Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

ERN1 dependent impact of glutamine and glucose deprivations on the pyruvate dehydrogenase genes expression in glioma cells.

Objective. The aim of the present study was to investigate the expression of pyruvate dehydrogenase genes such as PDHA1, PDHB, DLAT, DLD, and PDHX in U87 glioma cells in response to glutamine and glucose deprivations in control glioma cells and endoplasmic reticulum to nucleus signaling 1 (ERN1) knockdown cells, the major endoplasmic reticulum (ER) stress signaling pathway, to find out whether there exists a possible dependence of these important regulatory genes expression on both glutamine and glucose supply as well as ERN1 signaling. Methods. The expression level of PDHA1, PDHB, DLAT, DLD, and PDHX genes was studied by real-time quantitative polymerase chain reaction in control U87 glioma cells (transfected by empty vector) and cells with inhibition of ERN1(transfected by dnERN1) after cells exposure to glucose and glutamine deprivations. Results. The data showed that the expression level of PDHA1, PDHB, DLAT, and DLD genes was down-regulated (more profound in PDHB gene) in control glioma cells treated with glutamine deprivation. At the same time, ERN1 knockdown modified the impact of glutamine deprivation on the expression level of all these genes in glioma cells: suppressed the sensitivity of PDHB and DLD genes expression and removed the impact of glutamine deprivation on the expression of PDHA1 and DLAT genes. Glucose deprivation did not significantly change the expression level of all studied genes in control glioma cells, but ERN1 knockdown is suppressed the impact of glucose deprivation on PDHX and DLD genes expression and significantly enhanced the expression of PDHA1 and PDHB genes. No significant changes were observed in the sensitivity of PDHX gene expression to glutamine deprivation neither in control nor ERN1 knock-down glioma cells. The knock-down of ERN1 removed the sensitivity of DLAT gene expression to glucose deprivation. Conclusion. The results of this investigation demonstrate that the exposure of control U87 glioma cells under glutamine deprivation significantly affected the expression of PDHA1, PDHB, DLAT, and DLD genes in a gene specific manner and that impact of glutamine deprivation was modified by inhibition of the ER stress signaling mediated by ERN1. At the same time, glucose deprivation affected the expression of PDHA1, PDHB, PDHX, and DLD genes in ERN1 knockdown glioma cells only. Thus, the expression of pyruvate dehydrogenase genes under glutamine and glucose deprivation conditions appears to be controlled by the ER stress signaling through ERN1.

ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells.

Objective. The aim of the present investigation was to study the impact of glucose and gluta-mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in response to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up-regulated in control U87 glioma cells exposure under glucose deprivation condition in comparison with the glioma cells, growing in regular glucose containing medium. We also observed up-regulation of ECE1 gene expression in U87 glioma cells exposure under glutamine deprivation as well as down-regulation of the expression of EDN1 and EDNRA mRNA, being more significant for EDN1. Furthermore, the knockdown of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose and glutamine deprivation conditions. Thus, the ERN1 knockdown led to a strong suppression of EDN1 gene expression under glucose deprivation, but did not change the effect of glutamine deprivation on its expression. At the same time, the knockdown of ERN1 signaling introduced the sensitivity of EDNRB gene to both glucose and glutamine deprivations as well as completely removed the impact of glucose deprivation on the expression of ECE1 gene. Conclusions. The results of this study demonstrated that the expression of endothelin-1, its receptors, and ECE1 genes is preferentially sensitive to glucose and glutamine deprivations in gene specific manner and that knockdown of ERN1 significantly modified the expression of EDN1, EDNRB, and ECE1 genes in U87 glioma cells. It is possible that the observed changes in the expression of studied genes under nutrient deprivation may contribute to the suppressive effect of ERN1 knockdown on glioma cell proliferation and invasiveness.