ABSTRACT
Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.
Subject(s)
Endoplasmic Reticulum Stress , Endoribonucleases , Gene Expression Regulation, Neoplastic , Glioblastoma , Glycine Hydroxymethyltransferase , Humans , Glycine Hydroxymethyltransferase/genetics , Glycine Hydroxymethyltransferase/metabolism , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Endoplasmic Reticulum Stress/physiology , Endoplasmic Reticulum Stress/genetics , Cell Line, Tumor , Endoribonucleases/genetics , Endoribonucleases/metabolism , Glucose/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Hypoxia/physiology , Cell Hypoxia/genetics , Glutamine/metabolism , Gene Knockdown TechniquesABSTRACT
Objective. Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling) significantly suppressed the glioblastoma cell proliferation and modified the hypoxia regulation. The present study is aimed to investigate the impact of hypoxia on the expression of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine aminotransferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) in U87MG glioblastoma cells in relation to knockdown of ERN1 with the intent to reveal the role of ERN1 signaling pathway on the endoplasmic reticulum stress-dependent regulation of expression of these genes. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed to hypoxia introduced by dimethyloxalylglycine for 4 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH, PSAT1, PDPH, SHMT1, and ATF4 genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that hypoxia up-regulated the expression level of PHGDH, PSAT1, and ATF4 genes in control U87MG cells, but PSPH and SHMT1 genes expression was down-regulated. The expression of PHGDH, PSAT1, and ATF4 genes in glioblastoma cells with knockdown of ERN1 signaling protein was more sensitive to hypoxia, especially PSAT1 gene. At the same time, the expression of PSPH gene in ERN1 knockdown cells was resistant to hypoxia. The expression of SHMT1 gene, encoding the enzyme responsible for conversion of serine to glycine, showed similar negative sensitivity to hypoxia in both control and ERN1 knockdown glioblastoma cells. Conclusion. The results of the present study demonstrate that the expression of genes responsible for serine synthesis is sensitive to hypoxia in gene-specific manner and that ERN1 knockdown significantly modifies the impact of hypoxia on the expression of PHGDH, PSAT1, PSPH, and ATF4 genes in glioblastoma cells and reflects the ERN1-mediated reprograming of hypoxic regulation at gene expression level.
Subject(s)
Glioblastoma , Protein Serine-Threonine Kinases , Humans , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Glioblastoma/genetics , Cell Hypoxia/genetics , Serine/genetics , Serine/metabolism , Endoribonucleases/genetics , Hypoxia/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/geneticsABSTRACT
Objective. The aim of the current study was to investigate the expression of genes encoded homeobox proteins such as MEIS3 (Meis homeobox 3), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, and LHX6 in U87 glioma cells in response to glutamine deprivation in control glioma cells and cells with knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of a possible dependence on the expression of these important regulatory genes from glutamine supply and ERN1 signaling. Methods. The expression level of MEIS3, SPAG4, LHX, LHX2, and LHX6 genes was studied by real-time quantitative polymerase chain reaction in control U87 glioma cells (transfected by vector) and cells with ERN1 knockdown after exposure to glutamine deprivation. Results. It was shown that the expression level of MEIS3 and LHX1 genes was up-regulated in control glioma cells treated by glutamine deprivation. At the same time, the expression level of three other genes (LHX2, LHX6, and SPAG4) was down-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on LHX1 gene expression in glioma cells, but did not change significantly the sensitivity of all other genes expression to this experimental condition. Conclusion. The results of this investigation demonstrate that the exposure of U87 glioma cells under glutamine deprivation significantly affected the expression of all genes studied encoding the homeobox proteins and that this effect of glutamine deprivation was independent of the endoplasmic reticulum stress signaling mediated by ERN1, except LHX1 gene.
