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
* Author for correspondence (e-mail: higginp{at}mail.amc.edu)
Accepted 9 July 2002
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Summary |
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Key words: Keratinocytes, PAI-1, Reepithelialization, Gene targeting
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Introduction |
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Growth factor-initiated changes in the expression, focalization and/or
relative activity of uPA/PAI-1 may modulate cell migration either by
controlling the rate and extent of ECM barrier proteolysis or altering
cellular adhesive interactions with the ECM
(Pepper et al., 1992;
Seebacher et al., 1992
;
Stefansson and Lawrence, 1996
;
Mignatti and Rifkin, 2000
).
Co-expression of uPA, its surface-anchored receptor (uPAR) and PAI-1, for
example, are required for optimal Matrigel invasion by lung tumor cells
(Liu et al., 1995
). Peptides
that inhibit binding of uPA to its receptor ablated transforming growth
factor-ß1 (TGF-ß1)-induced planar motility by transformed
keratinocytes and significantly attenuated invasion across Matrigel barriers
(Santibanez et al., 1999
).
PAI-1 levels are consistently elevated, moreover, in aggressive tumor
phenotypes and PAI-1 expression is a major molecular feature of the
TGF-ß1-initiated epithelial-to-mesenchymal transition in various cell
systems (Santibanez et al.,
1999
; Akiyoshi et al.,
2001
; Zavadil et al.,
2001
). Variances in PAI-1 synthesis
(Providence et al., 2000
)
and/or site-localization (Kutz et al.,
1997
), therefore, would be expected to specifically impact on
cellular migration by affecting uPA activity as well as uPAR/vitronectin- or
integrin/vitronectin-dependent contacts
(Ciambrone and McKeown-Longo,
1990
; Deng et al.,
1996
; Chapman,
1997
; Stefansson and Lawrence,
1996
; Loskutoff et al.,
1999
). Targeted downregulation of PAI-1 synthesis with antisense
expression vectors and use of function-blocking antibodies, in fact, inhibited
basal as well as TGF-ß1-stimulated epithelial cell motility in both 2-
and 3-D model systems (Providence et al.,
2000
; Providence et al.,
2002
; Brooks et al.,
2001
; Kutz et al.,
2001
; Chazaud et al.,
2002
).
PAI-1 gene expression under conditions of induced migration is
predominantly transcriptional (Pawar et
al., 1995; Providence et al.,
2000
; Kutz et al.,
2001
). Multiple promoter elements mediate stimulus-specific
controls on PAI-1 transcription (e.g.
Westerhausen et al., 1991
;
Ryan et al., 1996
;
Slack and Higgins, 1999
). One
prominent regulatory sequence is the hexanucleotide E-box motif (CACGTG) that
is recognized by several members of the helix-loop-helix family of
transcription factors (e.g. MYC, MAX, TFE3, USF-1, USF-2, HIF-1)
(Riccio et al., 1992
;
Hua et al., 1998
;
Hua et al., 1999
;
Dennler et al., 1998
;
White et al., 2000
) and the
snail zinc-finger superfamily (Nieto,
2002
; Hajra et al.,
2002
). A consensus E-box, located at nucleotides -160 to -165
upstream of the transcription start site in the PAI-1 gene, is required for
PAI-1 promoter-directed reporter gene activation in growing EC-1 cells as well
as in hepatocytes subjected to mild hypoxia
(Kietzmann et al., 1999
;
White et al., 2000
). Using an
in vitro model of the epidermal response to injury, we now report that
monolayer wounding stimulates nuclear protein binding to a PAI-1
E-box-specific probe in the same cohort of cells induced to express PAI-1 mRNA
transcripts/protein. Deoxyolignucleotide affinity chromatography revealed
USF-1 to be a major PAI-1 E-box-binding factor. Monolayer wounding, moreover,
stimulated USF-1 nuclear translocation and PAI-1 E-box occupancy in
wound-proximal cells. These data suggest that the E-box may function as a
major transcriptional control element for migration-associated genes in much
the same manner as it has been implicated in the regulation of cell cycle
progression genes (e.g. Cogswell et al.,
1995
; White et al.,
2000
).
