Kelch-like 20 up-regulates the expression of hypoxia-inducible factor-2α through hypoxia- and von Hippel–Lindau tumor suppressor protein-independent regulatory mechanisms

► KLHL20 interacted with HIF-2α but not with HIF-1. ► KLHL20 up-regulated HIF-2α expression and HIF-2 activity. ► KLHL20 enhanced HIF-2 activity through a pVHL-independent pathway. Despite their structural similarity, hypoxia-inducible factor (HIF)-1α and HIF-2α have distinct functional properties and exhibit distinct spatiotemporal expression patterns, suggesting that the expressions of the two proteins are regulated by different mechanisms. To clarify the HIF-2α-specific regulatory mechanism, we screened HIF-2α-associated proteins in a yeast two-hybrid system and identified kelch-like 20 (KLHL20). HIF-2α, but not HIF-1α, interacted with KLHL20. siRNA-mediated knockdown of KLHL20 decreased HIF-2α protein, but not HIF-2α mRNA or HIF-1α protein. Depletion of KLHL20 decreased hypoxia-induced HIF activity, and consequently resulted in decreased expression levels of HIF-2α-responsive genes such as VEGF and CITED2. In contrast, overexpression of KLHL20 increased the expression levels and transcriptional activities of the O2-sensitive wild-type and O2-insensitive mutant forms of HIF-2α. KLHL20 siRNA also inhibited HIF-2 activity in von Hippel–Lindau tumor suppressor protein (pVHL)-deficient 786-O cells. These results indicate that KLHL20 is a novel player that regulates HIF-2α protein expression through mechanisms independent of hypoxia and pVHL.