Multiple downstream signaling pathways (mainly ERK, Akt, and STAT5) of EPO receptor (EPOR) were reported to be involved in EPO protection. The expression of EPOR was found in primary retinal and R28 cells (
Supplementary Fig. S2). However, the exact signaling pathways involved in EPO protection against glyoxal-AGEs remained largely unknown. To elucidate the signaling pathways in the current model, the major downstream pathways of EPOR signaling were selectively blocked by specific inhibitors. Unexpectedly, we did not observe the activation of STAT5 with EPO treatment (
Supplementary Fig. S3), indicating that STAT5 signaling might not have been involved under our experimental conditions. Therefore, the functions of ERK and Akt signaling in the current model were investigated. In the absence of glyoxal, EPO itself significantly enhanced the phosphorylation of ERK and Akt in a time-dependent manner in R28 cells (
Fig. 3A, left) and in primary retinal cells (
Fig. 3A, right). In R28 cells, glyoxal also stimulated ERK phosphorylation, but the elevated phosphorylation returned to normal level at 5 hours. Coincubation with EPO and glyoxal revealed no significant difference in ERK phosphorylation at 1 hour and 3 hours; however, the phosphorylation became much stronger at 5 hours in comparison with glyoxal treatment alone. For Akt, glyoxal gradually downregulated its phosphorylation, and this effect was rescued by EPO. It was noticed that 0.2 U/mL EPO was more powerful than 1 U/mL in enhancing ERK and Akt phosphorylation (
Fig. 3B, upper). In primary retinal cells, glyoxal was able to stimulate ERK phosphorylation, whereas such phosphorylation was not further enhanced by EPO application. Of note, very weak phosphorylation of Akt was found in the control; however, EPO was able to upregulate the Akt phosphorylation compared with glyoxal alone. In contrast to the R28 cells, 0.2 U/mL EPO showed effects on ERK and Akt similar to those of 1 U/mL EPO in the primary cells (
Fig. 3B, lower). Based on these observations, it was speculated that ERK and Akt pathways played important roles in glyoxal toxicity and EPO neuroprotection but with different temporal dependency. The ERK or Akt pathway was further studied with the use of specific inhibitors, such as U0126 (for ERK) or wortmannin (WM; for Akt). Viability assay(s) demonstrated that glyoxal reduced cell viability to 67.2% ± 0.3% (R28 cells) and to 74.6% ± 1.9% (primary cells); this was significantly rescued by EPO (to 78.2% ± 1.7% in R28 cells and to 84.4% ± 1.8% in primary cells;
P < 0.05). In R28 cells, the blockage of ERK signaling pathway by U0126 caused minor effects on glyoxal toxicity (glyoxal + DMSO, 67.2% ± 0.3% vs. glyoxal + U0126, 64.8% ± 1.2%;
P > 0.05) while largely abolishing EPO protection (glyoxal + EPO + U0126, 64.1% ± 1.1% vs. glyoxal + EPO + DMSO, 80.0% ± 1.7%;
P < 0.01). Of note, inhibition of the Akt pathway by WM led to more severe viability loss (glyoxal + WM, 55.6% ± 0.8%;
P < 0.01) compared with glyoxal + DMSO. Furthermore, EPO-enhanced viability was also eliminated by WM (glyoxal + EPO + WM, 55.3% ± 0.8%;
P < 0.01). Similar to the findings in R28 cells, the protection of EPO in primary cells was also inhibited by U0126 (EPO + DMSO, 84.4% ± 1.8% vs. EPO + U0126, 76.7% ± 1.8%;
P < 0.05) or WM (EPO + WM, 73.8% ± 1.3%;
P < 0.05). Of note, neither inhibitor affected primary cell viability when applied with glyoxal alone (
Fig. 3C). These phenomena were confirmed by using different inhibitors of ERK (PD98059) and Akt (LY294002) signaling (data not shown) in R28 cells. None of these inhibitors caused significant toxicity to R28 cells (
Supplementary Fig. S4). In addition, the viability results were supported by TUNEL assay: in R28 cells, glyoxal significantly increased apoptosis (7.9% ± 0.5%;
P < 0.01). In contrast, only 0.23% ± 0.06% of apoptosis was found in untreated control. The apoptotic cell population caused by glyoxal was significantly reduced by EPO (1.7% ± 0.2%;
P < 0.01). Coapplication of glyoxal with WM rather than U0126 further increased apoptosis to 10.3% ± 0.9% compared with DMSO. Both U0126 and WM were capable of eliminating the antiapoptotic effect of EPO: glyoxal + EPO + DMSO (1.7% ± 0.2%) compared with glyoxal + EPO + U0126 (7.8% ± 0.5%) compared with glyoxal + EPO + WM (8.2% ± 0.5%) (
P < 0.01). In primary cells, glyoxal also significantly increased apoptosis (41.8% ± 2.2%;
P < 0.01), superimposing the background cell death (5.6% ± 1.2%) in control. The application of EPO, however, significantly alleviated the apoptosis induced by glyoxal (glyoxal + EPO + DMSO, 11.1% ± 1.2%;
P < 0.01). EPO neuroprotection in primary cells was antagonized by either U0126 (glyoxal + EPO + U0126, 35.3% ± 1.9%;
P < 0.01) or WM (glyoxal + EPO + WM, 42.4% ± 2.4%;
P < 0.01). Neither U0126 nor WM affected the cell death induced by glyoxal alone (glyoxal + U0126, 41.9% ± 2.4% vs. glyoxal + WM, 44.4% ± 2.8%;
P > 0.05) (
Figs. 3D,
3E).