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Materials and Methods |
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Motility assessments and collection of cell subpopulations
Denudation zones were created by pushing the narrow end of a sterile P1000
plastic pipette tip through the quiescent, contact-inhibited, epithelial
monolayer. Wound closure rates, a function of planar motility in this
directional 2D migration assay (Kutz et
al., 2001), were calculated from measurements made using an
inverted microscope fitted with a calibrated ocular grid
(Providence et al., 2000
).
`Activated' cells (i.e. those immediately adjacent to the denudation site,
including cells that locomoted into the wound `bed') were harvested by pushing
the wide end of a P1000 pipette tip along the original injury tract,
displacing cells directly at, and 5 mm from, the migratory edge.
Scrape-released cells were aspirated and collected by centrifugation at 1400
g. Cells located 40 mm from the original wound border (i.e. in
the intact distal monolayer) were similarly harvested. To assess the effect of
targeted perturbation of PAI-1 synthesis on wound-induced motility, RK cells
were transfected with PAI-1 antisense and sense constructs created in the
Rc/CMV expression vector (Higgins et al.,
1997
).
Northern blot analysis
Total cellular RNA was isolated and denatured at 55°C for 15 minutes in
1x MOPS, 6.5% formaldehyde, and 50% formamide prior to electrophoresis
on agarose/formaldehyde gels (1.2% agarose, 1.1% formaldehyde, 1x MOPS).
RNA was transferred to Nytran membranes by capillary action in 10x SSC
(3 M NaCl, 0.3 M sodium citrate, pH 7.0), UV crosslinked and incubated for 2
hours at 42°C in 50% formamide, 5x Denhardt's solution, 1% SDS, 100
µg/ml sheared/heat-denatured salmon sperm DNA (ssDNA) and 5x SSC. RNA
blots were hybridized simultaneously with 32P-labeled cDNA probes
to PAI-1 and A-50 (RK and EC-1 cells) or PAI-1 and GAPD (HaCaT cells) for 24
hours at 42°C in 50% formamide, 2.5x Denhardt's solution, 1% SDS,
100 µg/ml ssDNA, 5x SSC and 10% dextran sulfate. Membranes were
washed three times in 0.1x SSC/0.1% SDS for 15 minutes each at 42°C
followed by three washes at 55°C prior to exposure to film.
Nuclear extracts
Cells were trypsinized, harvested by centrifugation, resuspended in 400
µl of cold buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT), placed on ice for 15 minutes, then vortexed for 10 seconds
after addition of 25 µl 10% Nonidet NP-40. Nuclei were collected by
centrifugation for 30 seconds at 14,000 g, resuspended in 50
µl of cold lysis buffer (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT) containing leupeptin, aprotinin, chymostatin, pepstatin A,
antipain (each at a final concentration of 10 µg/ml), rocked at 4°C for
15 minutes and extracts clarified at 10,000 g for 5 minutes.
For phosphatase treatments, isolated nuclei were lysed
(Cheung et al., 1999) and 5
µg nuclear extract protein incubated with potato acid phosphatase in
PIPES/KOH digestion buffer, pH 6.5, for 2 hours at 37°C prior to
electrophoresis on SDS/12% acrylamide gels and western blotting for USF-1 (as
indicated below).
Mobility shift assay
Double-stranded deoxyoligonucleotides (3-5 pM) were incubated at 37°C
for 10 minutes with T4 polynucleotide kinase (5-10 units/µl) in 70 mM
Tris-HCl buffer, pH 7.6, containing 10 mM MgCl2, 5 mM DTT and
[-32P]dATP (3000 Ci/mmol). Probes were purified by
filtration through 10 kDa cellulose spin columns. Constructs used were as
follows (only the coding strand is indicated):
Nuclear extracts were incubated with 50,000-100,000 cpm 32P
end-labeled target deoxyoligonucleotides in 5x gel shift buffer (20%
glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 0.4 mg/ml dIdC). Following room temperature incubation for
20 minutes, gel loading buffer (25 mM Tris-HCl, pH 7.5, 0.02% bromophenol
blue, 0.02% xylene cyanol, 4% glycerol) was added. Complexes were separated on
Tris/glycine gels (Tris/glycine buffer: 5 mM Tris-HCl, 2 mM EDTA, 100 mM
glycine) containing 4% acrylamide, 0.5% bisacrylamide, 2.5% glycerol, 0.75%
ammonium persulfate and 0.085% TEMED). Antibodies (1-2 µg per reaction)
were added to the formed extract protein/32P-labeled DNA probe
complexes and maintained at room temperature for 20 minutes prior to
electrophoresis for supershift assays
(White et al., 2000).
