RESUMO
OBJECTIVE: The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations. METHODS: The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction. RESULTS: It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3'-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells. CONCLUSIONS: Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.