No abstract

Summary The synthetic phospho‐ceramide analogue‐1 (PCERA‐1) down‐regulates production of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α) and up‐regulates production of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) in lipopolysaccharide (LPS) ‐stimulated macrophages. We have previously reported that PCERA‐1 increases cyclic adenosine monophosphate (cAMP) levels. The objective of this study was to delineate the signalling pathway leading from PCERA‐1 via cAMP to modulation of TNF‐α and IL‐10 production. We show here that PCERA‐1 elevates intra‐cellular cAMP level in a guanosine triphosphate‐dependent manner in RAW264.7 macrophages. The cell‐permeable dibutyryl cAMP was able to mimic the effects of PCERA‐1 on cytokine production, whereas 8‐chloro‐phenylthio‐methyladenosine‐cAMP, which specifically activates the exchange protein directly activated by cAMP (EPAC) but not protein kinase A (PKA), failed to mimic PCERA‐1 activities. Consistently, the PKA inhibitor H89 efficiently blocked PCERA‐1‐driven cytokine modulation as well as PCERA‐1‐stimulated phosphorylation of cAMP response element binding protein (CREB) on Ser‐133. Finally, PCERA‐1 activated cAMP‐responsive transcription of a luciferase reporter, in synergism with the phosphodiesterase (PDE)‐4 inhibitor rolipram. Our results suggest that PCERA‐1 activates a Gs protein‐coupled receptor, leading to elevation of cAMP, which acts via the PKA–CREB pathway to promote TNF‐α suppression and IL‐10 induction in LPS‐stimulated macrophages. Identification of the PCERA‐1 receptor is expected to set up a new target for development of novel anti‐inflammatory drugs.

Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. Previous reports showed that the natural sphingolipid ceramide 1-phosphate (C1P) stimulates macrophages migration, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses production of the key pro-inflammatory cytokine TNFα and amplifies production of the key anti-inflammatory cytokine IL-10 in LPS-stimulated macrophages, via one or more unidentified G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFκB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via the cAMP-PKA-CREB signaling pathway, while C1P neither mimicked nor antagonized these activities. Interestingly, both compounds have the capacity to additively inhibit TNFα secretion; PCERA-1, but not C1P, suppressed LPS-induced TNFα expression in macrophages in a CREB-dependent manner, while C1P, but not PCERA-1, directly inhibited recombinant TNFα converting enzyme (TACE). Finally, PCERA-1 failed to interfere with binding of C1P to either the cell surface receptor or to TACE. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.