1 Department of Anatomy and Cell Biology, The George Washington University
Medical School, Washington DC 20037, USA
2 Department of Ophthalmology, The George Washington University Medical School,
Washington DC 20037, USA
3 Division of Developmental and Newborn Biology, Department of Pediatrics,
Harvard Medical School, Boston, MA 02115, USA
4 Department of Pathology, The George Washington University Medical School,
Washington DC 20037, USA
* Author for correspondence (e-mail: mastepp{at}gwu.edu)
Accepted 22 August 2002
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Summary |
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Key words: Syndecan-1, Wound healing, Cell proliferation, Integrins
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Introduction |
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Previous studies have shown that the expression and localization of
keratinocyte integrins are upregulated in response to both skin
(Hertle et al., 1992;
Kainulainen, T. et al., 1998
)
and corneal (Stepp et al.,
1996
; Stepp and Zhu,
1997
) wounding. Integrins within the cell membrane associate with
many different molecules, including tetraspanins, growth factor receptors,
membrane-associated proteinases and syndecans. These associations can alter
integrin function either by clustering signaling molecules, altering integrin
internalization and turnover and/or by directly increasing ligand binding. The
primary syndecan expressed by keratinocytes is synd1 with lower amounts of
syndecan-4 (synd4) also expressed. Since syndecans can act as co-receptors
with integrins for matrix molecules, the loss of synd1 could affect
integrin-mediated functions.
The heparan sulfate side chains present on syndecan ectodomains associate
with growth factors and extracellular matrix molecules. Extracellular growth
factor:syndecan associations can either stimulate or inhibit tissue-specific
cellular functions depending on the cellular context. Normally syndecans bring
growth factors close to the cell surface and increase their association with
receptors; however, if syndecan is shed from the cell surface by the action of
proteases, the growth factor:syndecan interaction will reduce the
concentration of growth factors at the cell surface. Shed syndecan ectodomains
are found in the wound fluid after skin wounding
(Park et al., 2000a), and the
shedding of synd1 and synd4 has recently been demonstrated to be regulated by
the action of TIMP-3-specific metalloproteinases
(Fitzgerald et al., 2000
). The
most extensively studied syndecan:growth factor associations are those
involving fibroblast growth factor (Filla
et al., 1998
; Kusano et al.,
2000
) and hepatocyte growth factor
(Cornelison et al., 2001
;
Derksen et al., 2002
). Although
the integrin-syndecan interaction has been suggested by several studies
(Couchman and Woods, 1999
;
Saoncella et al., 1999
;
Kusano et al., 2000
), direct
association between synd1 and integrin has not been demonstrated. Syndecans
can also interact with proteins of the ADAMs (a disintegrin and
metalloproteinase) family; the ADAM-12syndecan interaction leads to
integrin activation and cell adhesion (Iba
et al., 2000
).
Others have studied the roles played by the cytoplasmic domain of the
syndecan core protein (Grootjans et al.,
1997; Hsueh and Sheng,
1999
; Grootjans et al.,
2000
; Hsueh et al.,
2000
). All four syndecan family members have cytoplasmic tails
that contain binding motifs of the PDZ type. PDZ domains, named after the
first three protein families shown to contain these motifs the PSD-95,
Disc-large (dlg) and Zonula occludens are known to organize protein
complexes at the cytoplasmic face of the cell membrane and to mediate the
interaction of the actin cytoskeleton with these complexes.
The dogma that heavily modified core proteins, including syndecans and
other HSPGs, are functional exclusively outside cells is beginning to be
questioned. It is clear that cells expressing synd1, including epidermal and
corneal keratinocytes, retain most of their synd1 within intracellular
compartments. The retention of large heavily glycosylated proteins and
proteoglycans was once believed to be due to their long transit time through
the endoplasmic reticulum and Golgi apparatus, but several lines of evidence
suggest intracellular functions. Brockstedt and colleagues have begun to look
at the role of synd1 in tubulin-mediated events
(Brockstedt et al., 2002),
including stabilization of the mitotic spindle and tubulin-mediated transport
of growth factors to the nucleus. The mechanism whereby synd1 mediates
transport within the cytoplasm probably involves transmembrane and cytoplasmic
domain sequences that remain available to cytosolic adaptors and docking
proteins after endocytic vesicles have budded off from the ER to make their
way to the plasma membrane. The cytoplasmic and transmembrane domains of synd1
have been shown recently to mediate a non-coated pit endocytic pathway
involving detergent-insoluble rafts (Fuki
et al., 2000
). The kinetics of the synd1-mediated pathway are
rapid, with a t1/2 of 1 hour and requiring intact actin
microfilaments and active tyrosine kinases. Detergent-insoluble rafts mediate
integration of signal transduction by growth factor receptors and integrins
after activation (Zajchowski and Robbins,
2002
). The idea that rafts might also regulate endocytic traffic,
in part, via synd1-mediated events, is interesting and requires further study.
The absence of synd1 might well affect the overall ability of cells to
regulate raft-mediated signal integration, which is essential for the
propagation of
6ß4-integrin-mediated signals
(Mainiero et al., 1997
;
Mariotti et al., 2001
).
Using our knowledge of the synd1 mouse gene
(Hinkes et al., 1993), we
designed a targeting vector to disrupt the expression of synd1 in mice. Given
the abundance of synd1 on stratified epithelia and the known roles syndecans
play in cell migration, we postulated that the loss of synd1 from the surface
of corneal and skin epithelial cells would affect the ability of these tissues
to respond to injury. Syndecan-1-knockout (synd1ko) mice appear healthy and
otherwise normal; however, when we studied the ability of these mice to heal
wounds, we observed healing defects after both corneal and skin wounding. To
begin to determine the mechanism behind the healing defects, additional in
vivo experiments were performed to assess cell proliferation, barrier function
and the localization and expression of several molecules implicated in
epithelial cell migration. In addition, mRNA expression profiles of unwounded
and wounded wild-type (wt) and synd1ko keratinocytes were assessed in the
cornea.
Additional studies using synd1ko mice have shown synd1 mediates
Wnt-1-induced mammary tumor progression
(Alexander et al., 2000),
resistance to bacterially mediated lung infections
(Park et al., 2001
), leukocyte
adhesion to endothelia (Gotte et al.,
2002
) and increased susceptibility to growth-factor-induced
angiogenesis (Gotte et al.,
2002
). This report is the first providing a description of the
generation of synd1ko mice and defects present within their epithelial
tissues. A previous report showed skin wound healing and cell migration
defects in the syndecan-4 knockout (synd4ko) mouse
(Echtermeyer et al., 2001
).
Although healing problems in the synd4ko mouse appear to result primarily from
defects in fibroblast migration, results presented in this study show that the
delayed healing in the synd1ko mouse results from impaired functioning of
keratinocytes.
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Materials and Methods |
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Construction of the synd1-targeting vector and generation of
synd1-deficient mice
The mouse synd1 gene structure was described previously
(Hinkes et al., 1993). The
targeting vector used was a 7.1 kb MboI/SalI fragment
isolated from a mouse 129 synd1 genomic DNA fragment containing DNA homologous
to the endogenous gene that included 1.4 kb of the 5' flanking sequence,
the entire 0.3 kb of the first exon and 5.3 kb of the large first intron. A
PKG neo-positive selection marker was inserted into a unique restriction site
to disrupt the signal peptide sequence, making it unlikely that the mature
proteins would reach the cell surface. A negative selection marker, MC1
thymidine kinase, flanked the first intron sequences at the 3' end of
the construct.
