Anoxia, acidosis, and intergenic interactions selectively regulate methionine sulfoxide reductase transcriptions in mouse embryonic stem cells

Abstract

Methionine sulfoxide reductases (Msr) belong to a gene family that contains one MsrA and three MsrBs (MsrB1, MsrB2, and MsrB3). We have identified all four of the genes that are expressed in mouse embryonic stem cell cultures. The vital cellular functions of the Msr family of genes are to protect cells from oxidative damage by enzymatically reducing the oxidized sulfide groups of methionine residues in proteins from the sulfoxide form (--SO) back to sulfide thus restoring normal protein functions as well as reducing intracellular reactive oxygen species (ROS). We have performed studies on the Msr family genes to examine the regulation of gene expression. Our studies using real-time RT-PCR and Western blotting have shown that expression levels of the four Msr family genes are under differential regulation by anoxia/reoxygenation treatment, acidic culture conditions and interactions between MsrA and MsrB. Results from these in vitro experiments suggest that although these genes function as a whole in oxidative stress protection, each one of the Msr genes could be responsive to environmental stimulants differently at the tissue level.

Fig. 1

Confirmation of stem cell identity and multipotent differentiation capability of the cultured mouse embryonic stem cells (MESCs) in feeder-layer-free culture system. A: Phase-contrast image of cultured MESCs without a feeder layer maintaining their typical round colonies with smooth edges indicating lack of differentiation. B: Positive staining with stem cell specific marker SSEA-1. C: Positive staining for the neuronal cell marker: neurofilament (NF). Antibodies to NF stain both cell bodies (arrowhead) and axons (large arrow). Nuclei are stained blue with DAPI (small arrow). D: Positive staining for the cardiac cell marker: cardiac Troponin T (cTnT) (green stain). Organized myofibrils are seen. E: Positive staining for the skeletal muscle cell marker: fast skeletal Troponin T (fsTnT) (green stain). DAPI, a blue nuclear fluorescent dye shows nuclear staining. F: Positive staining for both a-actinin (green) and desmin (red), markers for mesoderm-derived cell types. Magnifications for Figure 1 taken are: (A) 100×; (B) 250×; (C) 100×; (D) 600×;(E) 250×;(F) 250×.

Fig. 2

Real-time RT-PCR studies on Msr gene expression under oxidative stress induced by anoxia/reoxygenation. A: MsrA; (B) MsrB1; (C) MsrB2; (D) MsrB3; (E) MMP2; (F) MMP9. MsrB3 shows similar expression responses to MMP2 and MMP9 with their transcription levels downregulated when oxygen is depleted but regaining their normal expression levels quickly after a resupply of oxygen. Numbers show the hours of anoxia and reoxygenation respectively applied to MESCs. For example, 4+0 indicates 4 h of anoxia followed by 0 h of reoxygenation. Results with a statistically significant difference from the 0+0 group were labeled with asterisks (*)(P ≤0.05). Triplets were used in each experiment and two independent experiments were performed. Results were averaged with standard errors of mean (SEM) presented as error bars.

Fig. 3

Real-time RT-PCR studies on MsrB gene expression at the mRNA level after downregulation of MsrA expression by siRNA (168) transfection. A: MsrA mRNA levels; (B) MsrB1 mRNA levels; (C) MsrB2 mRNA levels. D: MsrB3 RNA levels. MsrB3 is the only one showing significantly increased mRNA expression after MsrA knockdown. E: MsrA protein levels from densitometry of Western blots shown in F. F: Western blotting assays on MsrA protein and β-actin after treating MESCs with MsrA specific siRNA (168) or negative control siRNA (882) for days 1, 2, and 3. Triplets were used in each experiment. Two independent experiments were performed for real-time RT-PCRs and three Western blotting experiments were done. Results were averaged with standard errors of mean (SEM) presented with error bars. 168-1 represent samples collected on day 1 with siRNA (168) treatment. Results from MsrA specific siRNA (168) treated samples were labeled with asterisks (*) if there are statistically significant differences from negative control siRNA (882) treated groups (P ≤0.05).

Fig. 4

Real-time RT-PCR studies on Msr gene expression in culture media with different pH conditions (pH 6.4, 6.8, and 7.5) at 1, 2, and 3 days in culture. All Msr genes, except for MsrB2, show increased expression at the mRNA level after 3 days of culture in acidic media with MsrB3 showing the most dramatic responses. A: MsrA mRNA levels; (B) MsrB1 mRNA levels; (C) MsrB2 mRNA levels; (D) MsrB3 mRNA levels. E: Western blotting assays on MsrA protein and β-actin after culturing MESCsin media with different pH for days 1, 2, and 3. F: Densitometry of MsrA bands normalized by β-actin after Western blotting shows equal levels of MsrA protein expression in cells cultured in media with different pH on the same days. In all comparisons, data from cells cultured at pH 7.5 are arbitrarily set as unit 1. Results with statistically significant differences from 0+0 group were labeled with asterisks (*)(P ≤0.05). Triplets were used in each experiment and at least two independent experiments were performed. Results were averaged with standard errors of mean (SEM) presented as error bars. Results are labeled with asterisks (*) if there are statistically significant differences (P ≤0.05) comparing samples treated with acidic culture media and cells cultured at pH 7.5.