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doi: 10.1242/10.1242/jcs.00051

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

¹ Center for Cell Biology & Cancer Research, Albany Medical College, Albany, NY 12208, USA
² Department of Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA

View larger version (62K): [in a new window]   Fig. 1. Monolayer injury stimulates PAI-1 synthesis specifically in wound-edge cells. Quiescent contact-inhibited cultures of RK, HaCaT and EC-1 cells were maintained either intact or scrape-wounded with a pipette tip. RK cultures were fixed and PAI-1 protein visualized by immunocytochemistry (A,B). Control quiescent RK cells expressed relatively low levels of PAI-1 (A). Within 6 hours after scraping, PAI-1 was readily detected in motile RK cells immediately juxtaposed to the injury site (B; arrow indicates direction of migration into the denuded area). Western analysis of lysates of HaCaT cells differentially harvested 24 hours after scrape-trauma confirmed a significant increase in PAI-1 expression by epithelial cells bordering the injury site compared with cells in the distal uninvolved monolayer (C). PAI-1 mRNA transcripts were upregulated in cells harvested from the wound edge within 5 hours after scrape-injury. PAI-1 mRNA abundance (normalized to A-50 and GAPD hydridization signal for EC-1/RK and HaCaT cells, respectively) remained elevated over the time course of wound repair (D). RK total RNA was not isolated at the 7 hour post-wounding time point in the analysis series summarized in D. The difference in PAI-1 mRNA kinetic profiles for HaCaT versus EC-1/RK cells reflects the relatively protracted time frame for HaCaT monolayer injury site closure compared with RK/EC-1 populations (i.e. 48-72 hours versus 24-36 hours).

View larger version (38K): [in a new window]   Fig. 2. Visualization of PAI-1-GFP in cellular migration tracks. Schematic of a pEGFP-1-based vector in which a chimeric transcript consisting of 1.3 kb of PAI-1 coding sequences and GFP is expressed under the control of a 0.8 kb PAI-1 `promoter' (A). Transfection of RK cells and reseeding in serum-containing medium resulted in synthesis of PAI-1-GFP detected initially in perinuclear Golgi-like structures (B). Approximately 6-12 hours later, PAI-1-GFP can be found in the matrix (probably vitronectin)-rich undersurface region upon removal of cells with saponin (C). The green `footprint' of a single cell is shown in C. Seeding of PAI-1 promoter-PAI-1 coding-GFP transfectants at low density in EGF-containing medium provides for the clear visualization of the chimeric PAI-1-GFP protein in cellular migration trails (D). The small bright image at the extreme right of the trail is the cell body. Transfection of RK cells with the PAI-1 promoter-PAI-1 coding-GFP vector followed by growth to confluency and subsequent scrape injury indicated that the resulting motile population deposited GFP-'tagged' PAI-1 into the cellular migration trails similar to that illustrated in D.

View larger version (38K): [in a new window]   Fig. 3. Targeted PAI-1 downregulation inhibits wound-stimulated cell motility. RK cells were transfected with a non-insert-bearing vector (Rc/CMV) or with constructs in which full-length PAI-1 coding sequences were cloned in antisense (Rc/CMVIAP) or sense (Rc/CMVPAI) orientation (A). Cultures were grown to confluency and scrape-wounded. Extent of repair-associated migration (% wound closure) was measured over a 24 hour period (B). There was no difference in stimulated motility among Rc/CMV- or Rc/CMVPAI-transfectants compared with non-transfected controls (RK). The rate of monolayer scrape repair by Rc/CMVIAP (antisense PAI-1)-transfected cells, in contrast, was significantly impaired (asterisk) relative to control RK cultures or to sense (Rc/CMVPAI) or empty vector (Rc/CMV) transfectants. Data plotted is mean±standard deviation of three independent wound repair determinations. Inset in B is a western blot of PAI-1 levels in the various cell types at the 24 hour time point illustrating downregulation of PAI-1 expression in the Rc/CMVIAP transfectants.

