Upstream stimulatory factor regulates E box‐dependent PAI‐1 transcription in human epidermal keratinocytes
Authors: Allen Rosalie R. , Qi Li , Higgins Paul J. 
Published: September 2004

AbstractCertain growth factors (e.g., TGF‐β1) 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‐β1. 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‐β1‐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‐β1‐stimulated cells. Transfection of a dominant‐negative USF construct effectively attenuated serum‐ and TGF‐β1‐induced PAI‐1 synthesis as well as TGF‐β1‐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. © 2004 Wiley‐Liss, Inc.