Protein phosphatase PPP3CA (calcineurin A) down-regulates hypoxia-inducible factor transcriptional activity

Hypoxia-inducible factors (HIF) are master regulators of the response to hypoxia. Although several kinases are known to modify their oxygen sensitive HIF-alpha subunits or affect indirectly their function, little is known about the role of phosphatases in HIF control. To address this issue, a library containing siRNAs for the 25 known catalytic subunits of human phosphatases was used to screen for their effect on HIF transcriptional activity in HeLa cells. Serine-threonine phosphatase PPP3CA (calcineurin A, isoform a) was identified as the strongest candidate for a negative regulator of HIF activity. Indeed, independent silencing of PPP3CA expression stimulated HIF transcriptional activity under hypoxia, without increasing the protein levels of HIF-1 alpha or HIF-2 alpha. Overexpression of a constitutively active PPP3CA form, but not its catalytically inactive counterpart, inhibited HIF activity and expression of HIF target genes but did not affect HIF-1 alpha or HIF-2 alpha expression. These results were phenocopied by treatment with the ionophore ionomycin, that activates endogenous PPP3CA. The effect of ionomycin was mediated by PPP3CA as it was largely abolished by PPP3CA silencing. Furthermore, ionomycin enhanced the down-regulation of HIF activity by wild-type PPP3CA overexpression. Overall, our results suggest the involvement of PPP3CA in fine-tuning the HIF-dependent transcriptional response to hypoxia.