* Divisions of Basic Sciences and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109; Department of Pathobiology and § Division of Dermatology, University of Washington, Seattle, Washington 98105; and
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
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Abstract |
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Wounding of skin activates epidermal cell
migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional
basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the
individual motile cells into a synchronized colony. We
found that outgrowths of human keratinocytes in
wounds or epibole cultures display parallel changes in
the expression of laminin 5, integrin 3
1, E-cadherin, and the gap junctional protein connexin 43. Adhesion
of keratinocytes on laminin 5, collagen, and fibronectin
was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold
over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin
3
1 with
laminin 5 was sufficient to promote GJIC. Mapping of
intermediate steps in the pathway linking
3
1-laminin
5 interactions to GJIC indicated that protein trafficking
and Rho signaling were both required. We suggest that
adhesion of epithelial cells to laminin 5 in the basement
membrane via
3
1 promotes GJIC that integrates individual cells into synchronized epiboles.
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Introduction |
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WOUNDING of the epidermis activates cell migration across interstitial connective tissue components exposed in the wound bed, initiates assembly of a provisional basement membrane (BM),1 increases
proliferation, and promotes changes in cell-cell adhesion and cell-cell communication (Clark, 1985; Grinell, 1992
;
Gailit and Clark, 1994
; Zambruno et al., 1995
; Martin,
1997
). It has been suggested that gap junctional intercellular communication (GJIC) may regulate certain aspects of
the wound healing process including synchronization of
the migration of cells (Larson, 1990
; Goliger and Paul,
1995
). Laminin 5 is the primary adhesive ligand present in
normal adult epidermal BM (Carter et al., 1991
; Rousselle et al., 1991
) and is secreted by keratinocytes in the provisional wound bed in tissue (Ryan et al., 1994
; Kainulainen
et al., 1998
) and in culture (Carter et al., 1990a
; Carter et al.,
1991
). Keratinocyte adhesion to laminin 5 is mediated by
two different integrin adhesion receptors (Carter et al.,
1990a
; Carter et al., 1991
; Xia et al., 1996
; Fuchs et al.,
1997
): integrin
6
4 mediates anchorage to laminin 5 in
hemidesmosomes (HDs) of homeostatic tissue (Carter et al., 1990a
; Stepp et al., 1990
; Jones et al., 1991
; Kurpakus et al., 1991
; Sonnenberg et al., 1991
; Fuchs et al., 1997
),
and integrin
3
1 mediates cell motility in wounds and
culture (Ryan et al., 1994
; Kainulainen et al., 1998
). Integrin-laminin 5 interactions mediate transmembrane signaling (Xia et al., 1996
; Giancotti, 1997
; Shaw et al., 1997
)
and can inhibit expression of differentiation markers (Watt
et al., 1993
; Symington and Carter, 1995
).
The BM performs a barrier function that separates
the epithelial cells from interstitial connective tissue in the
dermis. Wounding of the epithelium and BM exposes the
interstitial tissue and activates repair of the BM. Keratinocytes synthesize and deposit laminin 5 into the provisional BM of the wound bed (Ryan et al., 1994; Kainulainen et al., 1998
), redistribute HDs components (Gipson et al., 1993
), reorganize integrins, migrate across the dermis exposed in the wound bed (Grinell, 1992
; Hertle et al.,
1992
; Ryan et al., 1994
; Zambruno et al., 1995
; Kainulainen et al., 1998
), and alter intercellular adhesion and
communication (Goliger and Paul, 1995
; Saitoh et al., 1997
).
Based on in vitro studies, interaction of epithelial cells
with collagen-coated and fibronectin-coated substrates is
mediated by integrins
2
1 and
5
1, respectively (Carter
et al., 1990a
,b). However, migrating keratinocytes rapidly mask the fibronectin and collagen with deposited laminin
5 and shift their adhesion to
3
1 (Carter et al., 1990a
;
Carter et al., 1991
; Fuchs et al., 1997
). Studies with cultured human keratinocytes (Zhang and Kramer, 1996
) as
well as in mice in which expression of laminin 5 has been
disrupted (Ryan, M.C., K. Lee, Y. Myashito, S. Gil, and
W.G. Carter, manuscript in preparation) suggest that deposition of de novo synthesized laminin 5 is required for epithelial cell survival. The interactions of
1 and
4 integrins with ligands result in intracellular signals that are
transduced across the plasma membrane of epithelial cells
(Xia et al., 1996
; Giancotti, 1997
; Shaw et al., 1997
). Integrin-extracellular matrix (ECM) interactions modulate intracellular pH, activate protein kinase C, MAP kinase, and
focal adhesion kinase, and increase cytoplasmic calcium,
protein tyrosine phosphorylation, and gene expression (Clark and Brugge, 1995
). Conceivably, integrin-mediated
cell interactions with laminin 5, collagen, and fibronectin in
the wound bed may signal different epithelial cell functions.
