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.
Similar content being viewed by others
References
Chen R, Alvero AB, Silasi DA, Steffensen KD, Mor G (2008) Cancers take their Toll—the function and regulation of Toll-like receptors in cancer cells. Oncogene 27:225–233
Kelly MG, Alvero AB, Chen R, Silasi DA, Abrahams VM, Chan S, Visintin I, Rutherford T, Mor G (2006) TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer. Cancer Res 66:3859–3868
Szajnik M, Szczepanski MJ, Czystowska M, Elishaev E, Mandapathil M, Nowak-Markwitz E, Spaczynski M, Whiteside TL (2009) TLR4 signaling induced by lipopolysaccharide or paclitaxel regulates tumor survival and chemoresistance in ovarian cancer. Oncogene 28:4353–4363
Szczepanski MJ, Czystowska M, Szajnik M, Harasymczuk M, Boyiadzis M, Kruk-Zagajewska A, Szyfter W, Zeromski J, Whiteside TL (2009) Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res 69:3105–3113
He W, Liu Q, Wang L, Chen W, Li N, Cao X (2007) TLR4 signaling promotes immune escape of human lung cancer cells by inducing immunosuppressive cytokines and apoptosis resistance. Mol Immunol 44:2850–2859
Akira S, Takeda K (2004) Toll-like receptor signalling. Nat Rev Immunol 4:499–511
Theofilopoulos AN, Baccala R, Beutler B, Kono DH (2005) Type I interferons (alpha/beta) in immunity and autoimmunity. Annu Rev Immunol 23:307–336
Simonaro CM, Ge Y, Eliyahu E, He X, Jepsen KJ, Schuchman EH (2010) Involvement of the Toll-like receptor 4 pathway and use of TNF-alpha antagonists for treatment of the mucopolysaccharidoses. Proc Natl Acad Sci USA 107:222–227
Nozell S, Laver T, Moseley D, Nowoslawski L, De Vos M, Atkinson GP, Harrison K, Nabors LB, Benveniste EN (2008) The ING4 tumor suppressor attenuates NF-kappaB activity at the promoters of target genes. Mol Cell Biol 28:6632–6645
Dong J, Jimi E, Zeiss C, Hayden MS, Ghosh S (2010) Constitutively active NF-kappaB triggers systemic TNFalpha-dependent inflammation and localized TNFalpha-independent inflammatory disease. Genes Dev 24:1709–1717
Sharma V, Dixit D, Koul N, Mehta VS, Sen E (2011) Ras regulates interleukin-1beta-induced HIF-1alpha transcriptional activity in glioblastoma. J Mol Med 89(2):123–136
Semenza GL (2002) HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 8:S62–S67
Gupta SC, Sundaram C, Reuter S, Aggarwal BB (2010) Inhibiting NF-kappaB activation by small molecules as a therapeutic strategy. Biochim Biophys Acta 1799:775–787
Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3:721–732
Gupta SC, Kim JH, Prasad S, Aggarwal BB (2010) Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev 29:405–434
Jung YJ, Isaacs JS, Lee S, Trepel J, Neckers L (2003) IL-1beta-mediated up-regulation of HIF-1alpha via an NFkappaB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis. FASEB J 17:2115–2117
Kaur B, Khwaja FW, Severson EA, Matheny SL, Brat DJ, Van Meir EG (2005) Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis. Neuro Oncol 7:134–153
Zagzag D, Zhong H, Scalzitti JM, Laughner E, Simons JW, Semenza GL (2000) Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression. Cancer 88:2606–2618
Goto Y, Arigami T, Kitago M, Nguyen SL, Narita N, Ferrone S, Morton DL, Irie RF, Hoon DS (2008) Activation of Toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors. Mol Cancer Ther 7:3642–3653
Sato Y, Goto Y, Narita N, Hoon DS (2009) Cancer cells expressing Toll-like receptors and the tumor microenvironment. Cancer Microenviron 2(Suppl 1):205–214
Peyssonnaux C, Cejudo-Martin P, Doedens A, Zinkernagel AS, Johnson RS, Nizet V (2007) Cutting edge: essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis. J Immunol 178:7516–7519
Chen NJ, Chio II, Lin WJ, Duncan G, Chau H, Katz D, Huang HL, Pike KA, Hao Z, Su YW, Yamamoto K, de Pooter RF, Zuniga-Pflucker JC, Wakeham A, Yeh WC, Mak TW (2008) Beyond tumor necrosis factor receptor: TRADD signaling in toll-like receptors. Proc Natl Acad Sci U S A 105:12429–12434
Muzio M, Ni J, Feng P, Dixit VM (1997) IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278:1612–1615
Biswas S, Gupta MK, Chattopadhyay D, Mukhopadhyay CK (2007) Insulin-induced activation of hypoxia-inducible factor-1 requires generation of reactive oxygen species by NADPH oxidase. Am J Physiol Heart Circ Physiol 292:H758–H766
Sharma V, Tewari R, Sk UH, Joseph C, Sen E (2008) Ebselen sensitizes glioblastoma cells to tumor necrosis factor (TNFalpha)-induced apoptosis through two distinct pathways involving NF-kappaB downregulation and Fas-mediated formation of death inducing signaling complex. Int J Cancer 123:2204–2212
Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK, Sen E (2007) Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Mol Cancer Ther 6:2544–2553
An H, Yu Y, Zhang M, Xu H, Qi R, Yan X, Liu S, Wang W, Guo Z, Guo J, Qin Z, Cao X (2002) Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology 106:38–45
Covert MW, Leung TH, Gaston JE, Baltimore D (2005) Achieving stability of lipopolysaccharide-induced NF-kappaB activation. Science 309:1854–1857
