Abstract
The expression of retinoblastoma and several retinoblastoma-related genes was studied in glioma cell line U87 and its subline with knockdown of ERN1 (endoplasmic reticulum—nuclei-1), the main endoplasmic reticulum stress sensing and signaling enzyme. It was shown that a blockade of the ERN1 enzyme function increases the expression levels of retinoblastoma, retinoblastoma-like 1 and most retinoblastoma related genes: EID1, JARID1B, E2F1, E2F3, RBAP48 and CTIP, does not change RNF40 and RBAP46 and decreases KDM5A. We have also demonstrated that hypoxia reduces the expression levels of retinoblastoma, EID1, and E2F1 in ERN1-deficient glioma cells only. At the same time, the expression levels of retinoblastoma-like 1, E2F3, RBAP46, RBAP48 and CTIP decrease, while JARID1B and RBBP2 increase in both types of cells in hypoxic conditions, but the expression is much stronger in cells with suppressed function of ERN1. The expression level of JARID1B and KDM-5A mRNA is also enhanced in glutamine deprivation condition in both tested cell types, moreover, this effect is amplified by the blockade of the ERN1 enzyme function. The expression levels of retinoblastoma, EID1, RBAP48, and E2F3 are decreased in glutamine deprivation condition only in ERN1-deficient glioma cells, but RBL1, CTIP, RBAP46, and E2F1—in both tested cell types with more significant effect in ERN1-deficient cells. Glucose deprivation condition leads to a decrease of expression levels of retinoblastoma, RBL1, E2F3, RBAP46, and RBAP48 in both used cell types and of EID1 and E2F1 only in glioma cells with suppressed function of signaling enzyme ERN1. Thus, expression levels of retinoblastoma and most retinoblastoma-related genes are increased under a blockade of ERN1 enzyme function and significantly changed in hypoxia, glucose or glutamine deprivation conditions both in control U87 cells and ERN1-deficient cells, but inhibition of the unfolded protein response sensor ERN1 predominantly enhances these effects. Moreover, it is possible that the induction of the expression of retinoblastoma and most retinoblastoma-related genes after knockdown of ERN1 plays an important role in suppression of glioma proliferation.
Highlights
The endoplasmic reticulum is a key organelle in the cellular response to ischemia, hypoxia, and some chemicals which activate a complex set of signaling pathways named the unfolded protein response
The level of suppression of the enzymatic activity of ERN1 was estimated by analysis of the expression of XBP1 and its splice variant (XBP1s) in U87 glioma cells that overexpress a dominant-negative construct of endoplasmic reticulum-nuclei-1 as compared to control glioma cells transfected with a vector
Exposure of cells to hypoxia for 16 hrs as well as to glutamine or glucose deprivation conditions leads to a decrease of retinoblastoma mRNA expression levels, but only in glioma cells with suppressed function of the signaling enzyme ERN1: –21%, –46%, and –54%, respectively, as compared to control 2 (Figure 2)
Summary
The endoplasmic reticulum is a key organelle in the cellular response to ischemia, hypoxia, and some chemicals which activate a complex set of signaling pathways named the unfolded protein response. Minchenko et al / American Journal of Molecular Biology 2 (2012) 21-31 for survival or, alternatively, to enter cell death programs through endoplasmic reticulum-associated machineries [1,2,4]. As such, it participates in the early cellular response to the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, occurring under both physiological and pathological conditions
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