title: The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-alpha and interleukin-10 production in macrophages via the cAMP-PKA-CREB pathway in a GTP-dependent manner.
abstract: The synthetic phospho-ceramide analogue-1 (PCERA-1) down-regulates production of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) 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-alpha 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 G(s) protein-coupled receptor, leading to elevation of cAMP, which acts via the PKA-CREB pathway to promote TNF-alpha 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.
authors.affiliation: Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv, Israel.
authors.forename: Dorit
authors.initials: D
authors.lastname: Avni
authors.forename: Amir
authors.initials: A
authors.lastname: Philosoph
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meijler
authors.forename: Tsaffrir
authors.initials: T
authors.lastname: Zor
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D016753:Interleukin-10 / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000037:antagonists & inhibitors / Q000378:metabolism; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D002518:Ceramides; C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D007546:Isoquinolines; D008070:Lipopolysaccharides; C541330:PCERA-1; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D003994:Bucladesine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C116919:mitogen and stress-activated protein kinase 1; D017868:Cyclic AMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000262:Adenylyl Cyclases; D020889:Rolipram; D015232:Dinoprostone; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C093642:SB 203580; C064758:Ro 31-8220