The linearized targeting vector was transfected by electroporation into J1
mouse 129 embryonic stem (ES) cells (Li et
al., 1992). A positive-negative selection strategy using G418 and
gancyclovir was employed, and ES cell colonies were evaluated for homologous
recombination by Southern blot analysis (Thomas and Capechi, 1987). Following
karyotype analysis, one of the targeted ES cell clones (1G7) was microinjected
into C57B1/6 and BALB/c blastocysts. Chimeric mice were maintained, and
subsequently the synd1 mutation appeared in the germ line. Breeding of the
heterozygous offspring resulted in animals homozygous for the synd1-targeted
mutation. ES cell clones and mice were genotyped by standard Southern blot
techniques (Laird et al.,
1991
) using BstEII digests of genomic DNA and a 760 bp
synd1 genomic probe that is external to the targeting construct and located in
the first intron. This probe identifies a 7.1 kb fragment from the endogenous
allele and a 8.6 kb fragment from the PGK neo-containing targeted allele.
The expression of synd1 and synd4 mRNAs in synd1ko and wild-type mouse skin
was assessed by northern blots as described previously
(Kim et al., 1994). 10 µg
each of total RNA obtained from adult synd1ko and wild-type mouse skin were
run on a 1% formaldehyde gel and blotted onto nitrocellulose membranes, using
a vacuum blotter. Probes for northern blots comprised cDNAs encoding the
ectodomains of synd1 and synd4 labeled with P32 dNTPs, using the
Rediprime II Kit (Amersham Pharmacia; Piscataway, NJ) and were labeled to a
specific activity equal to or greater than 6x108 cpm/µg.
Blots were hybridized using Rapid-Hyb hybridization solution (Amersham
Pharmacia; Piscataway, NJ) for 1 hour at 68°C and were then washed twice
in 2xSSC-0.1% SDS at room temperature, and once in 0.1xSSC with
0.1% SDS at 60°C for 30 minutes. Blots were exposed overnight.
Animal protocols
For full-thickness skin wound-healing experiments, mice were anaesthetized
with Avertin, and two full-thickness, 3 mm diameter wounds were made using a
sterile biopsy punch on the shaved and disinfected backs of 25 synd1ko and 25
wild-type mice, at 4-6 weeks of age. Wounds were left undressed, and all mice
were housed separately after wounding. At various times after wounding, mice
were sacrificed using a Metofane anesthetic overdose, and wounds with a 1 cm
unwounded skin border were harvested and processed for histochemistry. The
time points analyzed included 2, 3, 4, 5 and 9 days post-injury.
For dermabrasion experiments, the procedure described by Wojcik and
colleagues was followed (Wojcik et al.,
2000). 10 synd1ko and 10 wt mice between 7-9 weeks-of-age were
anaesthetized and the fur on their backs trimmed with scissors and removed
using depilatory cream (Nair creme depilatory; 1 minute, Carter-Wallace Inc.,
New York, NY). Skin was disinfected and allowed to dry. Cellophane tape
(Scotch brand; 0.5 in width) was then applied and removed quickly eight times
centrally on the backs of mice. Dermabraded areas were treated with Betadine
solution and allowed to recover for either 2 or 5 days. Two hours prior to
sacrifice, mice were injected with BrdU solution; after sacrifice by
anaesthetic overdose, full-thickness skin tissues were dissected free from
underlying fascia, rapidly frozen in liquid nitrogen and processed for either
immunofluorescence or immunoblotting. For immunofluorescence, the frozen skin
tissues were trimmed to approximately 1 cm2 and then embedded in
tissue-embedding medium. For Western blotting, frozen skin tissues were ground
to a fine powder using a mortar and pestle kept frozen with liquid nitrogen.
Powdered skin was extracted as described previously
(Stepp et al., 1996
). For
immunoblotting, control tissues were of two types: skin taken from mice whose
hair had been removed immediately after sacrifice (control) and skin taken
from mice whose fur had been removed 2 days prior to sacrifice but whose skin
had not been subjected to dermabrasion (unwounded). For wounded tissues, 2 mm
areas of skin tissue surrounding the involved skin were obtained.
All corneal wound healing experiments on mice were conducted in compliance
with the recommendations of the Association for Research in Vision and
Ophthalmology. Manual debridement wounds were created on the corneas of
8-week-old synd1ko and wild-type mice, as described previously
(Stepp and Zhu, 1997). Mice
were anaesthetized with general anesthesia and their eyes numbed with a
topical anesthetic. Corneas were then scraped with a dull scalpel to remove
the epithelium within a 1.5-mm central corneal area, which had been demarcated
with a dull trephine. Animals were sacrificed by lethal overdose at times
ranging from 12 hours to 12 weeks. To determine the timing of wound closure,
corneas were stained with a vital dye at various times after wounding, then
evaluated under the dissecting microscope and determined to be `opened' or
`closed' on the basis of whether the dye had stained the central cornea. The
numbers of animals used per time point for wound closure analyses were as
follows: 12 hours: (7 wildtype and 6 synd1ko), 18 hours (10 wildtype and 17
synd1ko), 24 hours (14 wildtype and 25 synd1ko), 36 hours (8 wildtype and 16
synd1ko), 48 hours (no wildtype and 13 synd1ko), and 60 hours (no wildtype and
4 synd1ko). Data were analyzed for statistical significance using the
Mantel-Haenszel Chi-Square Test. After the eyes were assessed under the
dissecting microscope for scarring and neovascularization, they were
enucleated and frozen in Tissue Tek II OCT compound for immunofluorescence and
confocal microscopy. For both whole mount and cryostat-sectioned
immunofluorescence studies, at least three eyes from three different animals
were used per time point.
For mRNA profiling, after sacrifice, corneal epithelial sheets were harvested from unwounded mice and from mice at 18 hours after wounding, by scraping and immediate freezing in liquid nitrogen. The debrided epithelia from no fewer than 20-25 unwounded and 15-20 wounded corneas were pooled to obtain sufficient RNA for RNA profiling. Frozen tissues were used to extract RNA for cDNA array analyses using the Atlas 1.2 mouse cDNA array (Clontech; Palo Alto, CA). Comparisons were made between unwounded and wounded wild-type RNAs, between unwounded wild-type and unwounded synd1ko RNAs, and between unwounded synd1 and wounded synd1 RNAs using Atlas Image 1.0 (Clontech; Palo Alto, CA).
To determine the barrier function of the corneal epithelium after wounding
in the synd1ko mice, a surface biotinylation procedure was used
(Xu et al., 2000). Wt and
synd1ko mice were wounded and allowed to heal for 4 weeks. After sacrifice,
eyes were removed and placed in 1 mg/ml sulfo-NHS-LC-biotin (Pierce Chemical,
Rockford, IL) in Hank's media supplemented with 1 mM CaCl2 and 2 mM
MgCl2. After 30 minutes at room temperature, tissues were rinsed in
PBS and frozen in OCT for sectioning. To determine the extent of penetration
of biotin, tissue sections were incubated with rhodamineavidin (Vector Labs,
Burlingame, CA) for 1 hour and slides coverslipped and viewed using the
confocal microscope. To visualize Zonula occludens (ZO-1) localization and
biotin penetration simultaneously, tissues were incubated first in primary
antibody alone followed by Alexa Fluor 488 (Molecular Probes, Eugene, OR)
conjugated secondary antibody and rhodamine-avidin.