Ii M, Matsunaga N, Hazeki K, Nakamura K, Takashima K, Seya T, Hazeki O, Kitazaki T, Iizawa Y (2006) A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling. Mol Pharmacol 69:1288–1295
Kravchenko VV, Kaufmann GF, Mathison JC, Scott DA, Katz AZ, Grauer DC, Lehmann M, Meijler MM, Janda KD, Ulevitch RJ (2008) Modulation of gene expression via disruption of NF-kappaB signaling by a bacterial small molecule. Science 321:259–263
Verstak B, Nagpal K, Bottomley SP, Golenbock DT, Hertzog PJ, Mansell A (2009) MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4-mediated NF-kappaB proinflammatory responses. J Biol Chem 284:24192–24203
Sato S, Sugiyama M, Yamamoto M, Watanabe Y, Kawai T, Takeda K, Akira S (2003) Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-kappa B and IFN-regulatory factor-3, in the Toll-like receptor signaling. J Immunol 171:4304–4310
Wang H, Garcia CA, Rehani K, Cekic C, Alard P, Kinane DF, Mitchell T, Martin M (2008) IFN-beta production by TLR4-stimulated innate immune cells is negatively regulated by GSK3-beta. J Immunol 181:6797–6802
Yarilina A, Park-Min KH, Antoniv T, Hu X, Ivashkiv LB (2008) TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes. Nat Immunol 9:378–387
Rhee SH, Jones BW, Toshchakov V, Vogel SN, Fenton MJ (2003) Toll-like receptors 2 and 4 activate STAT1 serine phosphorylation by distinct mechanisms in macrophages. J Biol Chem 278:22506–22512
Kuhlicke J, Frick JS, Morote-Garcia JC, Rosenberger P, Eltzschig HK (2007) Hypoxia inducible factor (HIF)-1 coordinates induction of Toll-like receptors TLR2 and TLR6 during hypoxia. PLoS One 2:e1364
Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, Nizet V, Johnson RS, Haddad GG, Karin M (2008) NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature 453:807–811
Vivarelli MS, McDonald D, Miller M, Cusson N, Kelliher M, Geha RS (2004) RIP links TLR4 to Akt and is essential for cell survival in response to LPS stimulation. J Exp Med 200:399–404
Pore N, Jiang Z, Shu HK, Bernhard E, Kao GD, Maity A (2006) Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway. Mol Cancer Res 4:471–479
Ghosh S, Tewari R, Dixit D, Sen E (2010) TNFalpha induced oxidative stress dependent Akt signaling affects actin cytoskeletal organization in glioma cells. Neurochem Int 56:194–201
Kopp EB, Ghosh S (1995) NF-kappa B and rel proteins in innate immunity. Adv Immunol 58:1–27
Kawai T, Akira S (2006) TLR signaling. Cell Death Differ 13:816–825
Juang YT, Lowther W, Kellum M, Au WC, Lin R, Hiscott J, Pitha PM (1998) Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc Natl Acad Sci U S A 95:9837–9842
Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, Major J, Hamilton TA, Fenton MJ, Vogel SN (2002) TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol 3:392–398
Honda K, Takaoka A, Taniguchi T (2006) Type I interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors. Immunity 25:349–360
Nozell S, Laver T, Patel K, Benveniste EN (2006) Mechanism of IFN-beta-mediated inhibition of IL-8 gene expression in astroglioma cells. J Immunol 177:822–830
Tewari R, Sharma V, Koul N, Ghosh A, Joseph C, Hossain Sk U, Sen E (2009) Ebselen abrogates TNFalpha induced pro-inflammatory response in glioblastoma. Mol Oncol 3:77–83
Brat DJ, Bellail AC, Van Meir EG (2005) The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro Oncol 7:122–133
Huettner C, Paulus W, Roggendorf W (1995) Messenger RNA expression of the immunosuppressive cytokine IL-10 in human gliomas. Am J Pathol 146:317–322
Hellums EK, Markert JM, Parker JN, He B, Perbal B, Roizman B, Whitley RJ, Langford CP, Bharara S, Gillespie GY (2005) Increased efficacy of an interleukin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model. Neuro Oncol 7:213–224
Platten M, Kretz A, Naumann U, Aulwurm S, Egashira K, Isenmann S, Weller M (2003) Monocyte chemoattractant protein-1 increases microglial infiltration and aggressiveness of gliomas. Ann Neurol 54:388–392
Grauer OM, Molling JW, Bennink E, Toonen LW, Sutmuller RP, Nierkens S, Adema GJ (2008) TLR ligands in the local treatment of established intracerebral murine gliomas. J Immunol 181:6720–6729
Acknowledgment
The work was supported by a research grant from the Department of Biotechnology–DBT (BT/PR12924/Med/30/235/09) to ES. We would like to thank Mr. Shanker Dutt Joshi for technical assistance and help with immunohistochemistry.
Disclosure
The authors declare no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Saurav Roy Choudhury and Sadashib Ghosh contributed equally to this work.
Rights and permissions
About this article
Cite this article
Tewari, R., Choudhury, S.R., Ghosh, S. et al. Involvement of TNFα-induced TLR4–NF-κB and TLR4–HIF-1α feed-forward loops in the regulation of inflammatory responses in glioma. J Mol Med 90, 67–80 (2012). https://doi.org/10.1007/s00109-011-0807-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00109-011-0807-6