Cromoglycate drugs suppress eicosanoid generation in U937 cells by promoting the release of Anx-A1

Samia Yazid,Egle Solito,Helen Christian,Simon Mcarthur,Nicolas Goulding,Roderick Flower

doi:10.1016/j.bcp.2009.03.010

Samia Yazid, Egle Solito + Show 4 more

Open Access

https://doi.org/10.1016/j.bcp.2009.03.010

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Abstract

Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the anti-inflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5min) concentration-dependent activation of PKCα/β (Protein Kinase C; EC 2.7.11.13) and phosphorylation of Anx-A1 on Ser27. Both phosphoproteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B2 generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCα/β phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB2 generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A (phosphoprotein phosphatase; EC 3.1.3.16), which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators.

Highlights

Epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of the anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the antiinflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment
Hydrocortisone, prednisolone, methylprednisolone and betamethasone in this system finding that all stimulated Anx-A1 Ser27 phosphorylation in a qualitatively comparable fashion, prednisolone, methylprednisolone and betamethasone were not examined further in our assay systems
The Anx-A1 system in undifferentiated myelomonocytic U937 cells does not respond to glucocorticoids and it is necessary to pre-treat the cells with low concentrations of phorbol 12-myristate 13-acetate (PMA) to render them responsive [43]

Summary

Introduction

Anx-A1, a 37kDa member of the annexin super-family (13 proteins in mammals), and its N-terminal peptide N-acetyl2-26 [1, 2], have been shown by us, and by other laboratories to possess powerful anti-inflammatory actions in a wide variety of animal models of acute [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17] or chronic [18,19,20] inflammation.The biologically active pool, in this context, is the extracellular protein. Anx-A1 is both induced and secreted from cells under the influence of GCs [21,22,23,24]. The release, as opposed to the induction, of cytosolic Anx-A1 is increased by GCs acting in a receptordependent, non-genomic manner. This GC-induced secretory event is preceded by Ser phosphorylation apparently as a result of PKC (Protein Kinase C; EC 2.7.11.13) activation [25,26,27]. The Anx-A1 Ser27-Ala mutant is not secreted from cells and has a different intracellular distribution [28]. Once on the cell surface, Anx-A1 can act in an autocrine (or paracrine) fashion to inhibit cell activation probably by interaction with receptors of the FPR family, FPR-L1 (ALXR; [29,30,31])

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