UV crosslinking
A PAI-1 wild-type (WT) E-box probe body-labeled with 32P for use
as a probe in mobility shift assays was generated by PCR using a primer set
corresponding to promoter region nucleotides -171 to -166 and -159 to -154 and
purified on a 10 kDa spin column. Nuclear extract-probe binding reactions were
incubated in a 96-well microtiter plate for 20 minutes at room temperature
prior to UV irradiation (4.8 to 24.0 µJoules/cm2) followed by
DNase-1 treatment (2 µg/ml). Sample buffer (50 mM Tris-HCl, pH 6.8, 10%
glycerol, 1% SDS, 1% 2-mercaptoethanol, 0.01% bromophenol blue) was added, the
complexes boiled and resolved on SDS/polyacrylamide slab gels (9% acrylamide,
0.24% bis-acrylamide, 0.375 M Tris-HCl, pH 8.8, 0.1% SDS, 0.03% ammonium
persulfate, 0.025% TEMED).
Immunocytochemistry
Media were aspirated and the cells washed 3x in PBS prior to fixation
for 10 minutes in 3% formaldehyde. After three PBS washes (5 minutes each),
fixed cells were permeabilized with 0.5% Triton X-100/PBS for 10 minutes at
4°C, washed three times in PBS, incubated in glycine (10 mg/ml) for 15
minutes, and incubated with antibodies to USF-1 (or preimmune IgG) followed by
fluorescein isothiocynate (FITC)-labeled secondary antibodies. Cells were
visualized by incubation in propidium iodide which yields red nuclear
fluorescence under UV light. Coverslips were mounted in anti-fade reagent for
confocal microscopy. For immunodetection of PAI-1, cultures were fixed in 100%
methanol for 20 minutes at -20°C then rehydrated by rinsing in PBS prior
to sequential incubations with PAI-1 antibody followed by FITC-conjugated
secondary antibody.
Tethered deoxyoligonucleotide protein binding and western
blotting
The PAI-1 18 bp wild-type E-box deoxyoligonucleotide was ligated to a
biotinylated 16-mer target sequence tethered to streptavidincoated magnetic
particles using the Boehringer Mannheim DNA-Binding Protein Purification kit.
Nuclear protein binding to the PAI-1 `bait' construct was done in the presence
of poly dIdC and poly L-lysine, the mixture vortexed, particles harvested with
a magnetic separator and washed, and bound proteins eluted by boiling in
electrophoresis sample buffer (50 mM Tris/HCl, pH 6.8, 10% glycerol, 1% SDS,
1% 2-mercaptoethanol) prior to separation on SDS/12% acrylamide slab gels.
Protein transfers were probed with antibodies to USF-1 (c-20) or c-FOS
(4-1D-G) (Santa Cruz Biotechnology) and antigen:antibody complexes visualized
by horseradish peroxidase-conjugated secondary antibodies and ECL detection
reagent (Amersham Pharmacia) using X-OMAT AR-5 film
(Li et al., 2000).
PAI-1-GFP chimera expression constructs
The human PAI-1 promoter sequence from -800 to +71 was PCR-amplified for 30
cycles using the p800-Luc reporter plasmid as a template and Platinum
Taq polymerase. The promoter fragment was gel-purified for subsequent
cloning into the SacI/KpnI sites of the promoter-less
expression vector pEGFP-1 (Clontech, Palo Alto, CA). The full-length human
PAI-1 coding sequence (approximately 1.3 kb) was derived by RT-PCR from total
RNA isolated from human foreskin fibroblasts, amplified by PCR using Platinum
Taq polymerase, gel-purified and expressed as a GFP fusion protein by
T4 ligase insertion into the KpnI/EcoRI sites of pEGFP-N3.