Gap junctions are specialized membrane domains that
are composed of nonspecific channels that connect neighboring cells. Gap junctions provide cell-to-cell pathways
for molecules less than ~1,000 D (Loewenstein, 1981). In
many cell types, the assembly of gap junctions depends on
prior cell-cell adhesions mediated by cadherins (Musil et al.,
1990
; Jongen et al., 1991
; Fujimoto et al., 1997
). Furthermore, proteoglycans and glycosaminoglycans have been
shown to induce gap junction synthesis and function in primary liver cells (Spray et al., 1987
). Several recent studies suggest that GJIC is important in cell growth control and
differentiation (Beyer, 1993
; Goodenough et al., 1996
; Kumar and Gilula, 1996
). Vertebrate gap junctions are composed of proteins from the "connexin" gene family (Beyer,
1993
) and are designated with numerical suffixes referring
to the molecular mass of the deduced sequence in kilodaltons (e.g., Cx43) or an
/
nomenclature (Kumar and Gilula, 1996
). The predominant connexin in human epidermis and in cultures of human keratinocytes is Cx43
(Fitzgerald et al., 1994
). Consistent with the role of GJIC
in cell growth control, connexin proteins are differentially
expressed in skin with lower expression in the proliferative
regions and more expression upon differentiation (Risek
et al., 1992
; Goliger and Paul, 1994
; Salomon et al., 1994
). At the wound edge, connexin expression is decreased but
expression is enhanced at unwounded adjacent areas and
upon wound closure when the cells differentiate (Goliger
and Paul, 1995
; Saitoh et al., 1997
). After healing, gap
junctional protein expression returns to normal levels.
These changes correlate with the perceived roles of gap
junctions and have led to the idea that gap junctions may play a regulatory role in wound repair (Larson, 1990
; Goliger and Paul, 1995
).
We have hypothesized that interactions of keratinocytes
with laminin 5 may promote specific cell functions in
wound repair and quiescent epithelium not promoted by
dermal ECM ligands (Carter et al., 1990a,b; Carter et al.,
1991
; Gil et al., 1994
; Xia et al., 1996
). Here, we examined
the interdependence of integrin-mediated adhesion to laminin 5 on downstream GJIC. We report that adhesion of
keratinocytes and other cells to laminin 5 via integrin
3
1 promotes GJIC when compared with adhesion on collagen
or fibronectin. We suggest that
3
1-laminin 5 interactions promote GJIC and integrate individual epithelial
cells into a synchronous epibole.
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Materials and Methods |
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Cells and Cell Culture
Normal human foreskin keratinocytes (HFKs) were prepared as described by Boyce and Ham (1983). FEPE1L8 cells are HFKs that were
transformed with the genome of human papilloma virus 16 (Kaur et al.,
1989
). Both HFKs and FEPE1L8 cells were maintained in serum-free keratinocyte growth medium (KGM; Clonetics Corp., San Diego, CA) containing insulin, EGF, hydrocortisone, and bovine pituitary extract. Parental and integrin-transfected CHO K1 cells were grown in DME medium
with 10% FBS.
Epidermal epibole cultures, used as wound models, were prepared as follows. Punch biopsies (2 mm) were taken from fresh neonatal foreskins and attached onto coverglass coated with collagen type I (100 µg collagen/ ml H2O dried down to a film) dermal-side down and fed with KGM containing 5% FBS. Both epidermal keratinocytes (~90%) and dermal fibroblasts (~10%) grew out of the explants onto the culture surface for over 2 wk and segregated into separate populations forming confluent epiboles of 2-3 cm in diameter.
