087 PAI-1 Gene Expression in Wound-Edge Keratinocytes is Upstream Stimulatory Factor-Dependent and Required for Cell Migration

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Expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor important in the control of cell-to-matrix adhesion is spatially regulated following epidermal injury. PAI-1 synthesis was induced early after monolayer wounding and restricted to keratinocytes comprising the motile cohort closely recapitulating, thereby, similar events during cutaneous healing. MEK blockade with PD98059 or U0126 attenuated keratinocyte migration by ≥60%, as did transient transfection of a dominant-negative ERK1 construct, and completely inhibited PAI-1 transcript expression. Antisense downregulation of PAI-1 synthesis (by 80–85%) inhibited injury site closure. Keratinocytes isolated from PAI-1−/− mice confirmed the antisense results. PAI-1−/− cells had a significant wound healing defect; addition of active PAI-1 protein rescued the migratory phenotype. An E box (5′-CACGTG-3′) in the PE2 region of the PAI-1 gene was required for transcription of a PAI-1 promoter-driven luciferase reporter. Mutation of the CACGTG hexanucleotide to CAATTG inhibited promoter function. Competing CACGTG-containing DNAs, regardless of the presence or absence of PAI-1 specific flanking sequences or lacking accessory sequences inhibited complex formation between nuclear factors and a PE2 region probe. A CGAT substitution 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. Chromatin immunoprecipitation confirmed that this site was occupied by USF-1 or a USF-1-containing complex. Transfection of a dominant-negative USF construct effectively attenuated growth factor-induced PAI-1 synthesis. Dominant-negative USF-expressing keratinocytes, moreover, had a significantly reduced capacity for Matrigel barrier invasion. USF elements, therefore, are important regulators of PAI-1 transcription and the associated migratory response. (Supported by NIH Grants GM57242 and GM42461)