RETICULUM–NUCLEI SIGNALING ENZYME-1 KNOCKDOWN MODULATES EFFECT OF HYPOXIA AND ISCHEMIA ON THE EXPRESSION OF CIRCADIAN GENES IN GLIOMA CELLS

The main molecular components of circadian clock system are proteins which play a significant role in the control of both metabolism and malignant tumor growth. Endoplasmic reticulum stress as well as hypoxia and ischemia are important factors for tumor neovascularization and growth. We have studied the expression of circadian genes in the glioma cell line U87 under knockdown of endoplasmic reticulum–nuclei-1 (ERN1) sensing and signaling enzyme. It was shown that blockade of ERN1 leads to increase in the expression levels of PER1, PER3 and CLOCK mRNA; but the CRY1, PER2, BMAL1, BMAL2 and DEC2 mRNA levels are decreased. Moreover, the expression levels of most of the studied genes are increased under glucose or glutamine deprivation conditions both in control and ERN1-deficient glioma cells; however knockdown of ERN1 modifies the effect of these ischemic conditions. Hypoxia has different effects on the expression levels of circadian genes and these effects are dependent on ERN1 function. Hypoxia induces the expression of PER1, BMAL1 and DEC2 and decreased – PER3, CLOCK, CRY1 and BMAL2 mRNA in control glioma cells, while in genetically modified cells it induces the expression of BMAL1 mRNA only. Thus, the expression of circadian genes is dependent on ERN1 signaling enzyme function in normal, hypoxic and ischaemic conditions.


INTRODUCTION
Circadian factors PER1, PER2, PER3, CLOCK, BMAL1, CRY1 and CRY2 are the main molecular components of the circadian clock system and play a significant role in Biol. Stud. 2011: 5(2); 37-50 • DOI: https://doi.org/10.30970/sbi.0502.151 www.http://publications.lnu.edu.ua/journals/index.php/biology control of both metabolism and malignant tumor growth [1][2][3][4][5][6]. The rapid growth of different solid malignant tumors generates micro-environmental changes in association to nutrient deprivation, hypoxia and acidosis [7,8]. Under those ischaemic conditions, cancer cells trigger an adaptive response and initiate the formation and attraction of new blood vessels inside the tumor. Hypoxia and ischemia as well as endoplasmic reticulum stress are the main factors which are necessary for tumor neovascularization and growth [7,9]. Ischemia and hypoxia have been shown to induce a set of complex intracellular signaling events known as the unfolded protein response, which is mediated by three sensor systems to adapt cells for survival or, alternatively, to enter cell death programs through endoplasmic reticulum-associated machineries [7,[10][11][12][13]. The endoplasmic reticulum-nuclei-1 signaling enzyme (also named by inositol requiring enzyme-1alpha) is a major sensor which controls hundreds of genes [14,15]. 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. Two distinct catalytic domains of the bifunctional signaling enzyme endoplasmic reticulum-nuclei-1 were identified: a serine/threonine kinase and an endoribonuclease which contribute to endoplasmic reticulum -nuclei-1 signalling. The endoplasmic reticulum-nuclei-1-associated kinase activity autophosphorylates and dimerizes this enzyme, leading to the activation of its endoribonuclease domain, degradation of a specific subset of mRNA and initiation of the pre-XBP1 (X-box binding protein 1) mRNA splicing [14,16]. Mature XBP1 mRNA splice variant encodes a transcription factor that has a different C-terminus amino acid sequence and stimulates the expression of hundreds of unfolded protein response-specific genes [16,17].
Recently the endoplasmic reticulum-nuclei-1 gene was proposed as a major contributor to tumor progression among protein kinases because single mutations were detected in different human cancers and encoded by this gene enzyme [18]. Moreover, the growing tumor requires the endoplasmic reticulum stress for own neovascularization and growth and the complete blockade of endoplasmic reticulum -nuclei-1 signal transduction pathway has anti-tumor effects [7,9]. The endoplasmic reticulum stress response-signalling pathway is linked to the neovascularization process, tumor growth and differentiation, and cell death processes [8,9].
Circadian clock system controls the cell proliferation process and carcinogenesis, thus circadian proteins PER1, PER2, CLOCK, BMAL1 and CRY1 function as tumor suppressors [19][20][21]. Therefore, the overexpression of the circadian gene PER1 or PER2 in cancer cells resulted in significant growth inhibition and in apoptosis. Circadian genes have an emerging role as key players in malignant transformation [21]. Deregulated expression of the PER1, PER2 and PER3 genes as well as inactivation or knockout PER2, PER1, CLOCK and BMAL1 leads to malignization, especially in the breast, as well as to the appearance of different disorders such as obesity [22][23][24][25]. A better understanding of the impact of circadian gene networks on cell cycle control as well as nutrient balance at the molecular, cellular and system levels promises to shed light on the emerging association between circadian timing and disorders of sleep, diabetes, obesity, and cancer.
In this study we have investigated the role of signaling enzyme endoplasmic reticulum-nuclei-1 in the expression of different circadian genes in the glioma cell line U87 for evaluation of circadian gene expression responsibility from this signaling enzyme function under hypoxic and ischemic (glucose or glutamine deprivation) conditions.