The same PAI-1 coding region was also transferred into the
BamHI/AgeI site of the PAI-1 promoter-derived pEGFP-1
vector. All constructs were sequence verified. RK cells were seeded into 35 mm
dishes and allowed to reach a density of 1x105
cells/cm2 prior to transfection with 1-2 µg DNA using
Lipofectamine-Plus. Transfected cells were trypinsized and plated at low cell
density in EGF-supplemented (1 ng/ml) growth medium. In some cases,
transfected cells were removed from the culture dish prior to microscopy by
incubation in 0.2% saponin in Ca2+/Mg2+-free PBS leaving
a substrate-attached PAI-1-rich matrix
(Higgins et al., 1997).
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Results |
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PAI-1 is highly expressed in migratory cells in vitro as well as in vivo
(Pepper et al., 1992;
Romer et al., 1991
;
Providence et al., 2000
).
Similarly, elevated de novo synthesis of PAI-1 by two keratinocyte cell lines,
RK (determined immunocytochemically) and HaCaT (by western blotting), in the
in vitro monolayer wound model is restricted to cells immediately juxtaposed
to the denudation site and which have acquired morphologic characteristics of
an early motile phenotype (Fig.
1). Leading-edge cells extending cytoplasmic projections into the
denuded area were particularly immunoreactive with PAI-1 antibodies.
Differential harvest of locomoting RK, HaCaT and EC-1 epithelial cells
indicated, moreover, that PAI-1 transcripts were significantly increased in
cells harvested along the site of the original wound track (13-to 27-fold
relative to the distal quiescent, unperturbed, monolayer cells) and remained
elevated throughout the repair process
(Fig. 1). The difference in
PAI-1 mRNA expression kinetics between RK/EC-1 and HaCaT populations was
attributable to the relatively protracted time course of wound resolution
(24-36 hours vs. 48-72 hours) by HaCaT cells. Visual examination of the motile
front population suggested that the newly synthesized PAI-1 was particularly
abundant at a plane of focus corresponding to the cellular
undersurface-culture substrate region. Transfection of RK cells with a
GFP-tagged PAI-1 expression vector, in which transcription of the chimeric
PAI-1-GFP insert is under the control of PAI-1 promoter sequences, provided
for the direct visualization of PAI-1-GFP protein in the `matrix' of
saponin-dislodged keratinocytes as well as in the migratory tracks of growth
factor-stimulated cells (Fig.
2). This approach to insert expression control (i.e. using PAI-1
upstream elements to drive PAI-1-GFP transcription) was selected since the
time course of PAI-1-GFP chimera induction closely approximated that of the
endogenous PAI-1 gene (e.g. Ryan et al.,
1996
). The PAI-1 promoter-PAI-1 coding-GFP construct was
transfected into confluent RK monolayers followed by incubation in serum-free
medium prior to monolayer scraping. By 5 hours, scrape-activated cells had
migrated well into the wound `bed' exhibiting PAI-1-GFP-decorated migration
tracks perpendicular to the long axis of the original scrape injury (not
shown). These findings (e.g. Fig.
2) suggested that PAI-1 might function as a component of the basal
epidermal cell motile apparatus as it appears to do in other cell types
(Stefansson and Lawrence,
1996
; Waltz et al.,
1997
; Brooks et al.,
2001
). To evaluate this possibility, RK cells were transfected
with the PAI-1 antisense expression vector Rc/CMVIAP, cultured under
quiescence conditions, then scrape injured. Two criteria were specifically
evaluated including effects on PAI-1 synthesis and on 2D planar motility. The
resultant attenuation of wound-induced PAI-1 protein synthesis in Rc/CMVIAP
transfectants (confirmed by western blotting) reflected a significant
inhibition of injury site closure (Fig.
3).
