EffEct of hypoxia on thE ExprEssion of CCN 2 , PLAU , PLAUR , SLURP 1 , PLAT and ITGB 1 gEnEs in Ern 1 knockdown U 87 glioma cElls

The endoplasmic reticulum stress is an important factor of tumor growth and is induced in cancer cells. We have studied the effect of erN1 knockdown as well as hypoxia on the expression of genes encoding factors, which control cell proliferation, in U87 glioma cells. It was shown that the complete blockade of erN1 enzyme function leads to an increase of the PlAT (tissue plasminogen activator), ccN2 (ccN family member 2), and ITGB1 (integrin β-1) as well as to a decrease of PLAU (plasminogen activator, urokinase), PLAUR (plasminogen activator, urokinase receptor), and SlUrP1 (secreted lY6/PlAUr domain containing 1) mrNA expressions. Moreover, we have shown that hypoxia does not affect the expression level of ITGB1 mrNA, but increases that of ccN2, PlAUr, SlUrP1, and PlAT mrNA and decreases the expression level of only PlAU mrNA in control glioma cells. At the same time, in erN1 knockdown glioma cells the expression level of PlAU, PlAUr, and SlUrP1 mrNA is decreased under hypoxia, but PlAT and ITGB1 mrNA expression levels are increased under these experimental conditions. Thus, results of this study have shown that the expression level of all studied genes is affected by erN1 knockdown as well as by hypoxia and that the effect of hypoxia mostly depends on erN1 signaling enzyme function.

T he endoplasmic reticulum stress is associa ted with accumulation of unfolded proteins in the endoplasmic reticulum and is an im portant factor of malignant tumor growth [1,2].It controls the neovascularization and proliferation processes [3,4].This adaptive unfolded protein re sponse is activated upon the accumulation of mis folded proteins in the endoplasmic reticulum and is mediated by several endoplasmic reticulumresident sensors named ERN1 (from endoplasmic reticulum to nuclei1), PERK (PRKlike ER kinase), and ATF6 (activating transcription factor 6); however, bifunc tional enzyme ERN1 is the dominant sensor and signaling system [2,4,5].The endoplasmic reticu lum stress participates in the early cellular response to the accumulation of misfolded proteins in the lu men of the endoplasmic reticulum [1,2].Activation of the unfolded protein response tends to adapt cells for survival or, alternatively, to enter cell death pro grams [2,6].Hypoxia is associated with glioma de velopment and locally induces an adaptive response which imparts to tumor cells an enhanced survival and a more agressive behaviour [3,7,8].
The signaling enzyme ERN1 has endoribonu clease activity which is responsible for degradation of a specific subset of mRNA and initiation of the alternative splicing of preXBP1 (Xbox binding pro tein 1) mRNA [6,9,10].Mature XBP1 mRNA splice variant encodes a transcription factor that stimulates the expression of more than six hundred unfolded protein response-specific genes [10,11].Moreover, the spliced form of XBP1 nuclear translocation is enhanced through phosphorylation by different pro tein kinases [12,13].It was also shown that the al ternative splicevariant of XBP1 interacts with the FOXO1 (forkhead box O1) transcription factor and directs it toward proteasomemediated degradation [14].
Malignant gliomas are highly aggressive tu mors and are characterized by marked angiogenesis and extensive tumor cell invasion into the normal brain parenchyma [2].Hypoxia as well as endoplas mic reticulum stress is associated with glioma de velopment and locally induces an adaptive response which imparts to tumor cells an enhanced survival and a more agressive behaviour [2].It was shown that the complete blockade of ERN1 signal transduc tion pathway has antitumor effects in lung cancer and glioma cells [6,15,16].A better knowledge of tumor responses to hypoxia and endoplasmic re ticulum stress is required to elaborate therapeutical strate gies of cell sensibilization and angiogenesis inhibition, based on the blockade of survival mecha nisms.
Recently it was shown that CCN2 (CCN fami ly member 2) protein also known as IGFBP8 (insulin like growth factor binding protein 8) plays a role in cell proliferation, differentiation, angiogenesis, as well as cell adhesion in many cell types, and is related to plateletderived growth factor [22].Fur thermore, loss of connective tissue growth fac tor activates miR18b and promotes cell growth in nasopharyngeal carcinoma.It was also shown that integrin beta1 is involved in a variety of processes including cell proliferation, migration and invasion [19,20,23].
The main goal of this study was to investigate the role of the expression of genes related to the con trol of cell proliferation (CCN2, PLAU, PLAUR, SLURP1, PLAT, and ITGB1) in glioma U87 cells with ERN1 knockdown and its regulation upon hy poxia.

