Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene

Abstract

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.

Plasminogen activator inhibitor-1 promoter activity in adipocytes is not influenced by the 4 G/5 G promoter polymorphism and is regulated by a USF-1/2 binding site immediately preceding the polymorphic region

“…These data provide the basis for a putative role of USF in regulating genes during adipocyte differentiation. The binding of USF-2 was demonstrated to inhibit PAI-1 expression in hepatocytes (Samoylenko et al 2001) whereas USF-1 increased PAI-1 transcription in epithelial cells (Providence et al 2002). We were able to demonstrate by competition experiments, and by the appearance of specific supershift bands in EMSA, that both USF-1 and USF-2 from adipocytic nuclear extracts can bind specifically to the PAI-1 promoter site.…”

SERPINE1 (PAI-1) is deposited into keratinocyte migration “trails” and required for optimal monolayer wound repair

“…Perhaps as importantly, PAI-1 not only stimulated keratinocyte adhesion and wound-initiated planar migration but also rescued keratinocytes from plasminogen-induced substrate detachment/anoikis. These effects may reflect the fact that PAI-1 accumulates specifically in the cellular undersurface region where it is wellpositioned to modulate integrin-ECM or uPA/uPAR-ECM interactions as well as stromal remodeling [2,3,6,[8][9][10]14,15,20,24,29,37,42]. Activation of the PAI-1 gene early after monolayer wounding and its prominence in the injury repair transcriptome are consistent with the present data suggesting a dual function for PAI-1 (i.e., as a keratinocyte survival factor and regulator of epithelial migration) in the cutaneous injury response program.…”

“…uPAR-associated uPA/PAI-1 complexes, moreover, are endocytosed by LRP family members potentially altering LRP and/or uPAR signaling events important in migratory decisions [6,7,9]. Collectively, PAI-1 may function within the global program of the tissue repair response to coordinate cycles of cell-to-substrate adhesion/detachment [7,8,10,24,27,29]. The association between the activated phenotype and targeted accumulation of PAI-1 in close proximity to newly formed focal adhesions [20] is consistent with this function.…”

“…One particular transcript induced in abundance in wound-stimulated keratinocytes encodes plasminogen activator inhibitor type-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) gene family and the major physiologic regulator of the urokinase plasminogen activator (uPA)-dependent pericellular plasmin-generating cascade [29,39]. PAI-1 mRNA/ protein increase rapidly in cultured epithelial cells immediately adjacent to experimentally-created wounds and remain elevated over the course of monolayer "healing" [12,25,29], closely recapitulating PAI-1 spatial/temporal induction in the wounded epidermis [18,34]. Several SERPINS (i.e., PAI-1, protease nexin-1), in fact, modulate the complex integrative process of injury resolution largely through the focalized regulation of plasmin-initiated matrix remodeling, cell-to-substrate adhesion/detachment, migration and apoptosis [3,[7][8][9][10]24,26,35,40].…”

Allelic Variants of Upstream Transcription Factor 1 Associate With Carotid Artery Intima-Media Thickness The Cardiovascular Risk in Young Finns Study

“…It has been reported that in relatives of FCHL patients there is an increased risk for CVD mortality, indicating that the predetermined genetic factors in these individuals may effect the disease progression independently of risk factors. 42 However, USF1 is biologically highly relevant, because it encodes a ubiquitously expressed transcriptional regulator of several genes, such as apo E, 43 CRP, 44 plasminogen activator inhibitor-1 45 and osteopontin, 46 which are functionally important for lipid accumulation, 47 inflammation, and thrombosis. All these proteins have been recovered directly from atherosclerotic plaques of artery wall, [48][49][50][51] and all of them have also been connected to the development of early atherosclerosis.…”