Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene.
Several proteases and their co-expressed inhibitors modulate the interdependent processes of cell migration and matrix proteolysis during wound repair. Transcription of the gene encoding plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor important in the control of barrier proteolysis and cell-to-matrix adhesion, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. Using a well-defined culture model of acute epidermal wounding and reepithelialization, PAI-1 mRNA/protein synthesis was induced early after monolayer scraping and restricted to cells comprising the motile cohort. PAI-1 levels in locomoting cells remained elevated (relative to the distal, contact-inhibited monolayer regions) throughout the time course of trauma repair. Targeted PAI-1 downregulation by transfection of antisense PAI-1 expression constructs significantly impaired keratinocyte migration and monolayer scrape wound closure. Injury-induced PAI-1 transcription closely paralleled growth state-dependent controls on the PAI-1 gene. An E-box motif (CACGTG) in the PAI-1 proximal promoter (located at nucleotides -160 to -165), previously shown to be necessary for serum-induced PAI-1 expression, was bound by nuclear factors from wound-stimulated but not quiescent, contact-inhibited, keratinocytes. UV crosslinking approaches to identify E-box-binding factors coupled with deoxyoligonucleotide affinity chromatography and gel retardation assays confirmed at least one major E-box-binding protein in both serum- and wound-activated cells to be USF-1, a member of the helix-loop-helix family of transcription factors. An intact hexanucleotide E-box motif was necessary and sufficient for USF-1 binding using nuclear extracts from both serum- and wound-simulated cells. Two species of immunoreactive USF-1 were identified by western blotting of total cellular lysates that corresponded to the previously characterized phosphorylated and non-phosphorylated forms of the protein. USF-1 isolated by PAI-1 promoter-DNA affinity chromatography was almost exclusively phosphorylated. Only a fraction of the total cellular USF-1 in proliferating cultures, by comparison, was phosphorylated at any given time. PAI-1 E-box binding activity, assessed by probe mobility shift criteria, increased within 2 hours of monolayer scrape injury, a time frame consistent with wound-stimulated increases in PAI-1 transcription. Relative to intact cultures, scrape site-juxtaposed cells had significantly greater cytoplasmic and nuclear USF-1 immunoreactivity correlating with the specific in situ-restricted expression of PAI-1 transcripts/protein in the wound-edge cohort. USF-1 immunocytochemical staining declined significantly with increasing distance from the denudation site. These data are the first to indicate that binding of USF-1 to its target motif can be induced by 'tissue' injury in vitro and implicate USF-1 as a transcriptional regulator of genes (e.g. PAI-1) involved in wound repair.
Providence
Kirwin M
KM
Center for Cell Biology & Cancer Research, Albany Medical College, Albany, NY 12208, USA.
White
Lisa A
LA
Tang
Jianzhong
J
Gonclaves
John
J
Staiano-Coico
Lisa
L
Higgins
Paul J
PJ
D000818:Animals; D002465:Cell Movement / Q000235:genetics*; D016764:Cell Polarity / Q000235:genetics; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D024721:E-Box Elements / Q000235:genetics*; D004817:Epidermis / Q000293:injuries* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008164:Luminescent Proteins; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051720:Upstream Stimulatory Factors; D014945:Wound Healing / Q000235:genetics*
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D049452:Green Fluorescent Proteins