MATERIALS AND METHODS
Cell Lines and Culture Conditions. The glioma cell line U87 was obtained from ATCC (U.S.A.) and grown in high glucose (4,5 g/l) Dulbecco's modified Eagle's minimum essential medium (Gibco, Invitrogen, USA) supplemented with glutamine (2 mM), 10% fetal bovine serum (Equitech-Bio,Inc., USA), penicillin (100 units/ml; Gibco) and streptomycin (100 µg/ml; Gibco) at 37 o C in a 5% CO 2 incubator. In this work we used two sublines of this glioma cell line. One subline has suppressed both protein kinase and endoribonuclease activities of sensor and signaling enzyme endoplasmic reticulum-nuclei-1, which was obtained by selection of stable transfected clones with overexpression of endoplasmic reticulum-nuclei-1 dominant/negative constructs (dnERN1) [13]. Second subline was obtained by selection of stable transfected clones with vector, which was used for creation of dnERN1. This subline was used as control 1. Control 2 represents U87 glioma cells overexpressed dominant-negative construct of endoplasmic reticulum-nuclei-1 ( fig. 1).
Full suppression of enzymatic activity of dnERN1 was estimated by the analysis of expression of XBP1 and its splice variant (XBP1 s ) in U87 glioma cells that overexpress a dominant-negative construct of endoplasmic reticulum-nuclei-1 as compared to control glioma cells transfected with vector. As shown in fig. 1, inductor of endoplasmic reticulum stress tunicamycin strongly induced the alternative splicing of XBP1 only in control glioma cells.
Hypoxic conditions were created in special incubator with 3% oxygen and 5% carbon dioxide levels and culture plates were exposed for 16 hrs. For glucose or glutamine deprivation the growing medium in culture plates was replaced with medium without glucose or glutamine and exposed for 16 hrs.
RNA isolation. Total RNA was extracted from different tumor tissues and normal tissue counterparts using Trizol reagent according to manufacturer protocol (Invitrogen, U.S.A.). RNA pellets were washed with 75% ethanol and dissolved in nuclease-free water.
The amplification of beta-actin cDNA was performed using primers: forward -5′-CGTACCACTGGCATCGTGAT-3′ and reverse -5′-GTGTTGGCGTACAGGTCTTT-3′. The expression of beta-actin mRNA was used as control of analyzed RNA quantity. The primers were received from "Sigma" (USA).
An analysis of quantitative PCR was performed using special computer program "Differential expression calculator" and statistic analysis -in Excel program. The amplified DNA fragments were separated on a 2% agarose gel and visualized by 5x Sight DNA Stain (EUROMEDEA).

RESULTS
In this study, we used human glioma cell line U87 and its genetically modified variant (with suppressed activity of signaling enzyme endoplasmic reticulum-nuclei-1) to investigate the involvement of endoplasmic reticulum stress system in the effect of hypoxia and ischemia (glutamine or glucose deprivation) on the expression of different genes of circadian clock. For this aim, the cells were incubated at 37°C before harvesting in regular DMEM medium (control) and in the medium without glucose or glutamine as well as regular DMEM medium in hypoxic conditions (3% oxygen) for 16 hours. The mRNA expression levels of different circadian genes were quantified by real time polymerase chain reaction of complementary DNA.
We have found that PER1, PER2, PER3, CLOCK, BMAL1, BMAL2, DEC2 and CRY1 mRNA are expressed in the human glioma cell line U87 and the level of its expression depends upon the signaling enzyme endoplasmic reticulum-nuclei-1 function in normal, hypoxic and ischemic conditions. As shown in fig. 2, the level of PER1 mRNA expression is increased more than two fold in glioma cells, deficient in signaling enzyme endoplasmic reticulum-nuclei-1, as compared to control cells. Exposure of glioma cells to hypoxia for 16 hours or to glutamine deprivation conditions leads to an increase in the level of PER1 mRNA expression, but only in control cells (+31 and +16%, correspondingly). At the same time cell exposure to glucose deprivation conditions leads to an increase of PER1 mRNA expression both in control and in genetically modified cells (+25 and +22%, correspondingly).
The expression level of PER2 mRNA is decreased in glioma cells with suppressed activity of signaling enzyme endoplasmic reticulum-nuclei-1 in two fold as compared to control glioma cells ( fig. 3). Effect of glucose or glutamine deprivation conditions on the expression level of PER2 mRNA was observed both in control (+18 and +11%, correspondingly) and genetically modified (+36 and +27%, correspondingly) glioma cells been less significant in control cells. However, hypoxia reduces the expression level of PER2 mRNA in cells without endoplasmic reticulum-nuclei-1 activity and does not change it in control glioma cells ( fig. 3).
As shown in fig. 4, the level of PER3 mRNA expression is increased (+69%) in glioma cells with suppressed endoplasmic reticulum-nuclei-1 activity as compared to control glioma cells. Hypoxia significantly reduced expression of this mRNA level in both studied cell types, however glutamine deprivation condition did not change the expression level of PER3 mRNA both in control and genetically modified glioma cells. At the same time, the effect of glucose deprivation conditions on the expression of PER3 mRNA was different in control glioma cells (+28%) and cells with suppressed activity of endoplasmic reticulum-nuclei-1 (-16%) ( fig. 4).

