during hypoxic preconditioning by repeated hypoxic exposure was associated with increased mRNA and protein levels of DNA-PK catalytic subunit (DNA-PKcs) and Ku70/Ku80, the DNA-PK components, in human hepatoma HepG2 cells, followed by upregulation of Hsp70/Hsp90 and Bcl-2 and concurrent downregulation of Bax. Additionally, loss of DNA-PKcs led to attenuated expression of Hsp70/Hsp90, accelerated hypoxia-inducible factor-1alpha degradation, and increased susceptibility to hypoxia-induced cell death. We also found that the mRNA and protein levels of heat shock factor-1 (HSF1) were progressively increased with DNA-PK activation during hypoxic preconditioning, and inhibition of HSF1 function by KNK437 resulted in a significant decrease in the level of protein kinase Akt as well as of DNA-PKcs, with downregulation of Hsp70/Hsp90 and HIF-1alpha. Our results suggest the possibility that DNA-PK-mediated signaling pathway is required for the increase in HIF-1alpha expression through activation of HSF1 and subsequent upregulation of heat shock proteins after hypoxic reconditioning.