A potential target for methotrexate in macrophages : AMP-activated protein kinase.

Abstract

Abstract Background Methotrexate (MTX) remains a cornerstone of treatment in multiple forms of inflammatory arthritis. AMPK is a highly conserved protein kinase present in all eukaryotic cells and is activated by an increasing intracellular ADP/ATP levels. Once activated, AMPK will promote ATP production by switching on catabolic and turning off anabolic pathways. We hypothesize that AMPK activation mediates a major portion of the anti-inflammatory effects of MTX. Methods We investigated the role of the anti-inflammatory effect of MTX via AMPK in human monocytes-derived macrophages (MDM) and mouse bone marrow-derived macrophages (BMDM) along with AICAR and A769662 (AMPK activators) and compound C (a selective AMPK inhibitor). AMPK phosphorylation and total AMPK were measured by Western blotting. Cells were then stimulated with LPS or TNF-α, and production of pro-inflammatory cytokines were measured in the supernatant. Results MTX induced AMPK activation with effects comparable to the AMPK activators (A769662, AICAR ) in hMDM and BMDM. MTX-induced AMPK activation was associated with a reduction in the production of IL-6, IL-1 β, and TNF-α in response to LPS and TNF stimulation. Compound C is able to partially reverse the effects of MTX. Conclusions MTX is able to induce AMPK activation in both MDM and BMDM, and suppress pro-inflammatory cytokines in a manner dependent on AMPK activity. These results have been confirmed genetically in macrophages deficient in AMPK subunits and in serum transfer arthritis. Our findings raise the possibility that some anti-inflammatory effects of MTX are mediated by AMPK, suggest that AMPK may be a target for the anti-inflammatory agents and a target for the development of new anti-inflammatory drugs.