Abstract
Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.
Highlights
Lipocortin 1 is a member of the annexin family of calcium and phospholipid-binding proteins of which 20 are known at present
We have demonstrated that lipocortin 1 modulates the ERK signaling cascade at an upstream site probably by associating with key signaling components including the adaptor protein Grb 2
Increasing the expression of lipocortin 1 leads to constitutive activation of ERK1/2 kinase in RAW macrophages
Summary
Generation of Lipocortin 1 Expression Plasmids—Total RNA was isolated from murine skin samples (Micro-scale Total RNA Separator Kit; CLONTECH). Stimulated cells were lysed on ice in buffer containing 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 20 mM NaF, 2.5 mM -glycerophosphate, 0.2 mM PMSF, and 0.5 g/ml leupeptin and aprotinin. The activity of JNK was quantified as for p38 activity except that cells were lysed in buffer containing 20 mM Hepes, pH 7.7, 50 mM NaCl, 1% (v/v) Triton X-100, 0.1 mM EDTA, 0.2 mM PMSF, 0.5 g/ml aprotinin, and leupeptin and circulated with recombinant glutathione Stransferase-tagged truncated N terminus of cJun (GST-c-Jun-(5– 89)) immobilized on GSH-Sepharose [30]. The cell pellets were resuspended in 50 l of buffer 2 (20 mM Hepes, pH 7.9, 25% (w/v) glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, and 10 g/ml leupeptin, pepstatin, and aprotinin) and, after light vortexing, were extracted on ice for 15 min.
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