The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells

Abstract

Hypoxia activates the expression of proangiogenic and survival promoting factors as well as proinflammatory cytokines that support tissue inflammation. Hypoxia and inflammation are associated with tumor progression. The identification of the factors participating in the hypoxia associated inflammation is essential to develop strategies to control tumor hypoxia. The transcription factor ZNF395 was found to be overexpressed in various tumors including glioblastomas particularly in the network of a hypoxic response pointing to a functional role of ZNF395. On the other hand, ZNF395 was suggested to have tumor suppressor activities which may rely on its repression of proinflammatory factors. To address these conflictive observations, we investigated the role of ZNF395 in the expression of proinflammatory cytokines in the astrocytoma cell line U87-MG under hypoxia. We show that ZNF395 is a target gene of the hypoxia inducible factor HIF-1α. By gene expression analysis, RT-PCR and ELISA, we demonstrated that the siRNA-mediated suppression of ZNF395 impairs the hypoxic induction of IL-1β, IL-6, IL-8, and LIF in U87-MG cells. At ambient oxygen concentrations, ZNF395 had no enhancing effect, indicating that this transcriptional activation by ZNF395 is restricted to hypoxic conditions. Our results suggest that ZNF395 contributes to hypoxia associated inflammation by superactivating proinflammatory cytokines.

Figure 1

RTS3b, C33A, HEK293, U87-MG, and U937 cells were either grown under ambient O₂ atmosphere or in the presence of 2% O₂ for 12 hours before total RNAs or total protein extracts were prepared. (a) Quantitative real time RT-PCR was performed with each RNA and primers specific for ZNF395 and HPRT, which served as house-keeping gene. The ZNF395 values for RTS3b grown at ambient atmosphere were arbitrary set as 1 and the fold activations obtained for all other cells were calculated according to the comparative threshold method as previously described [27]. (b) A Western blot with 60 μg protein extracts from each cell line grown under normoxia or hypoxia was developed with antibodies against HIF-1α, ZNF395, or actin, which served as loading control. (c) U87-MG cells were either left untransfected (lanes 1, 2) or transfected with control siRNA (lane 3) or siRNA against HIF-1α (lane 4). 36 hours later, the cells were set to hypoxia for 12 hours, as indicated, before protein extracts were prepared and a Western blot with antibodies against ZNF395 and actin was performed. One set of cells was transfected with siControl or siHIF-1α and 48 hours later total RNA was isolated, which was used to perform qRT-PCR with primers for HIF-1α and HPRT. The fold activation was calculated as in (a).

Figure 2

U87-MG cells were transfected with control siRNA or siRNA against ZNF395 and total RNA was isolated 48 hours after transfection (normoxia) or the cells were transferred 36 hours after transfection to 2% O₂ for additional 12 hours (hypoxia) before total RNA was isolated. cDNA was prepared and quantitative real time-PCRs were performed with primers specific for IL-6, IL-8, IL-1β, and LIF (a) and for MCP-1/CCL2, CA IX, and ZNF395 (b) RT-PCR of HPRT serving as internal control. The values obtained by dividing the Ct values for the cytokine and HPRT, respectively, from the siControl cells grown under normoxia were set as 1, and the fold activations were calculated. The standard deviations are given. Each PCR was performed six times in duplicate except that for ZNF395. (c) U87-MG cells were transfected with siControl or siZNF395; 24 hours later, one set of cells was incubated in the presence of DMOG, while the other set of cells obtained the equivalent amount of ETOH (which served as solvent for DMOG) as control. Another 24 hours later, the supernatant was used for ELISA to measure the level of IL-6 and IL-8. Each ELISA was performed two times in triplicate. QRT-PCR with RNA isolated from these cells was used to investigate the effect of DMOG on the expression of ZNF395. ^∗ p < 0.05; ^∗∗ p < 0.01 by Student's t-test.