Involvement of TNFα-induced TLR4–NF-κB and TLR4–HIF-1α feed-forward loops in the regulation of inflammatory responses in glioma

Abstract

The precise role of different toll-like receptor (TLR) superfamily members is just beginning to get elucidated in glioblastoma multiforme (GBM). In this study, we observed heightened TLR4 levels in GBM tumor samples as compared to adjacent normal tissue. Since the pro-inflammatory cytokine tumor necrosis factor (TNF)α induces NF-κB activation in GBM, and as several common signaling mediators are involved in TNFα and TLR4-mediated NF-κB activation, we investigated the role of TLR4 in the regulation of NF-κB activation and inflammatory responses in TNFα-treated glioma cells. TNFα elevated TLR4 expression and inhibition of TLR4 signaling by either signaling inhibitor, neutralizing antibody, or small interfering RNA (siRNA)-attenuated TNFα-induced NF-κB activation. TLR4-mediated NF-κB activation was independent of canonical myeloid differentiation factor 88 signaling but involved toll/IL-1R homology domain-containing adaptor protein-inducing interferon-β. Inhibition of TLR4 signaling abrogated TNFα-induced increase in (1) transcription factors interferon (IFN) regulatory factor 3 and STAT-1 and (2) IFNβ and inflammatory cytokines/chemokines expression. Furthermore, TNFα-induced TLR4-dependent increase in AKT activation and HIF-1α transcriptional activation suggested the existence of TLR4-AKT-HIF-1α axis. Importantly, TNFα-induced TLR4 was abrogated in cells transfected with dominant negative IκB and HIF-1α siRNA. Our studies indicate that TNFα triggered TLR4-HIF-1α and NF-κB-TLR4 feed-forward loops act in tandem to sustain inflammatory response in glioma.

Tumor Necrosis Factor Alpha-Induced Hypoxia-Inducible Factor 1α–β-Catenin Axis Regulates Major Histocompatibility Complex Class I Gene Activation through Chromatin Remodeling

“…Although HIF-1␣ has been established as an important contributor to glioma progression, its role in the regulation of immunomodulatory HLA is not well understood. Besides hypoxia, inflammation-induced HIF-1␣ also plays an important role in glioma progression under normoxia (17). Our finding that both inflammation and hypoxia increase MHC-I expression suggests that HIF-1␣, independently of its stimulus of induction, is an important contributor to MHC-I expression in glioma.…”

“…Cells were washed twice and resuspended in fluorescence-activated cell sorting (FACS) buffer (1% bovine serum albumin in PBS with 0.1% NaN 3 ). At least 20,000 events per sample were collected on a FACSCalibur flow cytometer (Becton Dickinson) and ana-lyzed with CellQuest Pro software (Becton Dickinson) as described previously (17).…”

“…A Renilla luciferase vector (pRL-TK; Promega) at 20 ng/well was included for normalization of transfection efficiency. Cells were harvested at the indicated time points, and cell lysates were analyzed with the Dual-Luciferase reporter assay system (Promega) as described previously (17). The pGL3-HLA-B7 and pGL3-HLA-B7⌬␣ luciferase reporters were a kind gift from Peter J. van den Elsen, Leiden University Medical Center, Leiden, Netherlands (29).…”

“…This, together with the ability of HIF-1␣ to regulate ␤-catenin activity, suggests the existence of a ␤-catenin-HIF-1␣ axis in TNF-␣-treated cells under normoxia. We have reported that TNF-␣-induced HIF-1␣ is Akt dependent (17). As Akt is involved in Wnt signaling (51), we investigated whether ␤-catenin activation is Akt dependent.…”

“…Hypoxic tumor microenvironment plays an important role in the enhancement of tumor escape from immune surveillance (39). Besides, inflammation, which is recognized as an indispensable participant in tumor progression, induces HIF-1␣ activation in gliomas (17). As MHC-I expression, either constitutive or inducible, is dependent on microenvironmental cues, we investigated whether TNF-␣-induced HIF-1␣ regulates MHC-I expression through the modulation of a distinct repertoire of transcription factors, coactivators, and chromatin remodelers.…”