DISCUSSION
Highly aggressive brain tumor glioma forms from the malignization of astrocytes which are the most abundant cell types in the brain. They play an important role in the maintenance of neuronal functions. The molecular mechanisms underlying the uniquely aggressive behavior of these tumors have not yet been elucidated. In this study we investigate the effect of suppression of enzymatic activity of the main signaling system of endoplasmic reticulum stress on the expression of basic circadian factors which play a significant role in the control of main metabolic processes as well as proliferation process and malignant tumor growth in normoxic, hypoxic and ischemic conditions [9].
Our results clearly demonstrate that the expression pattern of the main circadian genes is controlled by the endoplasmic reticulum stress system which is hyper active in malignant tumors. We have shown that swiching off the endoplasmic reticulum stress in glioma cells by suppressing the activity of endoplasmic reticulum-nuclei-1, which is the major sensor and signaling system of this stress, changes the expression pattern of most circadian genes which, as known, are disregulated in malignant tumors. In glioma cells that lack the activity of the main endoplasmic reticulum stress signaling enzyme the expression level of PER1, which has strong tumor suppressor function, is significantly increased [5]. Its overexpression in cancer cells resulted in significant growth inhibition and apoptosis. This data correlates with suppression of glioma growth.
Endoplasmic reticulum stress changes the expression of hundreds of genes for the purpose of cellular adaptation to stress conditions and cell survival via the initiation of the pre-XBP1 mRNA splicing. Mature XBP1 mRNA encodes a transcription factor that stimulates the expression of unfolded protein response-specific genes. At the same time the expression of most of the endoplasmic reticulum stress-dependent genes is up-regulated, but there is a group of genes the expression of which is down-regulated [9,17]. Thus, the expression of pro-angiogenic factors IL-6, IL-8 and VEGF-A is downregulated and the expression of the anti-angiogenic factors SPARC, decorin, thrombospondin-1 and some other proteins functionally linked to mesenchymal differentiation (MYRL2, FHL1, COLL1A2) is up-regulated. Results of our investigastion have shown that the blockade of ERN1 leads to a change in the expression levels of circadian genes: Showing an increase in the expression levels of PER1, PER3 and CLOCK mRNA and a decrease the expression levels of CRY1, PER2, BMAL1, BMAL2 and DEC2 mRNA. It is known that in the circadian clock system there are strong interactions between different circadian factors with feedback mechanisms [4,5].
The circadian genes have the emerging role as key players in malignant transformation, because factors, encoded by these genes, participate in cell cycle control and tumor growth. Deregulated expression of circadian genes as well as inactivation or knockout of PER1, CLOCK and BMAL1 leads to malignization, especially in the breast, or the appearance of various disorders obesity in particular. A better understanding of the impact of circadian gene networks on cell cycle control as well as nutrient balance at the molecular, cellular and system levels promises to shed light on the emerging association between circadian timing and various medical conditions such as disorders of sleep, diabetes, obesity, and cancer [23,24].
The rapid growth of different solid malignant tumors generates micro-environmental changes due to nutrient deprivation, hypoxia and acidosis. Under those ischemic conditions, cancer cells trigger an adaptive response and initiate the formation of new blood vessels inside the tumor which are needed for its proliferation [8]. Hypoxia and ischemia are the main factors necessary for tumor growth via induction of complex intracellular signaling events and endoplasmic reticulum stress [9]. We have shown that hypoxia as well as ischemia significantly changes the expression pattern of different circadian genes and that these changes are dependent on endoplasmic reticulum stress signaling, because complete blockade of the endoplasmic reticulum-nuclei-1 enzyme activity modifies the effect hypoxia and ischemia has on most circadian genes expression levels in control glioma cells.