View larger version (65K): [in a new window]   Fig. 4. Binding of a PAI-1 E-box probe by nuclear proteins from growing and wound-stimulated RK cells. The 18 bp 32P-end labeled PAI-1 E-box probe (see Materials and Methods) was incubated with 10 µg of the nuclear protein fraction isolated from growing (A) or quiescent as well as wound-edge (B) RK cells and complexes resolved by gel electrophoresis. Two closely-spaced, `dumbell-shaped', bands (arrows) were evident in shifts produced by nuclear extracts derived from growing cells in the absence (none) of competing sequences (A). Incubation with the unlabeled WT PAI-1 18 bp E-box deoxyoligonucleotide (self) or the unlabeled standard consensus (SC) E-box construct (i.e. a CACGTG motif flanked by non-PAI-1 sequences) (both at a 50- to 100-fold molar excess) effectively blocked complex formation with the labeled probe. The AP-1 deoxyoligonucleotide and a mutant E-box construct (5'-CACGGA-3'), the latter in the context of PAI-1 flanking sequences, each failed to compete for probe binding (A). In contrast to PAI-1 probe patterns developed with nuclear extracts from quiescent (Q) RK cultures and which failed to form complexes that co-migrated with the slower mobile (i.e. upper) band, extracts prepared from wound-edge keratinocytes 2 hours post-scrape injury (W) produced the characteristic two-band complex (B).

View larger version (47K): [in a new window]   Fig. 5. Identification of USF-1 as PAI-1 E-box probe-binding factor. A 32P-body labeled PAI-1 wild-type (WT) E-box deoxyoligonucleotide probe was generated by PCR. Gel-purified probe was incubated with nuclear extracts from serum-stimulated EC-1 cells prior to UV irradiation and treated with DNase-1; the complexes were then boiled in sample buffer and resolved on SDS/9% polyacrylamide slab gels (A). A single major band at 44-45 kDa was resolved in the lane containing probe crosslinked to EC-1 nuclear extract (Probe+Extract). This band was not detected upon electrophoresis of probe alone (Free Probe) or in reactions where the probe was DNase-digested prior to addition of nuclear extract and UV crosslinking (Probe Digest). One low molecular weight nonspecific (ns) band was evident with the latter control. PAI-1 E-box-binding proteins were isolated from the nuclear fraction of growing EC-1 cells by tethered deoxyoligonucleotide affinity chromatography (B). Bound proteins were eluted and separated by gel electrophoresis. Western blotting confirmed USF-1 as one PAI-1 E-box target sequence binding element (B, Column). Two USF-1 species, corresponding in mobility to USF-1 (44 kDa) and phospho-USF-1 (P-USF-1, 45 kDa), were resolved by western analysis of extracts derived from growing EC-1 cells (B, Growing Cells). The predominant form of USF-1 eluted from PAI-1 deoxyoligonucleotide affinity columns co-migrated with the slower mobility (i.e. phosphorylated) USF-1 species. Acid phosphatase treatment (Phos) of nuclear extracts from serum-stimulated cells prior to gel electrophoresis and western blotting significantly decreased the abundance of the anti-USF-1 immunoreactive 45 kDa (P-USF-1) band compared with non-phosphatase-treated (Control) extracts (C).

View larger version (64K): [in a new window]   Fig. 6. USF-1 in situ localization and PAI-1 E-box-binding activity in wound-proximal keratinocytes. Compared with quiescent intact monolayer regions (A), HaCaT cells juxtaposed to the wound site (arrow in B indicates direction of migration into a denuded zone) exhibit significant cytoplasmic and nuclear immunocytochemical reactivity for USF-1 (B). In both panels A and B, nuclei are stained (red) with propidium iodide while green speckles indicate immunoreactive USF-1. Cells situated more distal (d) from the wound edge had considerably lower cytoplasmic and nuclear USF-1 (B). Nuclear USF-1 accumulation could be detected as early as 2 hours post-monolayer scraping (preceeding the increase in PAI-1 transcripts) and remained evident throughout the period of wound repair. The protracted (48-72 hour) time course of injury resolution in HaCaT cultures reflected (even at 24 hours after wounding) continued PAI-1 expression (Fig. 1) and nuclear USF-1 localization (B) by the migrating epithelium. The typical upper and lower dumbell-shaped gel retardation pattern was resolved upon incubation of nuclear extracts from growing (G), but not quiescent (Q), HaCaT and RK cultures with the 32P-labeled 18 bp PAI-1 E-box probe (C). The upper band was specifically supershifted upon addition of antibodies to USF-1 after formation of the protein-probe complex. Similarly, the USF-1-containing upper complex was also resolved upon incubation of nuclear extracts from RK cells harvested from the wound site (D). The upper-lower doublet retardation pattern was evident as early as 2 hours after scrape injury (2 hr edge) and, like growing RK cultures (G), addition of USF-1 antibodies specifically supershifted this upper complex.

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