materials and methods
cell lines and culture conditions.The glioma cell line U87 was obtained from ATCC (USA) and grown in high glucose (4.5 g/l) Dulbecco's modified Eagle's minimum essential medium (DMEM; Gib co, Invitrogen, USA) supplemented with glutamine (2 mM), 10% fetal bovine serum (EquitechBio, Inc., USA), penicillin (100 units/ml; Gibco) and strepto mycin (0.1 mg/ml; Gibco) at 37 °C in a 5% CO 2 in cubator.In this work we used two sublines of this glioma cell line.One subline was obtained by selec tion of stable transfected clones with overexpression of vector (pcDNA3.1),which was used for creation of dominantnegative constructs (dnERN1).This un treated subline of glioma cells (control glioma cells) was used as control 1 in the study of hypoxic effects on the expression level of ccN2, PlAU, PlAUr, SlUrP1, PlAT and ITGB1 genes.The other subline was obtained by selection of stable transfected clones with overexpression of dnERN1 and has suppressed both protein kinase and endoribonuclease activities of ERN1 signaling enzyme.These cells were ob tained from prof.M. Moenner (France) [2,15].The expression level of PlAU, PlAUr, SlUrP1, PlAT, ccN2 and ITGB1 genes in these cells was compared with that in the cells, transfected by vector (con trol 1), but this untreated subline was also used as control 2 for investigation the effect of hypoxia on the expression level of these genes under blockade ERN1 function.Hypoxic condition was created in special incubator with 3% oxygen and 5% carbon dioxide levels; culture plates with complete DMEM were exposed to these conditions for 16 hrs.
The suppression level of ERN1 enzymatic ac tivity in glioma cells that overexpress a dominant negative construct of endoplasmic reticulum-nu clei1 (dnERN1) was previously shown by analysis of the expression of XBP1 alternative splice vari ant (XBP1s), a key transcription factor in ERN1 signaling , and phosphorylated isoform ERN1 using cells treated by tunicamycin (0.01 mg/ml during 2 hours) [24].
rNA isolation.Total RNA was extracted from glioma cells using Trisol reagent (Invitrogen) as de scribed [24].RNA pellets were washed with 75% ethanol and dissolved in nucleasefree water.For ad ditional purification RNA samples were precipitated with 95% ethanol and redissolved again in nuclease free water.
reverse transcription and quantitative Pcr analysis.QuaniTect Reverse Transcription Kit (QIA експериментальні роботи GEN, Germany) was used for cDNA synthesis as de scribed previously [25].
An analysis of quantitative PCR was performed using special computer program Differential expres sion calculator.Statistical analysis was performed according to Student's test using OriginPro 7.5 software.The values of PLAU, PLAUR, SLURP1, PLAT, CCN2, and ITGB1 mRNA expressions were normalized to the expression of β-actin mRNA and represented as percent of control (100%).All values are expressed as mean ± SEM from triplicate measu rements performed in four independent experiments.

results and discussion
As shown in Fig. 1, the blockade of ERN1 en zyme, the major component of endoplasmic reticu lum stress signaling, affects the expression level of CCN2, PLAU, PLAUR, SLURP1, PLAT, and ITGB1 mRNAs in U87 glioma cells, but in different ways.Thus, the expression level of CCN2, PLAT, and ITGB1 mRNAs is increased in glioma cells, being more significant for CCN2 and PLAT: more than 5 and 4 times, correspondingly.At the same time, the expression of three other genes (PlAU, PlAUr, and SlUrP1) is significantly decreased after blockade of ERN1 signaling enzyme function (Fig. 1).Thus, the expression levels of PLAU mRNA is decreased more than 3 times, but urokinase plasminogen activator receptor as well as SLURP1 mRNA expression level was more than twice less as compared to control glioma cells.
We have also studied the hypoxic regulation of these gene expressions.As shown in Fig. 2, the exposure of glioma cells under hypoxic condition leads to significant up-regulation of CCN2 mRNA expression level (+ 54%) in control U87 glioma cells, but in ERN1 knockdown cells the effect of hypoxia had a reverse effect: CCN2 mRNA expres sion level is decreased ( 32%) in these cells (Fig. 2).Another type of hypoxic regulation was shown for PlAU gene.Thus, as shown in Fig. 3, an exposure of glioma cells under hypoxia leads to a 2fold decrease of the expression level of PLAU mRNA in control U87 glioma cells; however, in ERN1 knockdown cells the effect of hypoxia was significantly less ( 27%).At the same time, the expression of PLAUR mRNA in control U87 glioma cells is also increased (+ 21%) like CCN2 mRNA and blockade of ERN1 signaling enzyme modifies the effect of hypoxia on this mRNA expression level (Fig. 4).Thus, in these glioma cells the expression level of PLAUR mRNA is decreased ( 31%).Investigation of SLURP1 (secreted LY6/PLAUR domain containing 1) mRNA expression in U87 glioma cells has shown that the level of this mRNA expression is also affected by hypoxia in control as well as in ERN1 knockdown glioma cells like PLAUR mRNA (+ 23% in control glioma cells and 19% in ERN1 knockdown cells (Fig. 5)).
We have also shown that hypoxia has sig nificantly increased the expression level of PLAT mRNA (+ 93%) but did not affect the expression level of ITGB1 mRNA in control glioma cells (Fig. 6  and 7).At the same time, the blockade of ERN1 signaling enzyme leads to significant up-regulation of the expression of both PlAT and ITGB1 genes in U87 glioma cells: + 74% and + 22%, corresponding ly.Moreover, the knockdown of signaling enzyme ERN1 in U87 glioma cells leads to some morpho logical changes in these cells (Fig. 8) as well as to suppression of cell proliferation and growth of tumor originated from these cells [15,16].

