Trapidil inhibits monocyte CD40 expression by preventing IFN-gamma-induced STAT1 S727 phosphorylation.
Trapidil is a triazolopyrimidine that has been found to prevent restenosis after vascular injury. Although its precise mode of action is still unclear, several biological effects have been described including inhibition of IFN-gamma-induced CD40 expression on monocytes. Herein, we investigated the molecular mechanisms by which Trapidil exerts this inhibitory action. First, we observed that the inhibition of CD40 expression is associated with the suppression of CD40 gene transcription, as demonstrated by a clear decrease of CD40 nuclear RNA (nRNA) levels and unchanged CD40 mRNA half-life. IFN-gamma-induced CD40 transcription has been shown to be mediated by STAT1alpha dimers (p91/p84) which, after nuclear translocation, bind to GAS elements present in the promoter of IFN-gamma responsive genes. Electrophoresis mobility shift assay (EMSA) with both STAT1 consensus and CD40 mGAS probes showed that Trapidil did not affect the DNA binding ability of STAT1 dimers. STAT1 dimerization and activation are conferred by upstream phosphorylation of two amino acid residues of the STAT1 protein. The subsequent studies on these two potential STAT1 phosphorylation sites (Tyr701, Ser727) revealed that Trapidil attenuated IFN-gamma-induced Ser727 but not Tyr701 phosphorylation. The inhibition of CD40 transcription by Trapidil could at least partially owing to the impaired Ser727 phosphorylation of STAT1, since IFN-gamma failed to trigger CD40 expression in U3A S727A cells, a cell line displaying a point mutation at the Ser727 site. Collectively, our results indicate that phosphorylation of STAT1 at the Ser727 site enhances CD40 transcription and that Trapidil might be used as a selective inhibitor that could differentially modulate STAT1 target genes.
Zhou
Ling
L
Department of Immunology-Hematology-Transfusion, Erasme Hospital, Université Libre de Bruxelles, route de Lennik, 808, Brussels, B-1070, Belgium.
Schandené
Liliane
L
Mordvinov
Viatcheslav A
VA
Chatelain
Pierre
P
Pradier
Olivier
O
Goldman
Michel
M
Stordeur
Patrick
P
D019013:CD40 Antigens / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D050794:STAT1 Transcription Factor; D012694:Serine / Q000378:metabolism; D015534:Trans-Activators / Q000276:immunology / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014192:Trapidil / Q000494:pharmacology*
D019013:CD40 Antigens; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D012694:Serine; D007371:Interferon-gamma; D014192:Trapidil