Expression of Dominant Negative (DN)-Rho and Integrins in Cultured Cells
FEPE1L8 cells were infected with DN-Rho as follows. Plasmid expression
vectors were prepared by standard methods of subcloning pEXV-RhoN19
(Qiu et al., 1995a,b; a gift from Dr. Frank Gertler, FHCRC, Seattle, WA)
into the EcoRI site of the retroviral vector, pLXSN (a gift from Dr. Dusty
Miller, FHCRC, Seattle, WA). The vector contains a SV-40 promoter and
a neomycin resistance gene. Infection of pLXSN-RhoN19 into cells was
done according to previously described methods (Miller, 1993). In brief,
purified pLXSN-RhoN19 plasmid at a concentration of 2.5 mg/ml was
transfected using the Lipofectamine standard protocol (GIBCO BRL,
Gaithersburg, MD) into NIH3T3 PA317 packaging cell line (a gift from
Dr. Dusty Miller, also available through American Type Culture Collection, Rockville, MD; CRL9078). Packaging cells were passed 2 d after transfection into selection media containing 100 mM geneticin (G418;
GIBCO BRL). Surviving cells were allowed to grow to 80% confluence
and culture media containing the packaged virus was then collected and
kept frozen. Culture media containing virus was layered onto an 80% confluent 10-cm plate of FEPE1L8 cells for 6 h at 37°C. Infection media was
then washed off and fresh KGM was added to the cells. The next day, cells
were passed and allowed to attach and spread. G418 at a concentration of
75 mM was added to FEPE1L8 cells for selection.
CHOs were transfected with cDNAs encoding integrin subunits 2,
3,
and
6/
4 (Symington et al., 1993
; Kamata et al., 1994
) as follows. In brief,
electroporation was used to cotransfect CHO K1 cells with integrin subunit cDNAs in PBJ-1 vector (10 µg) and pCDneo plasmid (1 µg). Cells
were selected in medium containing geniticin (G418; GIBCO BRL) at 700 µg/ml, and then maintained in medium containing 100 µg/ml geneticin.
Antibodies, ECM, and Antibody/ECM-coated Surfaces
The antibodies against integrin subunits 2 (P4B4, P1H5) and
3 (P1F2,
P1B5) have been described previously (Wayner and Carter, 1987
; Carter et
al., 1990a
,b). Anti-
6 integrin subunit G0H3 was from Amac, Inc. (Westbrook, ME). Noninhibitory mAbs to the
3 chain of laminin 5 (C2-5) and fibronectin (P1H11) have been described previously (Xia et al., 1996
). Monoclonal D2-1 binds an epitope in the carboxy-terminal G domain of the
3
chain of laminin 5 and will be described elsewhere (Gil, S., E. Harper, and
W.G. Carter, manuscript in preparation). Monoclonal anti-E cadherin antibody (HECD1) was a generous gift from Dr. Masatoshi Tacheichi (Kyoto
University, Kyoto, Japan). Monoclonal anti-type VII collagen antibody
(L3D) was a generous gift from Dr. Ray Gammon (Virginia Mason, Seattle,
WA). mAb 3068 against Cx43 (Chemicon, Temecula, CA) was used for
studies with human cells. Rabbit polyclonal antibodies against the amino-terminal 20 residues of connexin Cx43 (AT-2, a generous gift of Barbara
Yancey; described in Yancey et al., 1989
) and against the last 16 amino acids
of Cx43 (PNRF; Hossain et al., 1998
) were used for studies with CHO cells.
Poly-L-lysine (Sigma Chemical Co., St. Louis, MO) was bound to tissue
culture plates at 0.5 mg/ml in PBS overnight at 4°C. Human plasma fibronectin and human placental collagen type I and IV were prepared as
described previously (Wayner and Carter, 1987) and were bound to tissue
culture plates at 10 µg/ml in PBS. Collagen I and IV did not differ in their
ability to promote dye transfer and both bind to
2
1, so hereafter they
are simply referred to as collagen. The dishes were subsequently blocked
with 0.25% heat-denatured BSA for 2 h. Laminin 5-coated tissue culture
plates were prepared as described previously (Xia et al., 1996
). Inhibition
studies with anti-laminin 5 antibodies have shown that laminin 5 is the major adhesive ligand present on these plates (Xia et al., 1996
).
ECM-coated beads were prepared by one of two methods. Purified collagen and fibronectin were bound directly to 1-µm latex beads per manufacturer's instructions (Molecular Probes, Eugene, OR). Laminin 5- and
fibronectin-coated beads were also prepared by incubating goat anti-
mouse antibody-coated microspheres (0.9 µm; Bangs Laboratories, Fishers, IN) with a noninhibitory mAb to laminin 5 (C2-5) or fibronectin
(P1H11) for 1 h followed by three washes with PBS and incubation with
three changes of culture supernatant from HFK cultures or three changes
of 10 µg/ml fibronectin, respectively. HFK culture supernatant contains
soluble laminin 5 (Xia et al., 1996).