Immunohistochemical studies
Corneal or skin tissues were sectioned (8 µm), and unfixed frozen
sections used for immunofluorescence microscopy as described previously
(Stepp and Zhu, 1997). Synd1
localization was evaluated using a rat monoclonal antibody against the synd1
core protein (281-2, now commercially available from Pharmingen, cat # 09341D;
San Diego, CA) and the synd4 core protein. Polyclonal anti-peptide sera
recognizing
9 and ß4 integrins were characterized as described
previously (Sta. Iglesia et al.,
2000
). The rat monoclonal against ZO-1 was obtained from Chemicon
(cat # MAB1520; Temecula, CA). Secondary antibodies were obtained from
Molecular Probes (Eugene, OR); Alexa Fluor 488 and/or Alexa Fluor 568 were
used for double labeling experiments.
Whole mounts were performed on eyes that were fixed immediately after
sacrifice in 70% methanol/DMSO for 2 hours followed by 100% methanol overnight
(as a minimum length of time). After fixation, corneas were dissected away
from the back of the eye, rehydrated, blocked and processed for 9
integrins. After the final wash after the secondary antibody, tissues were
processed for with propidium iodine prior to mounting en face, for viewing via
confocal microscopy. Images were viewed and captured by confocal scanning
laser microscopy using an inverted Olympus BX60 fluorescence microscope
(OPELCO, Dulles, VA) equipped with the Biorad MRC 1024ES laser system, Version
3.2 (Bio-rad, Hercules, CA). All images were imported into Adobe Photoshop 5.0
(Adobe Systems; Palo Alto, CA).
Bromodeoxyuridine (BrdU) cell proliferation analyses
Cell proliferation was assessed using the BrdU Labeling and Detection Kit I
(Roche Diagnostics; Indianapolis, IN), as recommended by the manufacturer.
Corneas were wounded as described above, and after sacrifice, eyes were
immediately placed in a BrdU-containing labeling solution consisting of
complete MEM (Stepp et al., 1997). After 30 minutes in labeling solution, the
eyes were washed three times and allowed to incubate for an additional 15
minutes in MEM without BrdU.
For dermabrasion studies, skin was obtained from mice injected with BrdU 2 hours prior to sacrifice. Uninvolved and involved skin was removed from the backs of the mice, gently stretched and flattened on a glass surface and rapidly frozen with liquid nitrogen. For corneal studies, intact eyes, and for skin studies, 1-2 cm2 pieces of skin were embedded in OCT and 8 µm sections cut. Sections were hydrated and fixed in ice-cold 70% methanol in 50 mM glycine buffer, pH-2, for 20 minutes at -20°C. After fixation, sections were incubated in PBS twice for 2 minutes each, incubated with 2.5% trypsin in 0.1% CaCl2 in PBS for 3 minutes at room temperature, briefly washed in PBS and then transferred to a solution containing 4 M HCl for 3 minutes at room temperature. Sections were washed again twice for 5 minutes each in PBS and then transferred to blocking solution (1% BSA and 0.1% horse serum in PBS). Slides were then processed for immunofluorescence. To quantify proliferation rates after corneal wounding, the numbers of labeled cells per unit area of the basal membrane surface of the basal cells were determined using morphometry. No fewer than five visual fields were counted per time point. At least two corneas from two different experimental procedures and two slides for each time point studied were used for these studies. For skin, the numbers of labeled cells per visual field at 20x magnification were assessed. Five wild-type and five synd1ko mice were assessed for data obtained 2 days after dermabrasion; two wild-type and two synd1ko mice were used for data obtained 5 days after wounding. The numbers of labeled cells in at least three different sections from each mouse were counted. Data were analyzed for significance using the unpaired t-test.
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Results |
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The progeny resulting from n=23 matings of heterozygous parents from either C57BL/6 or BALB/c lines showed a Mendelian transmission pattern, with 24% homozygotes, 46% heterozygotes and 30% wildtype, suggesting that the disruption of the synd1 gene does not alter mouse viability or cause embryonic lethality. Reproductive capacity was unchanged, as no significant difference in litter size was observed between matings of homozygous-null and wild-type mice of both C57BL/6 and BALB/c strains. No histological differences between tissues of the synd1ko and wild-type mice were observed at either 6 weeks or 6 months of age. Routine serum chemistry and hematologic studies revealed no differences between wild-type and synd1ko mice.
The characteristics of synd1ko epidermis and corneal epithelia were
assessed by immunofluorescence (Fig.
1D). In wild-type skin and cornea, synd1 was most abundant within
the cytoplasm at perinuclear locations as well as at cell-cell membranes. It
was not present at the apical squames or at the basal cell basement membrane
zone. ß4 integrin, a component of the hemidesmosomes, was present
primarily at the basal cell basal membranes in both the wt and the synd1ko
cornea and skin. Synd1, as expected, was absent from the skin and cornea of
the synd1ko mice. Despite the presence of synd4 mRNA in wild-type and synd1ko
mouse skin, synd4 protein was below detection in both wild-type and synd1ko
skin and cornea, and was not upregulated in synd1ko epithelia (data not
shown). The epithelial integrins 3 and
9, as well as E-cadherin
and ZO-1, were also assessed in the skin and cornea of the wild-type and
synd1ko mice and were found to be localized to the expected sites in the
synd1ko mouse tissues. Although ß4 integrin localized to the basement
membrane zone, ZO-1 was most abundant at the apical-surface of stratified
epithelia.
3 and
9 integrins were localized to the apical,
lateral and basal cell membranes of basal keratinocytes in skin.
9
integrin localization in the cornea was restricted to the limbal basal cells,
whereas
3 integrin was expressed equally in the limbus and central
cornea. The similarity between these wild-type and synd1ko epithelial tissues
in terms of localization of markers for epithelial tissue polarity indicated
that the synd1ko tissues are normal.
Synd1ko mice do not re-epithelialize corneal wounds properly
Synd1 is highly regulated during the repair of skin wounds, with expression
increasing in both the epidermis and dermis, and shed ectodomain accumulating
in the granulation tissues and wound fluid
(Bernfield et al., 1999). To
determine whether the synd1ko mouse epithelial tissues can repair wounds
normally, we first used a corneal epithelial debridement model, which involves
the manual removal of a circular patch of epithelial cells at the center of
the cornea, leaving the underlying basement membrane zone intact and native
(Fujikawa et al., 1984
;
Gipson et al., 1984
). Corneal
debridement wounds in the synd1ko mice re-epithelialized at a slower rate than
those in the wildtype, with differences becoming statistically significant
(P<0.05) by 24 hours after wounding
(Fig. 2A,B). Data showed that,
whereas 50% of the corneal wounds were closed at 22 hours in the wild-type
mice, it took 30 hours for 50% of the synd1ko wounds to close. Routine
histology was performed to assess the morphology of the corneal epithelial
cells after wounding (Fig. 2D).
During migration, the leading edge extended normally in both wild-type and
synd1ko mice; however, there appeared to be more inflammatory cells beneath
the leading edge in the healing synd1ko corneas. Although the wild-type
corneas appeared morphologically normal 1 week after wounding, at 2 weeks, the
epithelium of the synd1ko corneas showed evidence of hypoplasia.
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Cell proliferation abnormalities are present in the synd1ko corneal
epithelial cells before and after wounding
To determine whether there were differences between cell proliferation in
the synd1ko and wild-type corneal epithelial cells, cell proliferation was
assessed using BrdU (Fig. 2C).
Surprisingly, the synd1ko corneal epithelial cells showed a higher cell
proliferation rate prior to wounding than did the wild-type cells. For
wild-type mice, the proliferation rate was significantly elevated 24 hours
after injury, when the majority of the wounds were closed; by 48 hours, the
proliferation rate was back to that observed in unwounded wild-type corneas.
