The Kruppel-like KLF4 transcription factor, a novel regulator of urokinase receptor expression, drives synthesis of this binding site in colonic crypt luminal surface epithelial cells.
The urokinase-type plasminogen activator receptor (u-PAR) plays a central role in cell migration, growth, and invasion and is regulated, in part, transcriptionally. In mice, u-PAR expression is restricted to a few tissues, one of which is the colon. We therefore screened a colon expression library for regulators of u-PAR promoter activity and identified a zinc finger protein bearing consensus sequences to the Kruppel-like family of transcription factors and showing partial homology with one of the members, KLF4. Like u-PAR, KLF4 expression is predominant in the luminal surface epithelial cells of the colonic crypt, and we hypothesized that u-PAR synthesis in these cells is directed by this transcription factor. Colon cells from KLF4 null mice showed a dramatic reduction in u-PAR protein compared with wild-type mice. Conversely, KLF4 expression in HCT116 colon cancer cells increased the amount of u-PAR protein/mRNA. Transient transfection of KLF4 with a reporter driven by 5'-deleted u-PAR promoter fragments indicated the requirement of the proximal 200 base pairs for optimal expression. Mobility-shifting experiments demonstrated binding of KLF4 to multiple regions of the u-PAR promoter (-154/-128, -105/-71, and -51/-24), and chromatin immunoprecipitation assays confirmed the binding of KLF4 to the endogenous promoter. Deletion of the -144/-123 promoter region diminished but did not eliminate the ability of KLF4 to transactivate the u-PAR promoter, suggesting cooperativity of these binding sites with respect to activation of gene expression. In conclusion, we have identified KLF4 as a novel regulator of u-PAR expression that drives the synthesis of u-PAR in the luminal surface epithelial cells of the colon.
Wang
Heng
H
Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.
Yang
Lin
L
Jamaluddin
Md Saha
MS
Boyd
Douglas D
DD
D000423:Alcian Blue / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002469:Cell Separation; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D003106:Colon / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D005434:Flow Cytometry; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D055293:Receptors, Urokinase Plasminogen Activator; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation; D016335:Zinc Fingers
D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins; C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000423:Alcian Blue