5 HIF-1α dependent and independent regulation of PP2A in human aortic smooth muscle cells under hypoxia

Aims Although hypoxia can modulate the phosphoprotein phosphates system, few studies have addressed if this is mediated through HIF. Therefore, we investigated the involvement of hypoxia-induced HIF-1α on: PP2A activity, post–translational modification of PP2Ac, and abundance of key enzymes involved in post–translational modification of PP2A in HASMC. Methods and results HASMC and HAEC were cultured in cell type specific media for 24 hour under normoxic or hypoxic conditions (1% O 2 ) or following exposure to DMOG (100 µM). Effects on mRNA expression, phosphatase activity, post-translational modification and involvement of HIF-1α were assessed using RT-PCR, immunoblotting, an immunoprecipitation activity assay, ELISA and siRNA transfection. Hypoxia and DMOG decreased mRNA expression of HIF-1α and PPP2CA in HASMC and HAEC without altering cell viability. In HASMC hypoxia decreased phosphatase activity (total and PP2Ac) without affecting PP2Ac abundance, an effect mimicked by DMOG. Interestingly, hypoxia increased the level of phosphorylated and demethylated PP2Ac. The latter was associated with increased and decreased abundance of PME-1 and LCMT-1 respectively. Knockdown of HIF-1α prevented the hypoxia-mediated decrease in total phosphatase activity and mRNA expression of PPP2CA. However, it did not alter the effect of hypoxia on the abundance of pPP2Ac, DPP2Ac, LCMT-1 or PME-1. Conclusion In HASMC, hypoxia inhibits PP2A activity through a HIF-1α dependent mechanism. In addition, PP2Ac undergoes HIF-1α independent phosphorylation and demethylation during hypoxia in keeping with changes in the abundance of PME-1 and LMCT-1. The post-translational modification of PP2Ac is consistent with altered assembly of the PP2A holoenzyme and inhibition of activity. Together these data indicate a complex interaction between hypoxia and the PP2A system which warrants further study.