Antibody-coated surfaces were prepared by incubating affinity-purified
rabbit anti-mouse IgG at 10 µg/ml PBS on tissue culture dishes for 2 h followed by one PBS wash and incubation of monoclonal antiintegrin antibodies for 2 h. The plates were then blocked for 1 h with 0.5% BSA in
PBS. The mAbs used were against integrin subunits 2 (P4B4, P1H5),
3
(P1F2, P1B5), and
6 (G0H3).
Microinjection of Epibole Cell Cultures
Injections were carried out on a Nikon Diaphot TE300 inverted microscope with phase and fluorescence optics using a Narashigi micromanipulator. Micropipettes were drawn to between 50 and 80 mOhm (in 3 M KCl) from World Precision Kwik-Fil borosilicate glass capillaries (1.0 mm OD × 0.75 mm). A filtered 3% solution of Lucifer yellow CH (lithium salt; Sigma Chemical Co.) in 0.15 M LiCl was injected until the injected cell was brightly fluorescent and then the pipette was removed. After 3 min, the epibole cultures were photographed (Tri-X film, Kodak, ASA 400) with phase and fluorescence optics (excitation 425/40, emission 540/40).
Dye Transfer Assay
A 10-cm plate of HFKs or CHO cells was labeled with 0.5 µM calcein-AM
(Molecular Probes), the cell-permeant ester of calcein which is cleaved to
membrane-impermeant calcein by cellular esterases. Three other culture
plates were labeled with 0.25 µM DiI. After washing twice with PBS, the
two populations of cells were each trypsin/EDTA suspended, treated with
trypsin inhibitor, and pelleted. The cells were suspended in the appropriate media, mixed, plated on appropriate ECM-coated dishes, and placed
in a 37°C incubator. Under these conditions, integrins are still capable of
rapid ECM binding. Cells were plated in excess to ensure confluency.
Usually after 2 h of incubation, unattached cells were removed by gentle washing and dye transfer was assayed with a Zeiss microscope equipped with fluorescence optics. Calcein was viewed with a fluorescein filter set,
whereas rhodamine filters were used to view DiI labeling. Phase and fluorescent views for calcein and DiI were recorded using a 35-mm camera
with TMax film (Kodak) at 800 ASA and digital images were generated
using a Nikon Coolscan film scanner. The assignment of a cell as an acceptor of dye via transfer rather than a poorly loaded or leaking donor is
checked by digitally overlaying scanned images of DiI and calcein fluorescence. If a cell adjacent to a calcein-loaded, DiI-negative cell contains both punctate DiI and more diffuse calcein fluorescence, gap junction assembly and dye transfer occurred. If a DiI-labeled cell adjacent to a calcein-loaded cell does not contain calcein, then dye transfer did not occur
at that interface. (For a more complete description of this assay see
Lampe, 1994.) The fraction of cells that transferred dye was determined
by dividing the number of DiI-labeled cells that contained calcein (i.e.,
transfers) by the number of cell interfaces between calcein-loaded and DiI
cells (i.e., total). Error bars represent the standard deviation as determined by ANOVA using the general linear model procedure (Statistical
Analysis System, version 6.08; SAS Instruments, Cary, NC).
Northern Analysis of Cx43 mRNA Levels
Total RNA from HFKs adherent on collagen or laminin 5 for 2 h was isolated using TRIzol reagent (GIBCO BRL) according to manufacturer's instructions. Northern blots were obtained by electrophoresis of total RNA, 20 µg/lane, in formaldehyde-agarose gels (1.0% agarose, 6.6% formaldehyde, 20 mM MOPS, pH 7.0, 2.5 mM sodium acetate, 1 mM EDTA). Gels were blotted to Zeta-Probe nylon membranes (Bio-Rad Laboratories, Richmond, CA) by capillary action using 20× SSC. Blots were hybridized in 0.25 M NaCl, 7% SDS, 1 mM EDTA, 0.25 M sodium phosphate, pH 7.0, 150 mg/ml salmon sperm DNA, 50% formamide at 48°C. Filters were washed at 55°C with 0.2× SSC, 0.1% SDS. Cx43 cDNA clone G2 (the entire rat heart Cx43 coding sequence, kindly provided by E. Beyer, University of Chicago, Chicago, IL) was radiolabeled using the Prime-it RmT random primer labeling kit (Stratagene Inc., La Jolla, CA) and used as a hybridization probe for Northern analysis.
