Plant-Produced Asialo-Erythropoietin Restores Pancreatic Beta-Cell Function by Suppressing Mammalian Sterile-20-like Kinase (MST1) and Caspase-3 Activation

Abstract

Pancreatic beta-cell death adversely contributes to the progression of both type I and II diabetes by undermining beta-cell mass and subsequently diminishing endogenous insulin production. Therapeutics to impede or even reverse the apoptosis and dysfunction of beta-cells are urgently needed. Asialo-rhuEPO, an enzymatically desialylated form of recombinant human erythropoietin (rhuEPO), has been shown to have cardioprotective and neuroprotective functions but with no adverse effects like that of sialylated rhuEPO. Heretofore, the anti-apoptotic effect of asialo-rhuEPO on pancreatic beta-cells has not been reported. In the current study, we investigated the cytoprotective properties of plant-produced asialo-rhuEPO (asialo-rhuEPO^P) against staurosporine-induced cell death in the pancreatic beta-cell line RIN-m5F. Our results showed that 60 IU/ml asialo-rhuEPO^P provided 41% cytoprotection while 60 IU/ml rhuEPO yielded no effect. Western blotting results showed that asialo-rhuEPO^P treatment inhibited both MST1 and caspase-3 activation with the retention of PDX1 and insulin levels close to untreated control cells. Our study provides the first evidence indicating that asialo-rhuEPO^P-mediated protection involves the reduction of MST1 activation, which is considered a key mediator of apoptotic signaling in beta-cells. Considering the many advantages its plant-based expression, asialo-rhuEPO^P could be potentially developed as a novel and inexpensive agent to treat or prevent diabetes after further performing studies in cell-based and animal models of diabetes.

Keywords: MST1; asialo-rhuEPO; cytoprotection; insulin secretion; pancreatic beta-cell death.

FIGURE 1

Current view on how diabetic stimuli lead to activation of MST1 causing apoptosis and beta-cell dysfunction adopted from Ardestani et al. (2014), and how EPO and asialo-EPO display their cytoprotective functions via potentially binding to homodimeric EPOR or EPOR and β-CR heterodimeric receptor summarized from Brines and Cerami (2005, 2008). Cl MST1: Cleaved MST1.

FIGURE 2

Dose-dependent increase in cytotoxicity (A), caspase-3 activation and MST1 activation (B) in RIN-m5F cells incubated with 0–0.6 μM STS for 24 h. The EC₅₀ for STS on RIN-m5F beta-cells was determined by their cytotoxicities.

FIGURE 3

Cytoprotective effects of asialo-rhuEPO^P against STS-induced apoptosis in RIN-m5F cells. (A) Cytoprotective effects of 20, 40, 60, 80, and 100 IU asialo-rhuEPO^P against 0.123 μM STS-induced cell death. (B) Cytoprotective effects of 60 IU asialo-rhuEPO^P and rhuEPO^M against 0.123 μM STS-induced cell death. Data represent the average ± SE. Different letters labeled represent significant difference at p<0.05 level.

FIGURE 4

Western blot of MST1 (A), Caspase-3 (B), Bax and Bcl-2 (C). The levels of these proteins were measured in cell lysates prepared from cells treated with PBS containing 0.1% BSA (vehicle control), 0.123 μM STS, 0.123 μM STS+60 IU/ml rhuEPO^M or 0.123 μM STS+60 IU/ml asialo-rhuEPO^P. Active MST1 and caspase-3 were detected using an anti-MST1 and anti-caspase-3 antibody, respectively, which also cross-react with proMST1 and procaspase-3. Bax and Bcl-2 specific antibodies were used to detect these proteins. β-Actin was used as internal control. The experiment was repeated twice. All data plotted are the average of two independent experiments ± SD. Different letters labeled represent significant difference at p < 0.05 level.

FIGURE 5

Western blot of AKT. The ratio of AKT and p-AKT were measured in cell lysates prepared from cells treated with PBS containing 0.1% BSA (vehicle control), 0.123 μM STS, 0.123 μM STS+60 IU/ml rhuEPO^M or 0.123 μM STS+60 IU/ml asialo-rhuEPO^P. For detection of p-AKT and AKT, the blot was probed with anti-p-AKT antibody first followed by stripping the blot and re-probing with anti-total AKT antibody. The experiment was repeated twice. All data plotted are the average of two independent experiments ± SD. Different letters labeled represent significant difference at p < 0.05 level.

FIGURE 6

Western blot of PDX1 (A) and the levels of secreted insulin in cultured medium (B). (A) The levels of PDX1 protein were measured in cell lysates prepared from cells treated with PBS containing 0.1% BSA (vehicle control), 0.123 μM STS, 0.123 μM STS+60 IU/ml rhuEPO^M, or 0.123 μM STS+60 IU/ml asialo-rhuEPO^P. (B) Secreted insulin in cultured medium. Secreted insulin levels were measured in media from above four cultures. The measurement was repeated three times. The experiment was repeated twice. All data plotted are the average of two independent experiments ± SD. Different letters labeled represent significant difference at p < 0.05 level.