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. 2006 Mar;117(3):340-9.
doi: 10.1111/j.1365-2567.2005.02307.x.

Differential modulatory effects of annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages

Tomoko Smyth  1 , Hayley J Harris, Andrew Brown, Sabine Tötemeyer, Belinda A Farnfield, Duncan J Maskell, Makoto Matsumoto, Robin Plevin, Louise C Alldridge, Clare E Bryant
Affiliations

Differential modulatory effects of annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages

Tomoko Smyth et al. Immunology. 2006 Mar.

Abstract

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-alpha) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-alpha production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1.

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Figures

Figure 1
Figure 1
Cells over-expressing ANXA1 have increased iNOS activity and protein expression independent of transcription. (a) Cells over-expressing ANXA1 show increased activity of LPS-induced iNOS. Cells transfected with the empty expression vector (open column) or cells over-expressing (closed column) or under-expressing (cross hatched-column) ANXA1 were treated with LPS (1 μg/ml for 24 hr). Cell culture medium was removed, assayed for nitrite utilizing the Griess reaction and normalized to 106 viable cells. The data represent mean ± SEM of at least nine experiments on each cell line. Statistical significance is indicated by *P < 0·05 or **P < 0·01. (b) Cells over-expressing ANXA1 show increased protein expression of LPS-induced iNOS. Cells transfected with the empty expression vector or cells over- or under-expressing ANXA1 were treated with LPS (1 μg/ml for 24 hr). Cells were lysed, the proteins were separated by SDS–PAGE and transferred to PVDF membranes, which were then probed with anti-iNOS antisera. This blot is representative of at least six experiments on each cell line. (c) Cells over-expressing ANXA1 show no increase in LPS-induced iNOS mRNA. Cells transfected with the empty expression vector (open column) or cells over-expressing ANXA1 (closed column) were treated with LPS (1 μg/ml for 6 hr). Cells were lysed and DNA-free RNA was prepared using the RNeasy mini kit (Qiagen). Quantitative RT-PCR was performed using the QantiTect SYBR Green RT-PCR kit (Qiagen). The data show two separate repetitions of experiments in each cell line. (d) Cells over-expressing ANXA1 show increased activity of interferon-γ-induced iNOS. Cells transfected with the empty expression vector (open column) or cells over-expressing (closed column) or under-expressing (hatched column) ANXA1 were treated with LPS (1 μg/ml), murine IFN-γ (10 IU) or both LPS and IFN-γ together for 24 hr. Cell medium was removed, assayed for nitrite using the Griess reaction and normalized to 106 viable cells. The data represents mean ± SEM of at least four experiments on each cell line.
Figure 2
Figure 2
Increased ANXA1 expression does not affect the inflammatory mediators PGE2, COX-2 and TNF-α. (a) ANXA1 has no effect on LPS-induced PGE2 release. Cells transfected with the empty expression vector or cells over- or under-expressing (R1A) ANXA1 were treated with LPS (10 μg/ml for 24 hr). Cell medium was removed, assayed for PGE2 production by radioimmunoassay and normalized to 106 viable cells. The data represents mean ± SEM of four experiments on each cell line. (b) Over-expression of ANXA1 has no effect on LPS-induced COX-2 expression. Cells transfected with the empty expression vector or cells over- or under-expressing ANXA1 were treated with LPS (1 μg/ml for 24 hr). The proteins were separated by SDS–PAGE and after transfer to PVDF membranes the blots were probed with anti-COX-2 antisera. This blot is representative of at least four experiments on each cell line. (c) ANXA1 has no effect on LPS-induced TNF-α release. Cells transfected with the empty expression vector (open column) or cells over-expressing (closed column) ANXA1 were treated with LPS (1 μg/ml for 24 hr). Cell medium was removed, assayed for TNF-α production by ELISA and normalized to 106 viable cells. The data represent mean ± SEM of four experiments on each cell line.
Figure 3
Figure 3
Cells over-expressing ANXA1 have increased iNOS activity and protein expression by an ERK-dependent post-transcriptional mechanism. (a)Inhibition of ERK by U0126 inhibits the enhanced LPS-induced expression of iNOS in cells over-expressing ANXA1. Control or ANXA1 over-expressing cells were pretreated for 1 hr with U0126 (10 μm) or 0·1% ethanol (vehicle control). LPS (1 μg/ml) was added to the cells and, after 24 hr, the expression of iNOS protein was measured by Western blot analysis. The figure shows a representative blot and densitometric analysis from four different experiments. (b) Cells over-expressing ANXA1 have altered levels of phospho-eIF4E expression. Control or ANXA1 over-expressing cells were treated with LPS (1 μg/ml) for 0–60 min and the levels of phospho- and native eIF4E and 4E-BP1 present in the cells lysates were measured by Western blot analysis. Protein loading was confirmed by re-probing the blot for actin expression. A representative blot is shown from four different experiments.
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References

    1. Raynal P, Pollard HB. Annexins. the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994;1197:63–93. - PubMed
    1. Flower RJ, Rothwell NJ. Lipocortin-1: cellular mechanisms and clinical relevance. Trends Pharmacol Sci. 1994;15:71–6. - PubMed
    1. Diakonova M, Gerke V, Ernst J, Liautard JP, van der Vusse G, Griffiths G. Localization of five annexins in J774 macrophages and on isolated phagosomes. J Cell Sci. 1997;110:1199–213. - PubMed
    1. Traverso V, Morris JF, Flower RJ, Buckingham J. Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm. J Cell Sci. 1998;111:1405–18. - PubMed
    1. Cirino G, Peers SH, Flower RJ, Browning JL, Pepinsky RB. Human recombinant lipocortin 1 has acute local anti-inflammatory properties in the rat paw edema test. Proc Natl Acad Sci USA. 1989;86:3428–32. - PMC - PubMed

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