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Monolayer wounding stimulates PAI-1 E-box-binding activity
Since PAI-1 expression was clearly a critical modulator of epithelial cell
migration, it was important to clarify molecular events involved in PAI-1
expression control in response to monolayer wounding. A search of the 5'
flanking region of the PAI-1 gene, originally to identify potential cis-acting
elements involved in growth state-dependent gene expression
(Ryan et al., 1996;
Boehm et al., 1999
), identified
a consensus E-box motif (CACGTG) at nucleotides -165 to -160 upstream of the
transcriptional start site (White et al.,
2000
). This region is protected from DNase I digestion in growing
epithelial cells (Johnson et al.,
1992
). E-box-binding activity consisting of two closely-spaced
`dumbell-shaped' upper and lower bands, assessed using an 18 bp PAI-1 sequence
as a target in which the CACGTG motif was flanked both 5' and 3'
by PAI-1-specific sequences, was evident in RK
(Fig. 4), EC-1 and HaCaT (see
below) cells. This probe shift pattern was maintained when an unlabeled AP-1
deoxyoligonucleotide
(5'-CGCTTGATGACTCAGCCGGAA-3'), in 100-fold
molar excess, was included in the reaction mixture. Band shifts were
successfully competed, however, upon simultaneous addition of a 100-fold molar
excess of an unlabeled wild-type (self) competitor or a standard consensus
(SC) E-box construct (i.e. an E-box hexanucleotide motif with non-PAI-1
flanking sequences). A mutant E-box motif (either CACGGA or TCCGTG) flanked by
PAI-1-specific sequences failed to compete for probe binding
(Fig. 4). These same mutant
constructs were also incapable of forming shifted complexes when
32P end-labeled and used as targets in gel retardation assays (see
below). Collectively, these findings indicate a requirement, and specificity,
for an intact consensus hexanucleotide E box for protein binding to the
homologous site in the PAI-1 gene. Once probe E-box site occupancy by nuclear
factors isolated from constitutively-growing, PAI-1-expressing, RK cells was
established, it was necessary to determine whether a similar binding activity
could be detected in wound-stimulated cultures and, if so, the kinetics of
site occupancy relative to wound-induced expression of the endogenous PAI-1
gene. Nuclear extracts from differentially harvested wound-edge and distal
quiescent monolayer cells were incubated with the 32P-labeled 18 bp
PAI-1 E-box probe and the formed complexes resolved by electrophoresis.
Relative to cells that are in immediate proximity to the denudation injury and
that have the demonstrable characteristic 2-band pattern (upper and lower)
probe-binding activity, nuclear extracts of distal monolayer isolates
generally formed only a single complex corresponding in mobility to the lower
band (Fig. 4). E-box-binding
activity was evident soon after scrape injury in wound-edge cells consistent
with the subsequent increase in PAI-1 transcripts in the migrating cohort
(Fig. 1).
|
USF-1 is a PAI-1 E-box binding protein
An intact E-box site at nucleotides -160 to -165 in the proximal promoter
of the rat PAI-1 gene is an important platform for protein binding in response
to proliferative stimuli, mild hypoxia as well as to individual growth factors
including TGF-ß1 (Kietzmann et al.,
1999; White et al.,
2000
) (L.A.W. and P.J.H., unpublished). In order to identify
specific transcriptional effectors capable of binding to the PAI-1 E-box site
(based on data summarized in Fig.
4), a 32P body-labeled, PCR-amplified, fragment of the
PAI-1 promoter containing the CACGTG motif was UV crosslinked to nuclear
proteins isolated from growing EC-1 cells. A major complex of approximately
44-45 kDa was resolved after DNase-1 digestion of the probe-extract reaction
products and electrophoresis on SDS-acrylamide gels
(Fig. 5). Addition of
proteinase K to the UV-irradiated nuclear extract/deoxyoligonucleotide binding
reaction for a 5 minute incubation before gel electrophoresis eliminated the
44-45 kDa band, suggesting involvement of a crosslinked nuclear protein in the
formed complex (not shown). Prominent among E-box-binding proteins in this
mass range are several helix-loop-helix transcription factors most notably
members of the USF1/2 family (Littlewood
and Evan, 1995
). Tethered deoxyoligonucleotide affinity
chromatography was used, therefore, to isolate PAI-1 E-box-binding proteins
from the nuclear fraction of growing EC-1 cells. Bound proteins were eluted
and western blotting, in fact, confirmed USF-1 as one PAI-1 E-box target
sequence binding element (Fig.
5). Two immunoreactive USF-1 species, corresponding in mobility to
USF-1 and phospho-USF-1 (Galibert et al.,
2001
), were resolved in extracts of growing EC-1 cells
(Fig. 5). Blot analysis
suggested an approximately threefold increase in USF-1 levels in growing cells
compared with quiescent cells (L.A.W. and P.J.H., unpublished).
