Differential modulatory effects of annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages.
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.
Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
Tomoko
T
Smyth
Hayley J
HJ
Harris
Andrew
A
Brown
Sabine
S
Tötemeyer
Belinda A
BA
Farnfield
Duncan J
DJ
Maskell
Makoto
M
Matsumoto
Robin
R
Plevin
Louise C
LC
Alldridge
Clare E
CE
Bryant
D000818:Animals; D017305:Annexin A1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D017305:Annexin A1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D048049:Extracellular Signal-Regulated MAP Kinases; D015232:Dinoprostone