Erythropoietin-induced neuroprotection requires cystine glutamate exchanger activity

Abstract

Erythropoietin (Epo) has been used for many years in neonates for the treatment of anemia of prematurity. Epo has also been proposed for treatment of neonatal brain injury, as mounting evidence suggests neuroprotective properties for Epo. However, Epo's neuroprotective mechanism of action is poorly understood. In this study we hypothesized that Epo may confer neuroprotection by enhancing cellular redox defense brought about by cellular glutathione (GSH). This was examined in cultures of differentiated cortical neural stem cells and using the B104 cell line as model systems. Our data shows that Epo causes a time- and dose-dependent increase in expression and activity of system Xc(-), the transporter responsible for uptake of cystine for the production of glutathione. Cystine uptake increases 3-5 fold in differentiated neural stem cells and B104 cells treated with Epo. Exposure of cells to 100 microM kainate suppressed cellular GSH and caused excitotoxicity, but GSH levels and cell viability were completely restored by Epo in the continued presence of kainate. This rescue effect of Epo vanished if system Xc(-) was inhibited pharmacologically using S4-CPG in the presence of Epo leading to marked cell death of B104 cells and cultured mouse cortical neural stem cells. This could also be achieved using xCT siRNA to decrease xCT expression. This data suggests that system Xc(-) activity and protein expression are positively regulated by Epo directly explaining its neuroprotective effect.

Figure 1

EpoR and xCT expression in B104 cells. (a.) RT-PCR of 2μg of total RNA collected from B104 cells (b.) Immunoblot of EpoR, xCT and Actin. B104 cells (c.) xCT (TRITC), (e.) EpoR (FITC), (g.) combined. Differentiated neural stem cells d.) xCT (TRITC), (f.) EpoR (FITC) and (h.) combined.

Figure 2

Glutamate Toxicity in NSCs and B104. (a.) Immunoblot of NSC protein extract probed for cleaved caspase 3 and actin at increasing concentrations of glutamate (1-0 glu, 2–100 μM glu, 3–250 μM glu, 4–500 μM glu, 5-1mM glu, 6-3mM glu). (b.) Immunoblot of B104 cell extract probed for cleaved caspase 3 and actin at increasing concentrations of glutamate (1-0 glu, 2–100 μM glu, 3–250 μM glu, 4–500 μM glu, 5-1mM glu, 6-3mM glu).

Figure 3

Cell viability and cystine uptake in differentiated neural stem cells. (a.) Total cell count in control, 3mM glutamate, 3mM glutamate + Epo (10ng/ml), 3mM glutamate + Epo (10ng/ml), 3mM glutamate + Epo (10ng/ml) + S4-carboxyphenylglycine (250μM). (b.) Cystine uptake in differentiated NSCs –control, 3mM glutamate and 3mM glutamate + Epo (10ng/ml). Cell viability and cystine uptake in B104 cells (c.) Total cell count in control, 3mM glutamate, 3mM glutamate + Epo (10ng/ml), 3mM glutamate + Epo (10ng/ml), 3mM glutamate + Epo (10ng/ml) + S4-carboxyphenylglycine (250μM). (d.) Cystine uptake in differentiated B104 cells –control, 3mM glutamate and 3mM glutamate + Epo (10ng/ml). * p ≤ 0.05 using the paired student t-test.

Figure 4

Erythropoietin Time Course. (a.) Representative Western Blot of system Xc⁻ and actin in B104 cells treated with 10ng/ml of Erythropoietin. (b.) Relative xCT concentration (normalized to actin) at 0, 4, 8, 12 and 24hr time points. 25μg of protein was added to each well. *p≤0.05 and **p≤0.01 using the paired student t-test.

Figure 5

Epo Dose Response. (a.) Representative Western blot of system Xc⁻ in B104 cells with 0, 5, 10, 25, 50 ng/ml Epo. (b.) Relative xCT concentration (normalized to actin) at same concentrations listed in (a.). * p ≤ 0.05 using the paired student t-test.

Figure 6

Epo effect on total glutathione. B104 cells treated with kainate in the presence (5ng/ml and 10ng/ml) or absence of Epo. Glutathione is expressed as fluorescence/mg protein. *p ≤ 0.05 using the paired student t-test.

Figure 7

xCT siRNA effect on cell viability. Lane 1–5 include (1) Control, (2) scrambled siRNA 50ng/ml, (3) xCT siRNA 10ng/ml, (4) xCT siRNA 25ng/ml and (5) xCT siRNA 50ng/ml. (a) Total Cell Count. *p<0.05 and **p<0.01. (b) xCT and actin immunoblot, 25μg protein loaded to each well.