Although the rate of proliferation in the syn1ko corneas was higher before
wounding, it did not increase significantly at any time after wounding. Thus,
the cellular signal required to trigger increased cell proliferation after
wounding was either not transmitted or not recognized in corneal epithelial
cells in the absence of synd1.
Proliferation rate differences among cells generally reflect differences in
steady-state mRNA levels. To determine if there were any overall differences
in steady-state mRNA levels, mRNA profiling was performed using a mouse cDNA
array on total RNA isolated from both unwounded and wounded wild-type and
synd1ko epithelial tissues (Table
1). mRNA was extracted from corneal epithelial tissues obtained by
manual debridement of the ocular surface of unwounded mice immediately after
sacrifice or mice at 18 hours after wounding a time when the majority
of epithelial cells were still migrating but prior to the onset of
wound-induced cell proliferation in wt corneas. Only mRNAs whose expression
varied three-fold or greater are listed in
Table 1. Several differences
between mRNAs expressed in unwounded synd1ko and wild-type corneal epithelia
were observed, including mRNAs that encoded proteins important in cell
signaling and transcriptional regulation
(Table 1A). These differences
probably underlie the increase in cell proliferation observed in the unwounded
synd1ko corneal epithelial cells. In wild-type corneal epithelial cells,
wounding resulted in a three-fold or greater induction of six mRNAs, some of
which encoded proteins implicated in cell signaling and migration, such as
6 integrin. There was also a reduction in expression of four mRNAs
(Table 1B). During migration,
only one mRNA, which encodes keratin 14, normally upregulated in wild-type
epithelial cells, was also upregulated in synd1ko epithelial cells. By
comparison, six mRNAs were upregulated in wild-type corneas, while only three
mRNAs were detected at levels three-fold or greater during migration in
extracts from the synd1ko corneas, with nine mRNAs found at levels at least
three-fold lower (Table 1C).
Among those mRNAs, whose expression was reduced in the synd1ko corneal
epithelial cells, were several whose proteins are involved in regulation of
gene transcription. Taken together, the mRNA profiling data and the lack of an
increase in cell proliferation in synd1ko epithelial tissues after wounding
indicate that synd1ko corneal epithelial cells fail to activate appropriately
after wounding. The altered expression of several mRNAs prior to injury
further suggests that the lack of the synd1 HSPG alters cellular homeostasis
and results in the elevated cell proliferation rate observed in quiescent
synd1ko corneal epithelial cells.
|
Transient differences in the localization of 9 integrin are
observed immediately prior to wound closure in the cornea
To begin to determine the specific proteins responsible for delayed cell
migration in the synd1ko mice, the localization of a number of proteins
implicated in cell migration and/or epithelial cell-cell adhesion were
evaluated during and after reepithelialization, when the tissue normally
restratifies and the cells repolarize. The proteins evaluated include:
cytoskeletal proteins (actin, vinculin, talin, and -actinin); integrins
(
2,
3,
4,
5,
6,
9,
v,
ß1, and ß4); molecules involved in mediating cell-cell adhesion
(E-cadherin, ZO-1, occludin, and connexin 43); and matrix proteins including
tenascin, laminin-5, perlecan, and entactin.
The localization of 6ß4 integrin and synd1 has been reported to
be altered during corneal and skin wound healing
(Hertle et al., 1992
;
Stepp et al., 1996
;
Kainulainen, T. et al., 1998
).
In our wild-type corneas, synd1 and ß4 integrin localization overlapped
within basal cells (Fig. 3A).
Synd1 in wild-type corneas was expressed in multiple cell layers but was
absent from the apical squames. Upon wounding, ß4 integrin was no longer
polarized exclusively to the basal cell membranes of basal cells but was found
instead at lateral and apical membranes and away from the leading edge, at
suprabasal cell locations. Although both ß4 integrin and synd1 were
excluded from the tip of the leading edge, synd1 was present a short distance
back from the leading edge, proving that shedding of synd1 into the wound site
was restricted to the first few cells at the leading edge after corneal
debridement wounding.
|
In the synd1ko mouse corneas, the localization of ß4 integrin was also polarized at the basal cell layer and enhanced at basal membranes (Fig. 3A). After wounding, the changes in ß4 integrin localization observed in the synd1ko corneas were identical to those seen in wild-type mice; the timing of the shift in localization of ß4 integrin from basal to more lateral cell membranes was also similar. Previously, we and others have shown that the shift of ß4 integrin from exclusively basal to basal and suprabasal cells was associated with the loss of hemidesmosomes at the basal cell membranes (Sta. Iglesia and Stepp, 2000).
Although no differences in the localization of ß4 integrin were
observed before, during or after migration, differences in cellular
organization and 9 integrin were seen in en face views of whole mounts
of corneal tissues immediately prior to wound closure
(Fig. 3B). Unwounded wild-type
corneas had no
9 integrin within the cornea but brightly labeled cells
were present at the limbal region. A similar pattern was observed for
9
integrin in unwounded synd1ko corneas. When wounds were matched not by time
after wounding but for similar sizes of remaining wound bed, it became
apparent that wild-type corneal epithelial cells expressed more
9
integrin in and around the leading edges and at sites where the margins of the
epithelial sheets merged (Fig.
3B). In addition, the propidium-iodide-stained nuclei are larger
than those of the wildtype, suggesting that the cells are flatter and more
spread out in the synd1ko cornea compared to the wildtype. Twelve hours after
wounding,
9 integrin was not observed in the epithelial cells of the
migrating sheet, and by 48 hours,
9 integrin localization in the
central cornea had decreased to background levels in the cornea in both
wild-type and synd1ko corneas (data not shown). Other than these differences
in
9 integrin localization immediately prior to wound closure, no
differences in the localization of other integrins (
2,
3,
4,
5,
v and ß1), cytoskeletal proteins (actin,
vinculin, talin, and
-actinin), E-cadherin or matrix proteins
(tenascin, laminin-5, perlecan, and entactin) accompanied the delayed
re-epithelialization rate in the synd1ko mouse corneas.
Defects persist in synd1ko corneas after injury
To determine whether corneal healing defects were transient or persistent,
we assessed the localization of ß4 and 9 integrins at times up to
12 weeks after wounding by double labeling using an antibody against ZO-1 to
indicate the apical-most cell layers. Typical data for tissues obtained 12
weeks after wounding are shown in Fig.
4A. Age-matched unwounded corneas were included in the analyses to
control for the possibility of age-related defects in the synd1ko mice. The
localization of
9 integrin at the limbal region was not affected in the
synd1ko mouse by aging or wounding. Aging in the wild-type and synd1ko corneas
resulted in an increase in
9 integrin within numerous corneal basal
cells that appears to be independent of wounding. ß4 integrin remained
polarized to the basal aspect of the corneal epithelial basal cells in the
wounded synd1ko mice. The localization of ZO-1 was polarized to the
apical-most cell layers of the limbus and cornea of both the age-matched
unwounded synd1ko and the wounded wild-type mice. However, in the wounded
synd1ko corneas, ZO-1 staining was diffuse and more wide spread. The failure
of ZO-1 to repolarize properly in the synd1ko central cornea after wounding
did not extend to the limbus. Tissues taken at time points ranging from 48
hours to 8 weeks confirmed defects in ZO-1 relocalization in the corneas of
the synd1 ko mice (data not shown). ß4 integrin remained polarized to the
basal aspect of the corneal epithelial basal cells in the wounded synd1 ko
mice.
|
The mislocalization of ZO-1 to multiple cell layers after wounding in synd1 ko corneas reflects a persistent loss of tight junctions in these tissues. To determine whether this was the case, the barrier function in these tissues was assessed using a biotin penetration assay. Typical data obtained from mice 4 weeks after wounding indicate that, despite the diffuse localization of ZO-1, the barrier function was intact, and biotin had not penetrated the corneal epithelium of the synd1 ko mice (Fig. 4B).
