NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition

Background Under conditions of hypoxia, cancer cells with hypoxia inducible factor-1 alpha (HIF-1 alpha) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1 alpha under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited.Methods In this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown.Results Here we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1 alpha. We also demonstrated that NADH functions as a signal for conveyance of HIF-1 alpha degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1 alpha via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1 alpha is recogonized by VHL, which leads to degradation of HIF-1 alpha via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1 alpha was independent of proline hydroxylation.Conclusions Our findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1 alpha stability. These results also demonstrate the involvement of VHL in degradation of HIF-1 alpha through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1 alpha acetylation in hypoxia.

Automated summary

This study reveals that the high concentration of pyruvate in the culture medium can affect the stability of HIF-1α, and NADH serves as a signal for the degradation of HIF-1α under chronic hypoxia through the SIRT1 and VHL signaling pathway. This degradation leads to a reduction in hypoxia-enhanced invasion and angiogenic activities. Additionally, the study demonstrates that VHL is involved in the degradation of HIF-1α through recognition of hydroxylation in normoxia and acetylation in hypoxia.