Grp75 Mechanism For The Cytoprotective Effect

Glucose deprivation(GD) is a key factor in the process of brain ischemia, and it also was reported to be involved in the development of neuron degeneration diseases such as Alzheimer disease and amyotrophic lateral sclerosis(ALS). Then, the investigation of molecular and cellular changes in the process of glucose deprivation will contribute to understand the pathogenesis of these diseases. Rat adrenal pheochromocytoma(PC12) cell line is a well established model for the nerve system due to its ability to synthesis and store the catecholamine neurotransmitters dopamine and norepinephrine. In this study, PC12 cell line was used to investigate the injurious effect of glucose deprivation. Morphological and cytoflowmetric methods were applied to evaluate the cell death; Intracellular ATP level was determined by luciferase-luciferin method; Mitochondrial transmembrane potential(MTP); Reactive oxygen species(ROS) was measured by fluorescent probe DCFH-DA. The data demonstrated that glucose deprivation could induce the cell death including both necrosis and apoptosis; Intracellular ATP level increased in the first 3 hours, followed by progressive decrease till the end of GD treatment; and the mitochondrial transmembrane potential (Δψm) dropped after 6 hours, the complete depolarization occurred after 48 hours for GD treatment; ROS increased immediately to the peak level in the first 4 hours upon the GD treatment, and decreased progressively in the following time. Glucose regulated protein 75(grp75) is an important molecular chaperon belonged to the heat shock protein family(HSP). It was reported to up-regulate in the ischemic brain. Then, the reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry were performed to investigate the expression profile in PC12 cells under glucose deprivation. PC12 cells over-expressed or low-expressed grp75 protein were obtained by the stable transfection of sense and antisense grp75 vector. Thereafter, the cellular response of the different PC12 cells to GD treatment was examined by the methods described previously. Our data demonstrated that, the expression of grp75 was up-regulated after 3 hours of GD and reached the peak level after 24 hours; there was no significant difference in cell viability upon glucose deprivation between PC12 cells low expressed grp75 and control cells, while the overexpression of grp75 showed a protective effect against GD treatment; The overexpression of grp75 did not show any significant effect on the changes of intracellular ATP level and mitochondrial potential during glucose deprivation; The accumulation of ROS upon GD was inhibited by the overexpression of grp75 protein. These results indicated that the cytoprotective effect of grp75 was mediatedby its ability to inhibit the accumulation of ROS in PC12 cells upon glucose deprivation. The regulation of gene expression plays an important role in the determination of cell fate during the stress. p53 gene and the genes from the bcl-2 family were the most important candidates involved in the stress response. Then, RT-PCR was applied to investigate the expression of bcl-2 and bax during GD; Western blot was applied to investigate the expression of p53; The time course of P53 expression was compared between the grp75 over-expressed PC12 cells and the control cells; Furthermore, the in vivo interaction of grp75 and p53 protein was investigated with co－immunoprecipitation. The results demonstrated that, the expression of bcl-2 increased till 12 hours after GD treatment, and declined progressively in the rest of time for GD; the expression of bax decreased all along after the onset of glucose deprivation; Glucose deprivation induced the expression of p53 protein in PC12 cells, and the overexpression of grp75 delayed the induction of p53 expression; The co-immunoprecipitation assay confirmed the interaction of grp75 and p53 protein in vivo. These data indicated that grp75 can regulate the expression of p53 protein in PC12 cells under glucose depriv...