Cell proliferation defects and altered integrin expression were also
observed during the healing of cutaneous wounds in the synd1 ko mouse
To determine whether the synd1 ko mice would also show defects in the
repair of skin wounds, we first made 3 mm full-thickness skin wounds on the
backs of synd1 ko animals and compared healing to that of wild-type animals.
Typical histological data for 5 and 9 days are shown in
Fig. 5. Although no differences
in the rate of wound closure could be observed before day 5, pronounced
hypoplasia of the epithelial tissues within the wound bed of the synd1 ko mice
was seen, which persisted 9 days after wounding. The adherent palisaded layer
of epithelial cells observed in the wild-type mouse skin 9 days after wounding
was absent from the synd1 ko mouse wound bed. The synd1 ko epidermis was thin,
and basal cells remained flattened and did not regain their normal columnar
morphology. No differences in the expression of -smooth-muscle actin
expression in cells in the healing dermis were observed at any time point
after wounding, and wound contraction appeared normal (data not shown). These
data show that while re-epithelialization took place, restratification of
closed wounds was defective in the synd1 ko skin.
|
The above results show that poor healing after corneal and skin wounding in the synd1 ko mouse is associated with hypoplasia. To assess cell proliferation and integrin expression after skin wounding, we focused our efforts on a dermabrasion rather than a full-thickness skin wound model. The rationale for changing wound healing models was to focus our attention on epithelial cell proliferation rather than migration. We found that dermabraded skin healed more slowly in the synd1 ko mice, at both 2 and 5 days after wounding. In addition, synd1 ko dermabrasions appeared deeper and tissues more inflamed than those of wild-type controls (Fig. 6A). Cell proliferation was assessed using BrdU, and epidermal keratinocytes from the synd1 ko mice were found to proliferate at a significantly (P<0.05) higher rate prior to dermabrasion (Fig. 6B). At 2 days after wounding, there was only a slight increase in cell proliferation in the synd1 ko skin cells, whereas, in the wt skin, cell proliferation increased over three-fold. These significant differences in cell proliferation between wt and synd1ko skin persisted at 5 days after wounding.
|
To determine whether any differences in integrin, E-cadherin or ZO-1
localization occurred after dermabrasion in synd1ko mouse skin, tissues taken
from unwounded and wounded skin 2 days after dermabrasion were used in
colocalization studies (Fig.
6C,D). As had occurred after wounding in both wild-type and
synd1ko corneas, 9 integrin localization increased in the keratinocytes
after dermabrasion. Although it was restricted to the basal cell layer in
unwounded epidermis,
9 integrin was also found in multiple cell layers
of healing wild-type epidermis. In the synd1ko skin, there was also an
increase in localization of
9 integrin adjacent to the involved skin,
but it appeared to be more modest, affecting fewer cell layers. This result
was consistent with the results observed immediately prior to wound closure in
the cornea, where there also appeared to be less
9 integrin
localization after wounding in the synd1ko than in the wild-type tissues
(Fig. 3B). Furthermore, there
were no changes observed in either
3 integrin or E-cadherin
localization after dermabrasion. Similarly, ß4 integrin remained
localized to the basal-most cell membrane adjacent to the basement membrane,
and we saw no evidence of increased localization of ß4 integrin in either
wild-type or synd1ko skin after dermabrasion.
To confirm biochemical changes in integrin expression in wild-type and
synd1ko mouse skin in response to dermabrasion, analyses of integrin
expression were conducted in wild-type and synd1ko wounded and unwounded
full-thickness skin tissues using immunoblots
(Fig. 6E). The relative
expression levels of 9 and
3 integrins were compared in protein
extracts obtained from three different variables. Proteins were extracted from
the control back skin (c), skin from which hair had been removed 2 days before
but had not been dermabraded (u), and involved skin dermabraded 2 days
previously (w). For samples that had been dermabraded (w), extracted tissues
included a 2 mm region surrounding the involved wound bed. Data show that
3 and
9 integrins were present in higher amounts in synd1ko skin
regardless of whether or not it had been subjected to hair removal 2 days
prior to sacrifice. Hair removal alone stimulated
3 integrin expression
in both wild-type and synd1ko skin but decreased
9 expression. At 2
days after wounding, there was no increase in either
3 or
9
integrin expression observed in extracts from the involved skin. Although
confocal microscopy does not show any difference in integrin expression and
localization in the skin of the synd1ko and wild-type mice
(Fig. 1D), these biochemical
studies suggest that expression of some integrins is enhanced in the absence
of syndecan-1. After dermabrasion, confocal microscopy data show increased
localization of
9 integrin in both wild-type and, to a lesser extent,
in synd1ko keratinocytes adjacent to the involved wound bed
(Fig. 6C). The immunoblots used
whole skin extracts. After dermabrasion, degeneration of the epidermis
(Fig. 6A) results in lower
levels of epithelial proteins overall in wounded tissues and probably masked
differences in integrin expression in the skin next to the wound site. Thus,
although the immunoblot data do not provide direct support for enhanced
expression of
9 integrin protein during healing, they do show that
integrin expression within skin is altered in the absence of syndecan-1.
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Discussion |
---|
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---|
Surprisingly, there were no differences detected in the localization of
cytoskeletal proteins, including filamentous actin, vinculin, talin and
-actinin in synd1ko corneas. Both
3ß1 and
6ß4
laminin-binding integrins have been implicated in mediating epithelial cell
migration in response to injury (Belkin and
Stepp, 2000
), and their ligand, laminin-5, and its cleavage play a
role in facilitating stable or transient integrin-mediated cellular attachment
(Gianelli et al., 1997; Goldfinger et al.,
1998
; Goldfinger et al.,
1999
).
6ß4 protein expression increases in wild-type
mice after corneal scrape wounding (Stepp et al., 1997), and in this study, we
show a comparable increase in the wounded synd1ko mouse corneas. However,
after induction of cell proliferation in skin by dermabrasion, ß4
integrin localization was unaltered in the wild-type and synd1ko basal cells,
suggesting that the enhanced expression of
6ß4 after full
thickness skin wounding maybe related more to the disassembly of
hemidesmosomes and induction of cell migration than to induction of cell
proliferation.
Although we demonstrated cell migration delays in the synd1ko corneal
keratinocytes, full-thickness skin wounds did not show delayed migration;
however, it is possible that the complexity of wound healing after
full-thickness skin wounding masked the difference in keratinocyte migration.
Re-epithelialization after a full-thickness skin wounding involves epithelial
cell migration as well as wound contraction and the growth of new blood
vessels into the wound site. Keratinocyte proliferation and differentiation
are known to be subject to regulation by paracrine secretion of factors by
both keratinocytes and dermal fibroblasts
(Werner and Smola, 2001).
Synd1 expression is normally upregulated in both the dermis and the epithelium
after skin wounding. Defects in both stromal cells and keratinocytes could
contribute to the overall wound healing phenotype observed in the synd1ko
mice. The use of the corneal model for studies of re-epithelialization
eliminates several extrinsic factors present in the skin-healing model that
complicate assessment of migration rates, since it was developed to minimize
activation of stromal fibroblasts and allow cells to migrate over a native
basement membrane zone in the absence of induction of neovascularization.
