The large GTPase GBP-1 (guanylate-binding protein-1) is a major IFN-γ (interferon-γ)-induced protein with potent anti-angiogenic activity in endothelial cells. An ISRE (IFN-α-stimulated response element) is necessary and sufficient for the induction of GBP-1 expression by IFN-γ. Recently, we have shown that in vivo GBP-1 expression is strongly endothelial-cell-associated and is, in addition to IFN-γ, also activated by interleukin-1β and tumour necrosis factor-α, both in vitro and in vivo [Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle et al. (2002) Am. J. Pathol. 161, 1749–1759; Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmle et al. (2001) EMBO J. 20, 5568–5577]. In the present study, we identified a NF-κB (nuclear factor κB)-binding motif that, together with ISRE, is required for the induction of GBP-1 expression by interleukin-1β and tumour necrosis factor-α. Deactivation of the NF-κB motif reduced the additive effects of combinations of these cytokines with IFN-γ by more than 50%. Importantly, NF-κB p50 rather than p65 activated the GBP-1 promoter. The NF-κB motif and ISRE were detected in an almost identical spatial organization, as in the GBP-1 promoter, in the promoter regions of various inflammation-associated genes. Therefore both motifs may constitute a cooperative inflammatory cytokine response module that regulates GBP-1 expression. Our findings may open new perspectives for the use of NF-κB inhibitors to support angiogenesis in inflammatory diseases including ischaemia.
- guanylate-binding protein-1 (GBP-1)
- human umbilical-vein endothelial cells (HUVEC)
- nuclear factor κB (NF-κB)
- tumour necrosis factor
Abbreviations used: BAY 11-7082, [(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile]; C/EBP, CCAAT/enhancer-binding protein; EBM, endothelial cell basal medium; EMSA, electrophoretic mobility-shift assay; FBS, foetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBP-1, guanylate-binding protein-1; GFP, green fluorescent protein; IFN, interferon; GAS, γ-IFN activation site; HEK-293T cells, human embryonic kidney-293T cells; HUVEC, human umbilical-vein endothelial cells; IC, inflammatory cytokine; IL, interleukin; IRF-1, IFN regulatory factor-1; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-α-stimulated response element; IκB, inhibitory κB; MMP-1, matrix metalloproteinase-1; NF-κB, nuclear factor κB; Oct-1, octamer-binding protein-1; RANTES, regulated upon activation, normal T-cell expressed and secreted; STAT, signal transduction and activators of transcription; TNF, tumour necrosis factor; Tos-Phe-CH2Cl, tosylphenylalanylchloromethane.
- The Biochemical Society, London ©2004