Immunoblotting
Control and treated cells were washed once with PBS and solubilized directly with 2% SDS Laemmli sample buffer (whole cell extract) or 1%
Triton X-100 in PBS, both of which contained 5 mM EDTA, 50 µM VO4,
10 mM NaF, and protease inhibitors (2 mM PMSF, 1 µg/ml pepstatin, 10 µg/ml aprotinin, and 1 µg/ml leupeptin). The Triton X-100 insoluble fraction was solubilized with 2% SDS sample buffer and the DNA was
sheared through a 26-gauge needle. SDS-PAGE was performed on 10%
polyacrylamide gels (Laemmli, 1970). Proteins were transferred to nitrocellulose and Cx43 was detected using either anti-Cx43 polyclonal antibody (AT-2; Yancey et al., 1989
) for CHO cells or mAb 3068 anti-Cx43
for HFKs. Primary antibody was detected using affinity-purified, peroxidase-conjugated goat anti-primary antibody (Chemicon International, Temecula, CA). Peroxidase detection used ECL (Amersham, Arlington
Heights, IL) chemiluminescence and direct exposure to Hyperfilm MP
(Amersham). Densitometry was performed on a Macintosh 6100/66 using
a Hewlett Packard ScanJet 3c to collect the image and the public domain NIH Image program (developed at the U.S. National Institutes of Health
and available on the Internet at http://rsb.info.nih.gov/nih-image/).
Immunofluorescence Microscopy
Immunofluorescence was performed on keratinocytes or CHO cells. Cells
were washed once in PBS, incubated for 10 min in PBS containing 1% Triton X-100, and then fixed for 20 min with 2% formaldehyde in 0.1 M sucrose, 0.1 M cacodylate, pH 7.2 buffer. The cells were permeabilized with
1% Triton X-100 in PBS for 10 min and then blocked for 1 h with 0.5%
BSA, 3% goat serum in PBS. PNRF (Cx43), C2-5 (laminin 5), or P1F2 (integrin 3
1) antibodies were incubated with the cells for 1 h. The cells
were washed and bound antibody was reacted with FITC-conjugated, goat
anti-rabbit or anti-mouse antibody. After washing, immunofluorescence
was visualized using a Zeiss microscope equipped with epifluorescence. Images were photographed with TMax 400 ASA film.
Preparation and Immunostaining of Human Wounds
Wounds of the skin were prepared in normal human donors as described
previously (Olerud et al., 1995), and as follows. After informed consent,
1-mm-deep incisional wounds were prepared in the medial forearm or lateral leg with a Simplate II bleeding time device (General Diagnostics,
Durham, NC). At times ranging from 1 h to 28 d after the incision, the
wounds were removed from the donors with a 4-mm punch biopsy performed using local 1% lidocaine for anesthesia. Biopsies were immediately
placed in OCT and snap frozen in isopentane cooled in liquid nitrogen. 6-µm
cryostat sections were cut, mounted on glass slides, fixed (2% vol/vol formaldehyde in cacodylate buffer for 10 min), permeabilized (0.5% vol/vol Triton X-100 in PBS for 10 min), reacted with the indicated primary antibodies, and detected with peroxidase-conjugated secondary antibodies as
described previously (Carter et al., 1990a
; Carter et al., 1991
).
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Results |
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Parallel Changes in Laminin 5, 3
1, E-cadherin, and
Connexin Cx43 in Wounds
We investigated the possible interdependence of changes
in the expression and localization of the junctional components laminin 5, integrin 3
1, E-cadherin, and Cx43 in response to activation by injury of human epidermis (Fig. 1).
Laminin 5, detected with mAb C2-5 against the
3 chain,
was expressed in the normal BM distant to the wound
(Fig. 1 A, unfilled arrow). Incisional wounding of the skin
disrupts the normal BM in the wound bed and exposes dermal fibronectin (Fig. 1 F) and collagen (not shown)
present throughout the dermis. By 24 h after wounding,
laminin 5 was deposited into the provisional BM of the
wound bed by migrating epithelial cells (Fig. 1 A, unfilled
arrowhead). The wound edge is marked with filled arrows.