Fig. 1. The expression level of ccN2, PlAU, PlAUr, SlUrP1, PlAT, and ITGB1 mrNAs in erN1 knockdown U87 glioma cells (dnerN1). control -cells, stable transfected by pcDNA3.1 (Vector). The level of these mrNA expressions was normalized to the β-actin and compared to the control (Vector, 100%); n = 4; * Р < 0.05 as compared to control
Results of this study clearly demonstrated that the expression levels of all tested genes (ccN2, PlAU, PlAUr, SlUrP1, PlAT and ITGB1) encoding important regulatory factor, which control cell proliferation and participate in malignant tu mor growth, is affected by ERN1 signaling enzyme knockdown.Moreover, the decrease of PLAU and PLAUR mRNA expression levels in glioma cells af ter blockade of ERN1 signaling enzyme function is argued with suppression of tumor growth from these cells [15,16], because both PLAU and PLAUR are upregulated in the tumors as well as are responsible for cell proliferation and migration [17,18].Further more, the decrease of SLURP1 mRNA expression in U87 glioma cells after ERN1 knockdown can also participate in the suppression of tumor growth be

Fig. 2. effect of hypoxia on the expression level of ccN2 (ccN family member 2) in U87 glioma cells, transfected with vector pcDNA3.1 (Vector) and erN1 knockdown cells (dnerN1), measured by quantitative
PCR.The level of CCN2 mRNA expression was normalized to the expression of β-actin.In Fig. 2-7 the changes in the expression of different mrNA in both types of glioma cells were compared to the control 1 (Vector, 100%); n = 4; * Р < 0.05 as compared to the control 1, ** Р < 0.05 as compared with the control 2 (dnerN1) Fig. 3 Relative mRNA expression, % of control 1 cause there is data that SLURP1 protein can activate protein kinases and upregulate the NFkB gene ex pression [21].

. effect of hypoxia on the expression level of PlAU (urokinase plasminogen activator) mrNA in U87 glioma cells, stable transfected with vector pcDNA3.1 (Vector) and cells, stable transfected with dominant/ negative constructs of erN1 signaling enzyme (dnerN1), measured by quantitative real-time Pcr. The level of PLAU mRNA expression was normalized to the expression of β-actin
Protein CCN2 is a multifunctional protein and plays a role in cell proliferation, differentia tion, and angiogenesis.At the same time, loss of CCN2 activates miR18b and promotes cell growth in nasopharyngeal carcinoma [22].Moreover, over expression of MYC promoted vigorous tumor vas cularization and growth without changes in VEGF Fig. 4 Relative mRNA expression, % of control 1 140 level, but enhanced neovascularization correlated with downregulation of antiangiogenic thrombos pondin1 and CCN2 which binds vascular endothe lial growth factor and inhibits VEGFinduced angio genesis [26,27].Thus, the increased level of CCN2 in our experiment with ERN1 knockdown glioma cells can also be responsible for the suppression of proliferation and growth of glioma, originated from these glioma cells.

Fig. 5. Effect of hypoxia on the expression level of SLURP1 (secreted LY6/PLAUR domain containing 1) in U87 glioma cells, transfected with vector pcDNA3.1 (Vector) and erN1 knockdown cells (dnerN1), measured by quantitative PCR. The level of SLURP1 mRNA expression was normalized to the expression of β-actin
We have also shown that PLAT mRNA expres sion was significantly up-regulated after the block ade of ERN1 signaling enzyme in glioma cells.These results are also agreed with data of Kenagy et al. [28] and Simard et al. [29].Thus, Kenagy et експериментальні роботи

Fig. 7. effect of hypoxia on the expression level of ITGB1 (integrin beta-1) in U87 glioma cells, transfected with vector pcDNA3.1 (Vector) and erN1 knockdown cells (dnerN1), measured by quantitative Pcr. The level of PLAUR mRNA expression was normalized to the expression of β-actin
al. [28] has shown that induction of cell death is as sociated with enhanced expression of several genes, including the PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die.It is pos sible, that overexpression of PLAT mRNA in ERN1 knockdown glioma cells is also in the suppression of these cells proliferation [15,16] through induction of cell death.Moreover, induction of PLAT by shark cartilage extract plays an essential role in its an tiangiogenic activity [30].Integrin beta1 (ITGB1) is also involved in a variety of processes including cell proliferation, migration and invasion as a compo nent of receptors for collagen, fibronectin, laminin, and thrombospondin [19,20,23].There is data that ITGB1 participates in PLAUmediated angiogenesis through PLAUR [20].It is possible that increase of ITGB1 expression in ERN1 knockdown glioma cells is connected with strong upregulation of antiangio genic thrombospondin1 and antiproliferative effect of ERN1 blockade [30].