ERN1 dependent impact of glucose and glutamine deprivations on PBX3, PBXIP1, PAX6, MEIS1, and MEIS2 genes expression in U87 glioma cells.

Objective. Homeobox genes play a fundamental role in the embryogenesis, but some of them have been linked to oncogenesis. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of homeobox genes such as PAX6 (paired box 6), PBX3 (PBX homeobox 3), PBXIP1 (PBX homeobox interacting protein 1), MEIS1 (MEIS homeobox 1), and MEIS2 in ERN1 knockdown U87 glioma cells with the intent to reveal the role of ERN1 (endoplasmic reticulum to nucleus signaling 1) signaling pathway on the endoplasmic reticulum stress dependent regulation of homeobox genes. Methods. The control (transfected by empty vector) and ERN1 knockdown (transfected by dominant-negative ERN1) U87 glioma cells were exposed to glucose and glutamine deprivations for 24 h. The cells RNA was extracted and reverse transcribed. The expression level of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes was evaluated by a real-time quantitative polymerase chain reaction analysis and normalized to ACTB. Results. It was found that glucose deprivation down-regulated the expression level of PAX6, MEIS1, and MEIS2 genes in control glioma cells, but did not significantly alter PBX3 and PBXIP1 genes expression. At the same time, ERN1 knockdown significantly modified the sensitivity of all studied genes to glucose deprivation. Other changes in gene expression were detected in control glioma cells under the glutamine deprivation. The expression of PBX3 and MEIS2 genes was down- while PAX6 and PBXIP1 genes up-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on the majority of studied genes expression in U87 glioma cells. Conclusion. The results of the present study demonstrate that the exposure of U87 glioma cells under glucose and glutamine deprivations affected the expression of the majority of the studied homeobox genes and that the sensitivity of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes expression under these experimental conditions is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling.

Cortisol controls endoplasmic reticulum stress and hypoxia dependent regulation of insulin receptor and related genes expression in HEK293 cells.

Objective. Glucocorticoids are important stress-responsive regulators of insulin-dependent metabolic processes realized through specific changes in genome function. The aim of this study was to investigate the impact of cortisol on insulin receptor and related genes expression in HEK293 cells upon induction the endoplasmic reticulum (ER) stress by tunicamycin and hypoxia. Methods. The human embryonic kidney cell line HEK293 was used. Cells were exposed to cortisol (10 µM) as well as inducers of hypoxia (dimethyloxalylglycine, DMOG; 0.5 mM) and ER stress (tunicamycin; 0.2 µg/ml) for 4 h. The RNA from these cells was extracted and reverse transcribed. The expression level of INSR, IRS2, and INSIG2 and some ER stress responsive genes encoding XBP1n, non-spliced variant, XBP1s, alternatively spliced variant of XBP1, and DNAJB9 proteins, was measured by quantitative polymerase chain reaction and normalized to ACTB. Results. We showed that exposure of HEK293 cells to cortisol elicited up-regulation in the expression of INSR and DNAJB9 genes and down-regulation of XBP1s, XBP1n, IRS2, and INSIG2 mRNA levels. At the same time, induction of hypoxia by DMOG led to an up-regulation of the expression level of most studied mRNAs: XBP1s and XBP1n, IRS2 and INSIG2, but did not change significantly INSR and DNAJB9 gene expression. We also showed that combined impact of cortisol and hypoxia introduced the up-regulation of INSR and suppressed XBP1n mRNA expression levels. Furthermore, the exposure of HEK293 cells to tunicamycin affected the expression of IRS2 gene and increased the level of XBP1n mRNA. At the same time, the combined treatment of these cells with cortisol and inductor of ER stress had much stronger impact on the expression of all the tested genes: strongly increased the mRNA level of ER stress dependent factors XBP1s and DNAJB9 as well as INSR and INSIG2, but down-regulated IRS2 and XBP1n. Conclusion. Taken together, the present study indicates that cortisol may interact with ER stress and hypoxia in the regulation of ER stress dependent XBP1 and DNAJB9 mRNA expression as well as INSR and its signaling and that this corticosteroid hormone modified the impact of hypoxia and especially tunicamycin on the expression of most studied genes in HEK293 cells. These data demonstrate molecular mechanisms of glucocorticoids interaction with ER stress and insulin signaling at the cellular level.

Insulin receptor substrate 1 gene expression is strongly up-regulated by HSPB8 silencing in U87 glioma cells.

Objective. The aim of the present investigation was to study the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other functionally active proteins in U87 glioma cells under silencing of polyfunctional chaperone HSPB8 for evaluation of the possible significance of this protein in intergenic interactions.Methods. Silencing of HSPB8 mRNA was introduced by HSPB8 specific siRNA. The expression level of HSPB8, IRS1, HK2, GLO1, HOMER3, MYL9, NAMPT, PER2, PERP, GADD45A, and DEK genes was studied in U87 glioma cells by quantitative polymerase chain reaction.Results. It was shown that silencing of HSPB8 mRNA by specific to HSPB8 siRNA led to a strong down-regulation of this mRNA and significant modification of the expression of IRS1 and many other genes in glioma cells: strong up-regulated of HOMER3, GLO1, and PERP and down-regulated of MYL9, NAMPT, PER2, GADD45A, and DEK gene expressions. At the same time, no significant changes were detected in the expression of HK2 gene in glioma cells treated by siRNA, specific to HSPB8. Moreover, the silencing of HSPB8 mRNA enhanced the glioma cells proliferation rate.Conclusions. Results of this investigation demonstrated that silencing of HSPB8 mRNA affected the expression of IRS1 gene as well as many other genes encoding tumor growth related proteins. It is possible that the dysregulation of most of the studied genes in glioma cells after silencing of HSPB8 is reflected by a complex of intergenic interactions and that this polyfunctional chaperone is an important factor for the stability of genome function and regulatory mechanisms contributing to the tumorigenesis control.