Similar results were observed with staining with mAbs
that interact with the
3 and
2 chains of laminin 5 (results
not shown). For comparison, type VII collagen, which has
been reported to interact with laminin 5 (Chen et al., 1997
; Rousselle et al., 1997
), was expressed in the mature BM
distant to (Fig. 1 B, unfilled arrow) and adjacent to the
wound edge (filled arrowhead) but was absent from the
provisional BM (unfilled arrowhead). Thus, laminin 5 does
not depend on interactions with type VII collagen for localization to the provisional BM. Significantly, we detected a precursor form of the
3 chain of laminin 5 with mAb D2-1 (Fig. 1 C and a subsequent section at higher
magnification in I). The precursor
3 chain is expressed in
the cytoplasm of epithelial cells in the wound bed (Fig. 1,
C and I, unfilled arrowheads) but is reduced or undetectable in the normal BM distant from the wound edge (Fig.
1, C and I, unfilled arrows). Most importantly, precursor
3 detected by mAb D2-1 is expressed in the BM immediately adjacent to the wound edge at 24 h (Fig. 1, C and I,
filled arrowheads) and as early as 8 h after wounding (not
shown).
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Connexin Cx43 is expressed at high levels in the more
differentiated spinous and granular cell layers and in lower
amounts in basal cells of the normal epidermis distant
from the wound (Fig. 1 D and at higher magnification in
G, unfilled arrow). Within 24 h of injury, expression of
Cx43 decreased in the wound bed (unfilled arrowheads)
and in all epithelial cell layers adjacent to the wound
(filled arrowheads). As with laminin 5, the altered expression of Cx43 was detected as early as 8 h after wounding (not shown). It has been suggested that cadherin-mediated
intercellular adhesion is required for GJIC (Musil et al.,
1990; Jongen et al., 1991
; Fujimoto et al., 1997
). Consistently, expression of E-cadherin (Fig. 1 H) in intercellular
contacts was highest in differentiated cell layers distal to
the wound but was still detectable in the basal cell layer
(unfilled arrow) and was downregulated in the wound bed
(unfilled arrowhead).
Integrin 3 subunit (Fig. 1 E) and integrin
2 subunit
(not shown) localized to the basal, lateral, and apical plasma
membrane in the normal basal cells distant from the
wound (unfilled arrow). After wounding, activated cells in
the wound bed localized the integrin
subunits to the basal
plasma membrane presumably in contact with ligands in
the wound bed. In summary, these results indicate that
laminin 5, Cx43,
3
1, and E-cadherin in wounds display
parallel changes in expression and/or localization in the
wound bed and in the basal cells immediately adjacent to
the wound within 24 h of wounding.
GJIC Is Differentially Regulated in Leading and Following Cells of Epibole Cultures
Parallel changes in cell junctional components were also observed in in vitro epibole cultures of skin (Fig. 2). Punches of human neonatal foreskin explanted onto collagen yielded outgrowths of primary keratinocytes that migrated as an integrated tongue of cells (Fig. 2 A). An assay of GJIC was performed by microinjection of Lucifer yellow into cells at both the leading edge of the tongue and the following cells (Fig. 2 B). In three separate experiments (with >10 injections/experiment), the cells at the leading edge did not transfer a detectable amount of dye to neighbors whereas the following cells transferred dye efficiently. Immunofluorescence of cells at the leading edge of the tongue localized Cx43 to the perinuclear cytoplasm (Fig. 2 C, arrows). In contrast, Cx43 assembled into apparent punctate gap junctions in cells three to four rows back from the leading edge (Fig. 2 C, arrowheads). The dramatic differences in transfer of dye and immunofluorescence established that cells at the leading edge of the migratory tongue did not assemble functional gap junctions whereas the following cells assembled gap junctions and transferred dye to neighboring cells.
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Localization of laminin 5 and integrin 3
1 in the epibole cultures was also consistent with the staining in
wounded tissue. Laminin 5 expression was increased in keratinocytes at the leading edge of the epibole (Fig. 2 D, arrows). Most of the laminin 5 staining within cells at the
leading edge could be extracted with 0.5% Triton X-100 detergent (results not shown) indicating that it was present in
the cytoplasm. Integrin
3
1 (Fig. 2 E, arrows) was present
in prominent protrusions of the plasma membrane of the
cells at the leading edge in contrast to staining at cell-cell junctions in the following cells (Fig. 2 E, arrowheads).
