STAT1 is required for IFN-gamma-mediated gut-enriched Krüppel-like factor expression.
Gut-enriched Krüppel-like factor (GKLF/KLF4), a member of the Krüppel-like factor family of transcription factors, has recently been shown to be associated with growth arrest in the colonic mucosa. Our laboratory has previously demonstrated that GKLF is one of the down-stream targets of interferon-gamma (IFN-gamma) in colon cancer cells, but the signaling pathway regulating GKLF expression is not known. In this report, we showed that IFN-gamma increased GKLF and interferon regulatory factor-1 mRNA levels in a parallel fashion and these effects were abolished in STAT1 knockout mouse fibroblast cell lines, indicating that STAT1 mediated IFN-gamma-stimulated GKLF gene expression. IFN-gamma treatment induced phosphorylation of STAT1 and IFN-gamma-induced GKLF mRNA expression was attenuated by tyrosine protein kinase inhibitors, suggesting that phosphorylation of STAT1 played an essential role in this process. To further evaluate the effect of STAT1 on GKLF gene expression, a 2622-bp mouse GKLF promoter was isolated from a liver genomic library. In a transient transfection system, IFN-gamma treatment increased GKLF promoter activity by 3.5-fold. Sequential deletion and mutation analysis of the GKLF promoter has identified the sequence between -1675 and -1580, a region containing a GAS element, to be essential for IFN-gamma function. By electrophoretic mobility gel shift assay, nuclear extracts from IFN-gamma-stimulated HT-29 cells were found to bind to the GAS motif on the GKLF promoter and this protein-DNA complex was supershifted by the STAT1 antiserum. These results indicate that IFN-gamma-induced GKLF expression required phosphorylated STAT1 and that these effects were mediated, in part, through interaction of STAT1 with the GAS element on the GKLF promoter.
Chen
Zhi Y
ZY
Section of Gastroenterology, VA Boston Healthcare System and Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Shie
Jue Lon
JL
Tseng
Chi Chuan
CC
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D015854:Up-Regulation
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D010750:Phosphoproteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D014443:Tyrosine; D007371:Interferon-gamma; D008156:Luciferases