Meprin B Expression Impacts the Mediators of the Protein Kinase A Signaling Pathway Under Hypoxia Conditions

Abstract

Meprins are metalloproteases that are expressed abundantly in the proximal kidney tubules. The level of expression and the localization of meprins are associated ischemia/reperfusion (IR)‐induced acute kidney injury (AKI). Previous studies have shown that meprin β deficiency protect mice from IR‐induced kidney injury, resulting in better overall preservation of kidney function. In vitro studies have shown that meprins are capable of proteolytically processing proteins associated with the hypoxia response (e.g OS‐9) and inflammation (e.g IL‐β1, IL‐6, IL‐18). Previous work from our lab showed that proteolytic processing of PKA C by meprins occurs in vivo (in IR‐induced AKI). In vitro studies with purified PKA C proteins further demonstrated that cleaving of PKA C by meprin B is isoform‐specific and significantly reduces the PKA C kinase activity; which could potentially affect downstream targets of the protein kinase A (PKA) signaling pathway and hypoxia response genes. The goal of the current study was to determine how meprin β expression affects downstream mediators of the PKA signaling pathway under hypoxia conditions. To this end, Madin‐Darby canine kidney (MDCK) cells stably transfected with meprin β cDNA and sham‐transfected control cells cultured to 90–95% confluence then subjected to hypoxia (1% O2) for 2h followed by 2h of recovery (under normoxia conditions). Proteins were extracted from the cells and fractionated into cytosolic‐, nuclear‐, and membrane‐enriched fractions. Nuclear levels of hypoxia inducible factor (HIF‐1α) were evaluated to confirm induction of hypoxia. Western blot analysis was used to determine the levels of PKA C, PKA Cα and PKA Cβ isoforms, Phospho‐PKA C, phospho‐extracellular signal‐regulated kinase (P‐ERK), and heat shock protein (Hsp27) in the cytosolic‐ and nuclear‐enriched fractions. Statistical analysis utilized 2‐way ANOVA. Our data showed that the levels of PKA C in cytosolic‐enriched fraction decreased in meprin β transfected cells subjected to hypoxia. No change was observed in the levels of PAK Cα in protein fractions of both cell genotypes. In contrast, the levels PKA Cβ in nuclear and cytosolic‐enriched fractions of meprin β transfected cells subjected to hypoxia decreased when compared to non‐transfected cells. P‐PKA C levels in nuclear and cytosolic‐enriched fractions decreased in meprin β expressing cells subjected to hypoxia when compared to non‐transfected control cells. The level of P‐ERK in meprin β‐transfected cells increased significantly compared to non‐transfected control cells, which showed a significant reduction after hypoxia induction. There was no change in the levels of HSP27 in cytosolic‐enriched fraction for either genotype of cells. These results suggest that meprin B expression has a direct impact on the levels of ERK and could thus affect downstream hypoxia response genes (e.g. ANKRD37, RCOR2, and NDRG1) which may impact IR‐induced AKI via modulation of the PKA pathway.Support or Funding InformationNIH/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and SC1GM118271 to E. Moige OngeriThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.