The ERN1 signaling pathway of unfolded protein controls the expression of EDEM1 and its hypoxic regulation in glioblastoma cells.

Objective. For the effective growth of malignant tumors, including glioblastoma, the necessary factors involve endoplasmic reticulum (ER) stress, hypoxia, and the availability of nutrients, particularly glucose. The ER degradation enhancing alpha-mannosidase like protein 1 (EDEM1) is involved in ER-associated degradation (ERAD) targeting misfolded glycoproteins for degradation in an N-glycan-independent manner. EDEM1 was also identified as a new modulator of insulin synthesis and secretion. The present study aims to investigate the regulation of the EDEM1 gene expression in U87MG glioblastoma cells by hypoxia and glucose or glutamine deprivations depending on the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1) with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Methods. The U87MG glioblastoma cells (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (4 h). For glucose and glutamine deprivations, the cells were exposed to DMEM medium without glucose and glutamine, respectively, for 16 h. The expression level of the EDEM1 gene was studied by quantitative RT-PCR and normalized to the ACTB mRNA. Results. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to down-regulation of EDEM1 gene expression in glioblastoma cells. Moreover, the expression of this gene was also decreased after silencing ERN1 in glioblastoma cells. At the same time, the expression of EDEM1 gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the EDEM1 gene was increased under hypoxia in control U87MG cells, but resistant to hypoxia in cells with ERN1 knockdown. Furthermore, the expression of this gene was up-regulated under glucose and glutamine deprivations in control glioblastoma cells. However, the ERN1 knockdown increased the sensitivity of EDEM1 gene expression to glucose and decreased to glutamine deprivations. Conclusion. The results of the present study demonstrate that inhibition of ERN1 down-regulated the expression of the EDEM1 gene through protein kinase activity of ERN1 and that the regulation of this gene expression by hypoxia and nutrient supply, especially glucose, is differently controlled by ERN1 in glioblastoma cells.