Liver knock-out of nuclear inhibitor of protein phosphatase-1 in mice decreases hepatic iron

Abstract The liver plays a critical role in iron homeostasis and serves as the iron recycling and storage place. It also produces hepcidin, which regulates iron uptake by degrading iron exporter ferroportin (FPN). Protein phosphatase 1 (PP1) is a serine/threonine phosphatase that is composed of a catalytic subunit (PP1α, PP1β or PP1γ) and a ‘regulatory interactor of PP1‘ (RIPPO) that directs PP1 to a specific substrate and location within the cells. In the nucleus, PP1 is primarily regulated by RIPPOs known as Nuclear Inhibitor of PP1 (NIPP1) and the Phosphatase Nuclear Targeting subunit (PNUTS). The active site of PP1 contains Fe2+, which is essential for catalysis, however the role of PP1 in iron metabolism has not been described well. Liver-specific NIPP1 knock-out mice (NIPP1-/-) showed an increase in bile-duct hyperplasia (Boens S et al., 2016, Stem Cells, 34:2256). In this study, we demonstrate that NIPP1-/- livers also exhibit significant changes in iron metabolism. We conducted global proteomic analysis of NIPP1-/- livers in comparison to wildtype (WT) livers and detected increased levels of the transferrin receptor (TFR) (5.8-fold; p=0.05) and decreased levels of FPN (0.61-fold; p=0.038) in the NIPP1-/- liver. Western blot analysis confirmed a 1.6-fold increase in TFR expression (p=0.05) and a 0.8-fold decrease in FPN expression (p=0.04) in the livers of NIPP1⁻/⁻ compared to control mice. The mRNA expression analysis showed increased TFR mRNA expression in NIPP1-/- livers (Tfr, ΔCt = -6.12 in ctrl vs -5.5 in NIPP1-/-, p=0.02) but decreased FPN mRNA (Slc40a1, ΔCt = -0.30 in ctrl vs -0.96 in NIPP1-/-, p=0.02). Several other factors involved in iron metabolism also showed decreased mRNA expression levels including the ferroxidase hephaestin (Heph; ΔCt = -8.90 in control vs -10.37 in NIPP1-/-, p=0.02), bone morphogenetic protein 2 (Bmp2; ΔCt = -6.5 in control vs -8.5 in NIPP1-/-, p=0.01), and bone morphogenetic protein 6 (Bmp6; ΔCt = -6.8 in control vs -8.0 in NIPP1-/-, p=0.03). The total iron concentration measured by ELISA was significantly lower in the livers of NIPP1-/- mice compared to the controls (0.75-fold reduction, p=0.04). These findings suggest that altered location or activity of PP1 in NIPP1-/- livers impact iron absorption leading to a reduction in the intracellular iron, as indicated by the increased TFR, reduced FPN levels and decreased intracellular iron as well as iron regulatory proteins. Thus, PP1 may play an unrecognized role in regulating iron metabolism and potentially serve as a therapeutic target in iron overload diseases such as alcohol-related liver disease or hereditary hemochromatosis. Acknowledgments: This study was supported by NIH Research Grants 1R01HL125005, 5U54MD007597, 1S10OD028681 and 1SC1HL150685.