However, even in the corneal model, defects in stromal fibroblast secretion of
cytokines could contribute to the healing phenotype observed.
A dermabrasion wound model was used as it is less invasive than a
full-thickness wound model and involves primarily keratinocytes, thus leaving
the basal cellbasement-membrane hemidesmosome interaction intact.
Moreover, dermal response following dermabrasion is minimal; therefore, this
type of wound is ideal for assessing keratinocyte cell proliferation. To
perform dermabrasion, the hair overlying the area to be wounded was removed,
inducing quiescent hair follicles to enter the active growth stage, anagen.
Thus, proliferation of cells from the hair follicles is a contributing factor
to the responses to dermabrasion we observed. Although only BrdU-labeled
interfolicular keratinocytes were counted for cell proliferation studies,
differences in hair follicle regrowth in the synd1ko mouse might contribute to
the healing phenotype. Given the epithelial cell migration and proliferation
defects revealed in our studies, the known roles played by EGFR and
6ß4 in mediating both migration and proliferation in keratinocytes
(Mainiero et al., 1997
;
Murgia et al., 1998
;
Dans et al., 2001
;
Mariotti at al., 2001
) and the
ability of syndecans to function as co-receptors with integrins for growth
factors and matrix proteins (Couchman and
Woods, 1999
; Saoncella et al.,
1999
; Woods,
2001
), it seems likely that altered signaling via the
6ß4 integrin is responsible for some of the phenotypic properties
displayed by synd1ko keratinocytes after wounding.
9 integrin localization is reduced in the wounded synd1ko corneal
and epidermal keratinocytes compared with wounded wild-type cells. Immunoblots
from wild-type dermabraded skin show a reduction in
9 integrin in
response to hair removal as well as no change or a slight increase in the
amount of
9 integrin after wounding. The immunoblots also showed that
in the synd1ko skin,
9 decreases with hair removal and during healing.
3 integrin is increased in both the wild-type and synd1ko cells after
hair removal, and wounding causes no change in
3 integrin in the
wild-type cells and a slight reduction in the synd1ko cells. The transient
increase in
9 integrin localization immediately prior to wound closure
observed using wounded corneal whole mounts extends results we first reported
several years ago (Stepp and Zhu,
1997
). In closed corneal wounds of wild-type mice 24 hours after
wounding, we reported a transient increase in
9 integrin mRNA and
protein in the corneal basal cells but no increase in
9 integrin 18 or
48 hours after wounding. Using whole mounts, we show, in the current study,
that the increased expression of
9 integrin does not occur after
migration is completed but immediately prior to its completion.
The distinct localization of the 9 integrin within corneal
keratinocytes just prior to wound closure suggests that it functions to seal
off the edges of the advancing wound margins. The ligand(s) recognized by
keratinocyte
9 integrin remain unknown. Known ligands for
9
integrin include tenascin-C (Yokosaki et
al., 1994
), osteopontin
(Yokosaki et al., 1999
),
VCAM-1 (Taooka et al., 1999
),
von Willibrand factor (Takahashi et al.,
2000
), tissue transglutaminase
(Takahashi et al., 2000
),
blood coagulation factor XIII (Takahashi
et al., 2000
), L1-CAM
(Silletti et al., 2000
) and
ADAMs-12 and -15 (Eto et al.,
2000
). The most recently characterized ligand is perhaps the most
intriguing, the EIIIA segment of fibronectin (FN); this alternatively spliced
region of FN was previously thought to be recognized only by
4 integrin
(Liao et al., 2002
).
4
and
9 integrins are close homologs, sharing 39% amino-acid identity,
and among the reported
9 ligands, all except tenascin-C also interact
with
4 integrin. Numerous studies have reported the upregulation of
EIIIA after wounding in skin
(ffrench-Constant et al.,
1989
) and cornea (Cai et al.,
1993
; Vitale et al.,
1994
; Nickeleit, 1996). However, given that
4 integrin is
not expressed by skin and corneal keratinocytes, the significance of FN to
corneal wound healing remains unclear
(Gipson et al., 1993
).
If 9 integrin can be shown to mediate cell migration after wounding
by interacting with the EIIIA domain of FN, then the function of FN
alternative splicing in skin and corneal wound healing might be revealed.
Further, the reduced localization of
9 integrin in the synd1ko corneas
prior to closure and in skin after dermabrasion may be contributing to the
healing delay. It is also tempting to suggest that the interaction between
9 integrin and ADAMs might be altered in the absence of synd1, given
that both
9 integrin (Eto et al.,
2000
) and synd1 (Iba et al.,
2000
) can interact with ADAM-12. Unfortunately,
9 integrin
expression is not maintained in cultured keratinocytes under standard
conditions. This explains not only why keratinocytes have not been shown to
interact with this alternatively spliced FN isoform but also why the ligand(s)
for this integrin within epithelial tissues remain unknown.
It was initially surprising that synd1ko mice were viable and had no
obvious phenotype. Yet our studies demonstrated that they are defective in
cutaneous and corneal wound healing. Our data add to those reporting defective
leukocyte adhesion and increased angiogenesis in the synd1ko mice
(Gotte et al., 2002).
Echtermeyer and colleagues deleted the gene encoding synd4 in mice and also
observed a cutaneous wound-healing defect
(Echtermeyer et al., 2001
).
Furthermore, these synd4ko mice showed signs of delayed closure and impaired
angiogenesis, with fibroblasts derived from the synd4ko mice showing slower
than normal migration rates as well as poorly forming focal adhesions
(Ishiguro et al., 2000
). In
the synd1ko mouse, the keratinocytes fail not only to activate cell
proliferation in response to wounding but also to revert completely back to
normal after migration is complete.
Improved primary epithelial cell culture models for both wild-type and
synd1ko keratinocytes that maintain 9 integrin expression would be a
great aid in determining the role played by
9 integrin in mediating the
healing defects in the synd1ko mouse. In addition, the generation of mice
lacking multiple syndecan family members will allow for the exact
determination of how each of the syndecan HSPGs interacts developmentally and
within tissues to maintain appropriate tissue- and organ-specific functions.
Delayed healing is a hallmark of aging, so a better understanding of the
functions of the molecules that mediate proper wound healing will allow us to
develop better treatments for those who suffer the consequences of impaired
healing.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander, C. M., Reichsman, F., Hinkes, M. T., Lincecum, J., Becker, K. A., Cumberledge, S. and Bernfield, M. (2000). Syndecan 1 is required for Wnt-1-induced mammary tumorigenesis in mice. Nat. Genet. 25,329 -332.[CrossRef][Medline]
Belkin, A. M. and Stepp, M. A. (2000). Integrins as receptors for laminins. Micro. Res. Tech. 51,280 -301.
Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. and Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68,729 -777.[CrossRef][Medline]
Brockstedt, U., Dobra, K., Nurminen, M. and Hjerpe, A. (2002). Immunoreactivity to cell surface syndecans in cytoplasm and nucleus: tubulin-dependent rearrangements. Exp. Cell Res. 274,235 -245.[Medline]
Cai, X., Foster, C. S., Liu, J. J., Kupferman, A. E., Filipec, M., Colvin, R. B. and Lee, S. J. (1993). Alternatively spliced fibronectin molecules in the wounded cornea: analysis by PCR. Invest. Ophthalmol. Vis. Sci. 34,3585 -3592.[Abstract]
Cornelison, D. D., Filla, M. S., Stanley, H. M., Rapraeger, A. C. and Olwin, B. B. (2001). Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev. Biol. 239,79 -94.[CrossRef][Medline]
Couchman, J. R. and Woods, A. (1999).
