Molecular mechanisms of IL-18BP regulation in DLD-1 cells: pivotal direct action of the STAT1/GAS axis on the promoter level.
Interleukin (IL)-18, formerly known as interferon (IFN)-gamma-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-gamma, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-gamma. Mutational analysis revealed that a proximal gamma-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-gamma-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-gamma-stimulated DLD-1 colon carcinoma cells.
Bachmann
Malte
M
Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe-University Frankfurt, Frankfurt am Main, Germany.
Paulukat
Jens
J
Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe-University Frankfurt, Frankfurt am Main, Germany.
Pfeilschifter
Josef
J
Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe-University Frankfurt, Frankfurt am Main, Germany.
Mühl
Heiko
H
Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe-University Frankfurt, Frankfurt am Main, Germany.
D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D007371:Interferon-gamma / Q000502:physiology; D011401:Promoter Regions, Genetic; D050794:STAT1 Transcription Factor / Q000502:physiology*
D017931:DNA Primers; D036341:Intercellular Signaling Peptides and Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C118370:interleukin-18 binding protein; D007371:Interferon-gamma