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

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)alpha induces NF-kappa B activation in GBM, and as several common signaling mediators are involved in TNF alpha and TLR4-mediated NF-kappa B activation, we investigated the role of TLR4 in the regulation of NF-kappa B activation and inflammatory responses in TNF alpha-treated glioma cells. TNF alpha elevated TLR4 expression and inhibition of TLR4 signaling by either signaling inhibitor, neutralizing antibody, or small interfering RNA (siRNA)-attenuated TNF alpha-induced NF kappa B activation. TLR4-mediated NF-kappa B activation was independent of canonical myeloid differentiation factor 88 signaling but involved toll/IL-1R homology domain-containing adaptor protein-inducing interferon-beta. Inhibition of TLR4 signaling abrogated TNF alpha-induced increase in (1) transcription factors interferon (IFN) regulatory factor 3 and STAT-1 and (2) IFN beta and inflammatory cytokines/chemokines expression. Furthermore, TNFa-induced TLR4-dependent increase in AKT activation and HIF-1 alpha transcriptional activation suggested the existence of TLR4-AKT-HIF-1 alpha axis. Importantly, TNF alpha-induced TLR4 was abrogated in cells transfected with dominant negative I kappa B and HIF-1 alpha siRNA. Our studies indicate that TNFa triggered TLR4-HIF-1 alpha and NF-kappa B-TLR4 feed-forward loops act in tandem to sustain inflammatory response in glioma.