Syndecan-4 and integrins: combinatorial signaling in cell adhesion.
J. Cell Sci. 112,3415
-3420.
Dans, M., Gagnoux-Palacios, L., Blaikie, P., Klein, S.,
Mariotti, A. and Giancotti, F. G. (2001). Tyrosine
phosphorylation of the ß4 integrin cytoplasmic domain mediates Shc
signaling to extracellular signal-regulated kinase and antagonizes formation
of hemidesmosomes. J. Biol. Chem.
276,1494
-1502.
Derksen, P. W., Keehnen, R. M., Evers, L. M., van Oers, M. H.,
Spaargaren, M. and Pals, S. T. (2002). Cell surface
proteoglycan syndecan-1 mediates hepatocyte growth factor binding and promotes
Met signaling in multiple myeloma. Blood
99,1405
-1410.
Dobra, K., Andang, M., Syrokou, A., Karamanos, N. K. and Hjerpe, A. (2000). Differentiation of mesothelioma cells is influenced by the expression of proteoglycans. Exp. Cell Res. 258,12 -22.[CrossRef][Medline]
Echtermeyer, F., Streit, M., Wilcox-Adelman, S., Saoncella, S., Denhez, F., Detmar, M. and Goetinck. P. (2001). Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4. J. Clin. Invest. 107,R9 -R14.[Medline]
Eto, K., Puzon-McLaughlin, W., Sheppard, D., Sehara-Fujisawa,
A., Zhang, X. P. and Takada, Y. (2000). RGD-independent
binding of integrin 9ß1 to the ADAM-12 and -15 disintegrin domains
mediates cell-cell interaction. J. Biol. Chem.
275,34922
-34930.
ffrench-Constant, C., van de Water, L., Dvorak, H. F. and Hynes, R. O. (1989). Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. J. Cell Biol. 109,903 -914.[Abstract]
Filla, M. S., Dam, P. and Rapraeger, A. C. (1998) The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity. J. Cell Physiol. 174,310 -321.[CrossRef][Medline]
Fitzgerald, M. L., Wang, Z., Park, P. W., Murphy, G. and
Bernfield, M. (2000). Shedding of syndecan-1 and -4
ectodomains is regulated by multiple signaling pathways and mediated by a
TIMP-3-sensitive metalloproteinase. J. Cell Biol.
148,811
-824.
Fujikawa, L. S., Foster, C. S., Gipson, I. K. and Colvin, R. B. (1984). Basement membrane components in healing rabbit corneal epithelial wounds: immunofluorescence and ultrastructural studies. J. Cell Biol. 98,128 -138.[Abstract]
Fuki, I. V., Meyer, M. E. and Williams, K. J. (2000). Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts. Biochemical. J. 351,607 -612.[CrossRef][Medline]
Giannelli, G., Falk-Marzillier, G., Schiraldi, O.,
Stetler-Stevenson, W. G. and Quaranta, V. (1997). Induction
of cell migration by matrix metalloprotease-2 cleavage of laminin-5.
Science 277,225
-228.
Gipson, I. K., Kiorpes, T. C. and Brennan, S. J. (1984). Epithelial sheet movement: effects of tunicamycin on migration and glycoprotein synthesis. Dev. Biol. 101,212 -220.[Medline]
Gipson, I. K., Watanabe, H. and Zieske, J. D. (1993). Corneal wound healing and fibronectin. Int. Ophthalmol. Clinics. 33,149 -163.
Goldfinger, L. E., Stack, M. S. and Jones, J. C.
(1998). Processing of laminin-5 and its functional consequences:
role of plasmin and tissue-type plasminogen activator. J. Cell
Biol. 141,255
-265.
Goldfinger, L. E., Hopkinson, S. B., deHart, G. W., Collawn, S.,
Couchman, J. R. and Jones, J. C. (1999). The 3 laminin
subunit,
6ß4 and
3ß1 integrin coordinately regulate
wound healing in cultured epithelial cells and in the skin. J. Cell
Sci. 112,2615
-2629.
Gotte, M., Joussen, A. M., Klein, C., Andre, P., Wagner, D. D.,
Hinkes, M. T., Kirchhof, B., Adamis, A. P. and Bernfield, M.
(2002). Role of syndecan-1 in leukocyte-endothelial interactions
in the ocular vasculature. Invest. Ophthalmol. Vis.
Sci. 43,1135
-1141.
Grootjans, J. J., Reekmans, G., Ceulemans, H. and David, G.
(2000). Syntenin-syndecan binding requires syndecan-synteny and
the cooperation of both PDZ domains of syntenin. J. Biol.
Chem. 275,19933
-19941.
Grootjans, J. J., Zimmermann, P., Reekmans, G., Smets, A.,
Degeest, G., Durr, J. and David, G. (1997). Syntenin, a PDZ
protein that binds syndecan cytoplasmic domains. Proc. Natl. Acad.
Sci. USA 94,13683
-13688.
Hertle, M. D., Kubler, M. D., Leigh, I. M. and Watt, F. M. (1992). Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. J. Clin. Invest. 89,1892 -1901.[Medline]
Hinkes, M. T., Goldberger, O. A., Neumann, P. E., Kokenyesi, R.
and Bernfield, M. (1993). Organization and promoter activity
of the mouse syndecan-1 gene. J. Biol. Chem.
268,11440
-11448.
Hsueh, Y. P., Wang, T. F., Yang, F. C. and Sheng, M. (2000). Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature 404,298 -302.[CrossRef][Medline]
Hsueh, Y. P. and Sheng, M. (1999). Regulated
expression and subcellular localization of syndecan heparan sulfate
proteoglycans and the syndecan-binding protein CASK/LIN-2 during rat brain
development. J. Neurosci.
19,7415
-7425.
Iba, K., Albrechtsen, R., Gilpin, B., Frohlich, C., Loechel, F.,
Zolkiewska, A., Ishiguro, K., Kojima, T., Liu, W., Langford, J. K. et al.
(2000). The cysteine-rich domain of human ADAM 12 supports cell
adhesion through syndecans and triggers signaling events that lead to beta1
integrin-dependent cell spreading. J. Cell Biol.
149,1143
-1156.
Ishiguro, K., Kadomatsu, K., Kojima, T., Muramatsu, H., Nakamura, E., Ito, M., Nagasaka, T., Kobayashi, H., Kusugami, K., Saito, H. et al. (2000). Syndecan-4 deficiency impairs the fetal vessels in the placental labyrinth. Dev. Dyn. 219,539 -544.[CrossRef][Medline]
Kainulainen, T., Hakkinen, L., Hamidi, S., Larjava, K.,
Kallioinen, M., Peltonen, J., Salo, T., Larjava, H. and Oikarinen, A.
(1998). Laminin-5 expression is independent of the injury and the
microenvironment during reepithelialization of wounds. J.
Histochem. Cytochem. 46,353
-360.
Kainulainen, V., Wang, H., Schick, C. and Bernfield, M.
(1998). Syndecans, heparan sulfate proteoglycans, maintain the
proteolytic balance of acute wound fluids. J. Biol.
Chem. 273,11563
-11569.
Kim, C. W., Goldberger, O. A., Gallo, R. L. and Bernfield, M. (1994). Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns. Mol. Biol. Cell 5, 797-805.[Abstract]
Kusano, Y., Oguri, K., Nagayasu, Y., Munesue, S., Ishihara, M.,
Saiki, I., Yonekura, H., Yamamoto, H. and Okayama, M. (2000).
