Calpain Mediates a von Hippel-Lindau Protein–independent Destruction of Hypoxia-inducible Factor-1α

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Abstract
Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1α requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1α under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1α expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2′-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1α protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1α degradation. Furthermore, chelating intracellular calcium attenuated HIF-1α destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1α even under normoxia. An active role of calpain in lowering HIF-1α amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1α that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1α destruction involving the calpain system.