Phosphorylation of USF-1 is necessary for DNA binding
(Cheung et al., 1999
) and,
consistent with this requirement, the predominant form of USF-1 eluted from
PAI-1 deoxyoligonucleotide affinity columns co-migrated with the 45 kDa
(phospho-USF-1) species (Fig.
5). That the slower migrating 45 kDa species was phospho-USF-1 was
confirmed by potato acid phosphatase treatment of nuclear extracts from
serum-stimulated cells (which have abundant levels of the 45 kDa USF-1
immunoreactive protein) prior to western analysis. Once identified, it was
important to assess whether E-box-dependent USF-1 binding could be resolved in
nuclear extracts of scrape injury-stimulated cells (since PAI-1 transcripts
were upregulated specifically in wound-edge keratinocytes;
Fig. 1). Initial analysis of
wounded monolayers indicated that increased levels of immunoreactive USF-1
were evident in cells immediately adjacent, and in close proximity, to the
denuded site. Compared with intact cultures, scrape injury-juxtaposed cells
had significantly greater cytoplasmic and nuclear USF-1 immunoreactivity
(Fig. 6) correlating with the
specific in situ-restricted expression of PAI-1 transcripts/protein in the
wound-edge cohort (Fig. 1).
Mobility shift studies were designed, therefore, to evaluate whether this
augmented USF-1 nuclear accumulation, at least following wound stimulation,
correlated with an increase in USF-1 PAI-1 E-box construct binding activity.
Nuclear extracts from constitutively growing HaCaT and RK cells produced the
typical upper and lower doublet band shift pattern with the target 18 bp PAI-1
E-box probe. The upper band was specifically supershifted by antibodies to
USF-1 indicating that at least one contributing factor in this slower
migrating complex was USF-1 (Fig.
6). Extracts from quiescent cells (i.e. contact-inhibited cultures
maintained in serum-free medium for 3 days) did not form the upper banding
component with the target PAI-1 probe and the complexes that were resolved
were generally unreactive with USF-1 antibodies. Comparison of the probe gel
retardation patterns obtained with extracts from growing RK cells to those
developed with nuclear extracts isolated from wound-edge harvested cells (2
hours post-scrape injury), in contrast, confirmed that the upper component in
the doublet complex resolved with extracts from injury site cells, like that
in proliferating keratinocytes, could also be supershifted by USF-1 antibodies
(Fig. 6).
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Discussion |
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De novo synthesized PAI-1 protein accumulates in the cellular undersurface
region likely in a complex with matrix vitronectin
(Higgins and Ryan, 1989;
Seiffert et al., 1994
;
Lawrence et al., 1997
),
although PAI-1 has been suggested to also associate with fibronectin and/or
laminin deposits in migration tracks
(Seebacher et al., 1992
). This
SERPIN is well-positioned, therefore, to modulate integrin-ECM or uPA/uPAR-ECM
interactions as well as ECM barrier proteolysis. PAI-1 may dissociate bound
vitronectin from the uPAR, detaching cells that use this receptor as a
vitronectin anchor (Deng et al.,
1996
; Deng et al.,
2001
; Kjoller et al.,
1997
; Loskutoff et al.,
1999
). Alternatively, PAI-1 may directly inhibit
v
integrin-mediated attachment to vitronectin by blocking accessibility to the
RGD sequence located proximal to the uPAR binding site
(Stefansson and Lawrence,
1996
; Loskutoff et al.,
1999
). uPAR-associated uPA/PAI-1 complexes, furthermore, are
internalized by endocytosis promoting uPA receptor recycling
(Andreasen et al., 1997
) and,
thereby, vitronectin-dependent cell movement. Transgenic approaches suggest,
however, that PAI-1 promotes vitronectin-independent angiogenesis specifically
by inhibiting plasmin proteolysis, and thus preserves an appropriate matrix
`scaffold' to support cell migration or provide required neovessel stability
(Bajou et al., 2001
). These
findings highlight the complexity of cellular motile controls that
collectively reflect the level of expression of participating elements, the
nature of the `matrix' encountered, the system context (i.e., 2D vs 3D
migration) and the growth factor environment. The rapid kinetics of
wound-stimulated PAI-1 induction and relatively short matrix-associated
half-life (Higgins and Ryan,
1989
) suggests that this protein may influence cellular adhesive
events for a specified duration during injury repair.
