Response to oxidative stress of AML12 hepatocyte cells with knockout of methionine sulfoxide reductases

Abstract

Methionine sulfoxide reductases are enzymes that reduce methionine oxidation in the cell. In mammals there are three B-type reductases that act on the R-diastereomer of methionine sulfoxide, and one A-type reductase (MSRA) that acts on the S-diastereomer. Unexpectedly, knocking out the four genes in the mouse protected from oxidative stresses such as ischemia-reperfusion injury and paraquat. To elucidate the mechanism by which lack of the reductases protects against oxidative stresses, we aimed to create a cell culture model with AML12 cells, a differentiated hepatocyte cell line. We employed CRISPR/Cas9 to create lines lacking the four individual reductases. All were viable and their susceptibility to oxidative stresses was the same as the parental strain. The triple knockout lacking all three methionine sulfoxide reductases B was also viable, but the quadruple knockout was lethal. We thus modeled the quadruple knockout mouse by creating an AML12 line lacking the three MSRB and heterozygous for the MSRA (Msrb3KO-Msra+/−). We measured the effect of ischemia-reperfusion on the various AML12 cell lines, using a protocol that modeled the ischemic phase by glucose and oxygen deprivation for 36 h followed by return of glucose and oxygen for 3 h as the reperfusion phase. This stress killed ∼50% of the parental line, an effect we chose to facilitate detection of either protective or deleterious changes in the knockout lines. Unlike the protection afforded the mouse, the knockout lines produced by CRISPR/Cas9 did not differ from the parental line in their response to ischemia-reperfusion injury or paraquat poisoning. In the mouse, inter-organ communication may be essential for protection induced by lack of methionine sulfoxide reductases.