Participation of syndecan 2 in the induction of stress fiber formation in
cooperation with integrin 5ß1: structural characteristics of
heparan sulfate chains with avidity to COOH-terminal heparin-binding domain of
fibronectin. Exp. Cell Res.
256,434
-444.[CrossRef][Medline]
Laird, P. W., Zijderveld, A., Linders, K., Rudnicki, M. A., Jaenisch, R. and Berns, A. (1991). Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19, 4293.[Medline]
Li, E., Bestor, T. H. and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69,915 -926.[Medline]
Liao, Y. F., Gotwals, P. J., Koteliansky, V. E., Sheppard, D.
and van de Water, L. (2002). The EIIIA segment of fibronectin
is a ligand for integrins 9ß1 and
4ß1 providing a
novel mechanism for regulating cell adhesion by alternative splicing.
J. Biol. Chem. 277,14467
-14474.
Mainiero, F., Murgia, C., Wary, K. K., Curatola, A. M., Pepe,
A., Blumemberg, M., Westwick, J. K., Der, C. J. and Giancotti, F. G.
(1997). The coupling of 6ß4 integrin to Ras-MAP
kinase pathways mediated by Shc controls keratinocyte proliferation.
EMBO J. 16,2365
-2375.
Mariotti, A., Kedeshian, P. A., Dans, M., Curatola, A. M.,
Gagnoux-Palacios, L. and Giancotti, F. G. (2001). EGF-R
signaling through Fyn kinase disrupts the function of integrin 6ß4
at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.
J. Cell Biol. 155,447
-458.
Murgia, C., Blaikie, P., Kim, N., Dans, M., Petrie, H. T. and
Giancotti, F. G. (1998). Cell cycle and adhesion defects in
mice carrying a targeted deletion of the integrin ß4 cytoplasmic domain.
EMBO J. 17,3940
-3951.
Nickeleit, V., Kaufman, A. H., Zagachin, L., Dutt, J. E., Foster, C. S. and Colvin, R. B. (1996). Healing corneas express embryonic fibronectin isoforms in the epithelium, subepithelial stroma, and endothelium. Am. J. Pathol. 149,549 -558.[Abstract]
Park, P. W., Pier, G. B., Preston, M. J., Goldberger, O. A.,
Fitzgerald, M. L. and Bernfield, M. (2000a). Syndecan-1
shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas
aeruginosa. J. Biol. Chem.
275,3057
-3064.
Park, P. W., Reizes, O. and Bernfield, M.
(2000b). Cell surface heparan sulfate proteoglycans: selective
regulators of ligand-receptor encounters. J. Biol.
Chem. 275,29923
-29926.
Park, P. W., Pier, G. B., Hinkes, M. T. and Bernfield, M. (2001). Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411,98 -102.[CrossRef][Medline]
Perrimon, N. and Bernfield, M. (2000). Specificities of hepraran sulphate proteoglycans in developmental processes. Nature 404,725 -728.[CrossRef][Medline]
Perrimon, N. and Bernfield, M. (2001). Cellular functions of proteoglycans an overview. Semin. Cell Dev. Biol. 12,65 -67.[CrossRef][Medline]
Rapraeger, A. C. (2000). Syndecan-regulated
receptor signaling. J. Cell Biol.
149,995
-998.
Rapraeger, A. C. (2001). Molecular interactions of syndecans during development. Semin. Cell Dev. Biol. 12,107 -116.[CrossRef][Medline]
Saoncella, S., Echtermeyer, F., Denhez, F., Nowlen, J. K.,
Mosher, D. F., Robinson, S. D., Hynes, R. O. and Goetinck, P. F.
(1999). Syndecan-4 signals cooperatively with integrins in a
Rho-dependent manner in the assembly of focal adhesions and actin stress
fibers. Proc. Natl. Acad. Sci. USA
96,2805
-2810.
Silletti, S., Mei, F., Sheppard, D. and Montgomery, A. M.
(2000). Plasmin-sensitive dibasic sequences in the third
fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate
homomultimerization and concomitant integrin recruitment. J. Cell
Biol. 149,1485
-1502.
Sta. Iglesia, D. D., Gala, P. H., Qiu, T. and Stepp, M. A.
(2000). Integrin expression during epithelial migration and
restratification in the tenascin-C-deficient mouse cornea. J.
Histochem. Cytochem. 48,363
-376.
Stepp, M. A. and Zhu, L. (1997). Upregulation
of 9 integrin and tenascin during epithelial regeneration after
debridement in the cornea. J. Histochem. Cytochem.
45,189
-201.
Stepp, M. A., Zhu, L. and Cranfill, R. (1996). Changes in ß4 integrin expression and localization in vivo in response to corneal epithelial injury. Invest. Ophthalmol. Vis. Sci. 37,1593 -1601.[Abstract]
Takahashi, H., Isobe, T., Horibe, S., Takagi, J., Yokosaki, Y.,
Sheppard, D. and Saito, Y. (2000). Tissue transglutaminase,
coagulation factor XIII, and the pro-polypeptide of von Willebrand factor are
all ligands for the integrins 9ß1 and
4ß1.
J. Biol. Chem. 275,23589
-23595.
Taooka, Y., Chen, J., Yednock, T. and Sheppard, D.
(1999). The integrin 9ß1 mediates adhesion to
activated endothelial cells and transendothelial neutrophil migration through
interaction with vascular cell adhesion molecule-1. J. Cell
Biol. 145,413
-420.
Thomas, K. R. and Capecchi, M. R. (1987). Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51,503 -512.[Medline]
Vitale, A. T., Pedroza-Seres, M., Arrunategui-Correa, V., Lee, S. J., DiMeo, S., Foster, C. S. and Colvin, R. B. (1994). Differential expression of alternatively spliced fibronectin in normal and wounded rat corneal stroma versus epithelium. Invest. Ophthalmol. Vis. Sci. 35,3664 -3672.[Abstract]
Werner, S. and Smola, H. (2001). Paracrine regulation of keratinocyte proliferation and differentiation. Trends Cell Biol. 11,143 -146.[CrossRef][Medline]
Wojcik, S. M., Bundman, D. S. and Roop, D. R.
(2000). Delayed wound healing in keratin 6a knockout mice.
Mol. Cell. Biol. 20,5248
-5255.
Woods, A. (2001). Syndecans: transmembrane
modulators of adhesion and matrix assembly. J. Clin.
Invest. 107,935
-941.
Xu, K.-P., Li, X.-F. and Yu, F.-S. (2000).
Corneal organ culture model for assessing epithelial responses to surfactants.
Toxicol. Sci. 58,306
-314.
Yokosaki, Y., Palmer, E. L., Prieto, A. L., Crossin, K. L.,
Bourdon, M. A., Pytela, R. and Sheppard, D. (1994). The
integrin 9ß1 mediates cell attachment to a non-RGD site in the
third fibronectin type III repeat of tenascin. J. Biol.
Chem. 269,26691
-26696.
Yokosaki, Y., Matsuura, N., Sasaki, T., Murakami, I., Schneider,
H., Higashiyama, S., Saitoh, Y., Yamakido, M., Taooka, Y. and Sheppard, D.
(1999). The integrin 9ß1 binds to a novel recognition
sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of
osteopontin. J. Biol. Chem.
274,36328
-36334.
Zajchowski, L. D. and Robbins, S. M. (2002).
Lipid rafts and little caves: Compartmentalized signalling in membrane
microdomains. Eur. J. Biochem.
269,737
-752.