Similar to PAI-1 induction under conditions of mitogenic stimulation
(White et al., 2000) is the
rapid wound-related recruitment of USF-1 to the same defined E-box site in the
PAI-1 promoter. Site occupancy, moreover, likely requires conservation of the
CANNTG motif as mutations outside of the two central nucleotides resulted in
loss of competitive binding activity. USF dimers as well as TFE3, HIF and
MYC/MAX family member homo- or heterodimers recognize E-box motifs within
certain genes including p53 and PAI-1 (e.g.
Riccio et al., 1992
;
Reisman and Rotter, 1993
;
Hua et al., 1998
;
Hua et al., 1999
;
Dennler et al., 1998
;
Kietzmann et al., 1999
;
White et al., 2000
) and
present data are consistent with the preference of USF proteins for CACGTG or
CACATG sequences (Littlewood and Evan,
1995
; Ismail et al.,
1999
). Successful PAI-1 probe competition by a CACGTG `core'
flanked by non-PAI-1 sequences and failure of specific E-box mutants with
PAI-1 homologous flanking DNA to similarly compete (or to produce band shifts
when used as targets) indicate, furthermore, that an intact hexanucleotide
E-box motif is necessary and sufficient for USF-1 binding in both serum- and
wound-simulated cells. The enrichment for phospho-USF-1 by DNA affinity
chromatography of extracts from growing cells compared with the relative
abundance of phospho- and non-phosphorylated species resolved by western
blotting of cell extracts indicated that USF-1 that bound to DNA was almost
exclusively phosphorylated, whereas only a fraction of the total cellular
USF-1 in proliferating cultures was phosphorylated at any given time. These
data are consistent with the known phosphorylation requirement of certain HLH
factors for E-box motif recognition
(Nozaki et al., 1997
;
Cheung et al., 1999
).
The mechanism of USF-1 functional mobilization (i.e. DNA-binding) in
response to wounding is speculative. Monolayer injury is associated with the
induced expression of several growth factors (e.g. FGF, HB-EGF, TGF-ß)
and with MAP kinase activation in cells bordering the denudation site
(Sato and Rifkin, 1988;
Dieckgraefe et al., 1997
;
Song et al., 2000
;
Ellis et al., 2001
). Certain
growth factors, particularly those of the TGF-ß family, stimulate
occupancy of E-box sequences in several genes including PAI-1
(Riccio et al., 1992
;
Hua et al., 1998
;
Hua et al., 1999
) as well as
activate MAP kinases (Kutz et al.,
2001
; Yue and Mulder,
2001
). Specific E-box-binding factors, including USF-1 and TFE3,
are phosphorylated at consensus MAP kinase target residues
(Galibert et al., 2001
;
Weilbaecher et al., 2001
)
facilitating DNA site interactions. At least one member of the stress family
of MAP kinases (p38) does, in fact, phosphorylate USF-1
(Galibert et al., 2001
),
although other growth-related kinases may also target USF-1. In synchronized
cells, for example, the DNA-binding activity of USF-1 is regulated by cyclin
A-p34cdc2- or cyclin B1-p34cdc2-dependent
phosphorylation within the USF-specific region (USR), the likely target site
(Cheung et al., 1999
).
Phosphorylation of residues within the USR appears to initiate a
conformational switch that exposes the DNA-binding domain
(Cheung et al., 1999
). Similar
to the requirements for interaction of MAX with its target E-box sequence,
USF-1 DNA-binding activity may be regulated, therefore, in a growth state- or
wound-responsive manner apart from direct controls on USF-1 or MAX synthesis
(Miltenberger et al., 1995
;
Lun et al., 1997
). One
possibility is that MAP kinase activation in the injured epithelium, dependent
or independent of an autocrine growth factor-initiated loop, results in USF-1
phosphorylation and subsequent trans-activation of specific USF-1 target genes
(e.g. PAI-1) as part of the switch from a sessile to motile phenotype.
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