title: Upstream stimulatory factor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes.
abstract: Certain growth factors (e.g., TGF-beta1) initiate a "plastic" response in human keratinocytes (HaCaT cells) characterized by changes in gene expression and increased cell motility. While microarray analyses identified a number of involved genes, plasminogen activator inhibitor type 1 (PAI-1) is among the subset most highly responsive to TGF-beta1. Previous antisense attenuation of PAI-1 synthesis confirmed an essential role for this protease inhibitor in cell motility (Providence et al., 2002, J Cell Sci 115:3767-3777; Providence and Higgins, 2004, J Cell Physiol 200:297-308). It was important, therefore, to clarify molecular mechanisms underlying PAI-1 expression control in human keratinocytes. A consensus E box motif (5'-CACGTG-3') at nucleotides -566 to -561 in the PE2 region of the PAI-1 gene was required for TGF-beta1-induced transcription of a PAI-1 promoter-driven luceriferase reporter. Truncation of the PE2 E box or mutation of the CACGTG hexanucleotide to CAATTG inhibited growth factor-stimulated promoter function confirming the importance of this site in inducible expression. A similar mutation at the PE1 E box (nucleotides -682 to -677), in contrast, did not result in reduced luciferase activity. Competing CACGTG-containing DNAs, regardless of the presence or absence of PAI-1-specific flanking sequences or lacking accessory sequences (i.e., Smad-binding sites, AAT trinucleotide spacer), inhibited complex formation between HaCaT cell nuclear factors and a 45-mer PE2 region probe. A deoxyoligonucleotide that differed from the consensus E box by a CG --> AT substitution (the same base change incorporated into the PAI-1p806-lucerifase reporter by site-directed mutagenesis) but with random (i.e., non-PAI-1) flanking sequences also failed to compete with the PE2 region probe for protein binding whereas the same construct with an intact CACGTG motif was an effective competitor. The major protein/DNA interactions in the PE2 segment, therefore, are E box-dependent. USF-1, a member of the upstream stimulatory factor family, bound the PE2 construct suggesting a role for USF proteins in E box residence and PAI-1 gene expression. Chromatin immunoprecipitation, using primers designed to amplify a 300-bp PE2-associated promoter fragment and containing no other E box motifs except the target CACGTG at nucleotides -566 to -561, confirmed that this site was occupied by USF-1 or a USF-1-containing complex in both quiescent and TGF-beta1-stimulated cells. Transfection of a dominant-negative USF construct effectively attenuated serum- and TGF-beta1-induced PAI-1 synthesis as well as TGF-beta1-stimulated Matrigel barrier invasion. Dominant-negative USF-expressing keratinocytes, moreover, specifically had a reduced capacity for Matrigel barrier invasion. USF elements, therefore, are important regulators of growth factor-initiated PAI-1 transcription (as predicted from the identification of PAI-1 as a direct USF target gene) and the associated epithelial migratory response.
authors.affiliation: Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York 12208, USA.
authors.forename: Rosalie R
authors.initials: RR
authors.lastname: Allen
authors.forename: Li
authors.initials: L
authors.lastname: Qi
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Higgins
mesh_terms: D001483:Base Sequence; D002461:Cell Line, Transformed; D003094:Collagen; D004338:Drug Combinations; D000078404:Epidermal Cells; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter / Q000235:genetics; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D007797:Laminin; D016350:Leucine Zippers; D008156:Luciferases / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D011509:Proteoglycans; D015533:Transcriptional Activation / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
chemical_list: D004338:Drug Combinations; D007797:Laminin; D017395:Plasminogen Activator Inhibitor 1; D011509:Proteoglycans; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C059485:matrigel; D003094:Collagen; D008156:Luciferases