Hypoxic regulation of insulin receptor substrate 2 gene expression is differently regulated by endoplasmic reticulum stress and hydrocortisone in normal human astrocytes and glioblastoma cells.

Abstract

Objective. The insulin receptor substrate 2 (IRS2) is phosphorylated by the tyrosine kinase activity of the insulin receptor and the insulin-like growth factor I (IGF-1) receptor upon receptor stimulation. It mediates insulin signaling controlling metabolism as well as cell proliferation and invasion in tumors. Hypoxia and endoplasmic reticulum (ER) stress are significant factors in regulating the growth of malignant tumors including glioblastoma. The present study aims to investigate the regulation of the IRS2 gene expression in normal human astrocytes and U87MG glioblastoma cells by hypoxia and ER stress in the context of the native stress hormone hydrocortisone, which is widely used for the co-treatment of glioblastoma. Methods. The normal human astrocytes (line NHA/TS) and U87MG glioblastoma cells were used. Hypoxia was introduced by the HIF1A prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG), which mimics the effects of hypoxia under normoxic conditions. Tunicamycin and thapsigargin were used for the induction of ER stress. Hydrocortisone-water soluble BioReagent, suitable for cell culture (cyclodextrin-encapsulated hydrocortisone) was used. Cells were treated with DMOG, tunicamycin, thapsigargin, and hydrocortisone for 4 h. RNA was extracted with TRIzol reagent. IRS2 gene expression was examined by quantitative real-time RT-PCR and normalized to beta-actin mRNA. Results. It was found that hypoxia decreased the IRS2 gene expression in normal human astrocytes, but upregulated it in glioblastoma cells. At the same time, hydrocortisone did not significantly change the expression of this gene in both normal astrocytes and glioblastoma cells. However, hypoxia in combination with hydrocortisone strongly increased IRS2 gene expression in both cell types. Tunicamycin decreased the expression of the IRS2 gene in normal astrocytes, but increased it in glioblastoma cells and this effect of tunicamycin was not significantly altered by hypoxia in both cell types. At the same time, thapsigargin did not significantly alter the expression of the IRS2 gene in normal astrocytes, but it strongly upregulated it in glioblastoma cells. Hypoxia modified the effect of thapsigargin on this gene expression in both cell types, but by different ways: decreased in normal astrocytes and increased in glioblastoma cells. In addition, the impact of tunicamycin and thapsigargin on IRS2 gene expression was significantly upregulated by hydrocortisone in normal astrocytes and especially in glioblastoma cells. At the same time, the combined effect of hypoxia and hydrocortisone enhanced the expression of the IRS2 gene in tunicamycintreated normal astrocytes, especially in the glioblastoma cells. Hydrocortisone also increased the effect of hypoxia on this gene expression in thapsigargin-treated normal astrocytes and decreased it in glioblastoma cells. Conclusion. Our findings provide evidence that hypoxic regulation of IRS2 gene expression is modified by inducers of ER stress and hydrocortisone, but differently in normal astrocytes and glioblastoma cells and that the combined effect of hypoxia with ER stress and hydrocortisone greatly enhanced this gene expression in both cell types, especially in the glioblastoma cells.