Gut-enriched Kruppel-like factor represses cyclin D1 promoter activity through Sp1 motif

Cancer cells differ from normal cells in many characteristics including los s of differentiation and uninhibited cell proliferation. Recent studies hav e focused on the identification of factors contributing to cell growth and differentiation, Gut-enriched Kruppel-like factor (GKLF or KLF4) is a newly identified eukaryotic transcription factor and has been shown to play a ro le in regulating growth arrest, We have previously shown that GKLF mRNA lev els were significantly decreased in colon cancer tissues, and that overexpr ession of GKLF in colonic adenocarcinoma cells (HT-29) resulted in reductio n of cyclin D1 (CD1) mRNA and protein levels. The current study was underta ken to determine the mechanisms by which GKLF inhibited CD1 expression, In a transient transfection system, GKLF suppressed CD1 promoter activity by 5 5%, Sequential deletion and site-directed mutation analysis of the CD1 prom oter have identified the sequence between -141 and -66, a region containing an Sp1 response element, to be essential for GKLF function, By electrophor etic mobility gel shift assay, recombinant GKLF and nuclear extracts from H T-29 cells were found to bind to the Spl motif on the CD1 promoter, The inh ibitory effect of GKLF on the CD1 promoter activity was completely abolishe d by excessive amount of Sp1 DNA and GKLF significantly reduced the stimula tory function of Spl suggesting that GKLF and Spl may compete for the same binding site on the CD1 promoter. These results indicate that GKLF is a tra nscriptional repressor of the CD1 gene and that the inhibitory effect of GK LF is, in part, mediated by interaction with the Sp1 binding domain on its promoter.