ABSTRACT
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.
Subject(s)
Annexin A1/agonists , Bucladesine/therapeutic use , Cyclic AMP/agonists , Neutrophil Infiltration/drug effects , Phosphodiesterase 4 Inhibitors/therapeutic use , Pleurisy/drug therapy , Rolipram/therapeutic use , Animals , Annexin A1/antagonists & inhibitors , Annexin A1/genetics , Annexin A1/metabolism , Apoptosis/drug effects , Bucladesine/antagonists & inhibitors , Cells, Cultured , Cyclic AMP/analogs & derivatives , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Lipopolysaccharides/toxicity , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/pathology , Phosphodiesterase 4 Inhibitors/chemistry , Phosphorylation/drug effects , Pleurisy/immunology , Pleurisy/metabolism , Pleurisy/pathology , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , RAW 264.7 Cells , Rolipram/antagonists & inhibitorsABSTRACT
Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity.