We conclude that keratinocytes at the leading edge of
skin wounds or epibole outgrowths display parallel changes
in expression or localization of laminin 5, 3
1, and Cx43.
Cells at the leading edge of the tongue versus the following cells are exposed to a different environment of cell-
substrate, cell-cell interactions, and intercellular communication. In one model, expression and deposition of laminin 5 in the provisional wound bed may partially mask dermal
fibronectin and collagen ligands exposed in the wound bed
as described previously in culture (Carter et al., 1990a
; Carter et al., 1991
). These results support the hypothesis
that GJIC and integrin-dependent substrate adhesion may
be interdependent functions.
Laminin 5 Promotes GJIC
The possible interdependence of cell-substrate adhesion
and GJIC was investigated in an in vitro model. Laminin 5, collagen, and fibronectin were chosen to examine their influence on GJIC. HFKs were assayed for GJIC by loading
separate plates of adherent cells with either of two different fluorescent dyes, calcein or DiI, suspending the
labeled cells with trypsin, mixing the cells in a 1:4 ratio
(calcein/DiI), and then adhering the cells onto laminin 5, collagen, or fibronectin ligands. Calcein, which can be
readily loaded into cells via its acetoxymethylester form, is
hydrophilic and small enough to pass between cells via gap junctions, and DiI is a lipophilic molecule that marks cells
that could function as dye acceptors (Lampe, 1994). Labeled HFKs plated at high density on either collagen- or
laminin 5-coated surfaces attached within 1-2 min and
spread within 5-20 min. After 30 min, no difference in the
amount of attachment or spreading could be observed on
either ligand. With fibronectin-coated plates, HFK attachment took 5-10 min. After 2.5 h of incubation, the HFKs had extensive cell-cell contacts on all three ECM ligands
(Fig. 3 A, top). Note that in all cases these cells were plated
in excess numbers so they would be essentially 100% confluent in order to ensure that extensive cell-cell interfaces
formed. By comparison, HFKs plated on tissue culture
plastic take >1 h to bind and much longer to spread (not
shown). After plating on the different ECM ligands for 2.5 h,
phase images (Fig. 3 A, top) and fluorescence images of
calcein (Fig. 3 A, bottom) and DiI (not shown) were collected. A representative example of HFKs plated on laminin 5 is presented in color in Fig. 3 B to illustrate the dye
transfer assay. Cells with bright green calcein fluorescence
in these views represent cells initially loaded with calcein
(donors) and cells with red DiI fluorescence are potential
recipients of dye via transfer at a cell-cell interface seen in
the phase image. The assignment of a cell as a recipient of
dye via transfer rather than a poorly loaded or leaking donor was checked by digitally overlaying scanned images of
red DiI and green calcein fluorescence. If a cell in contact
with a calcein-loaded, DiI-negative cell contained both
punctate red DiI and more diffuse green calcein yellow
punctate fluorescence was produced in a recipient cell. This indicated that gap junction assembly and dye transfer
had occurred (arrows). If a DiI-labeled cell adjacent to a
calcein-loaded cell did not contain calcein, then dye transfer did not occur at that interface (arrowheads). The ratio
of recipients to total cell interfaces was calculated for HFK
attachment to collagen, fibronectin, and laminin 5 in at
least three separate experiments and the results are shown
in Fig. 3 C. Dye transfer was approximately two- and fivefold better on laminin 5 than on collagen and fibronectin, respectively. These results clearly and reproducibly indicated that adhesion to laminin 5 promoted GJIC when
compared with collagen or fibronectin.
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We also determined whether exogenously added ECM components that interact with the apical surface of cells can elicit similar integrin-based responses when compared with substrate-coated ligands. We used latex microspheres coated with purified collagen, fibronectin, or laminin 5. Alternatively, we used fibronectin and laminin 5 bound to microspheres via linker antibodies. When cells were first attached to poly-L-lysine-coated substratum for 0.5 h and then treated for 2 h with ECM-coated beads, the extent of dye transfer was similar to that observed with the ECM-coated culture dishes; cells treated with laminin 5 beads showed three- and fivefold more interfaces showing dye transfer than cells treated with collagen or fibronectin, respectively (Fig. 3 C).
Laminin 5 Promotes Assembly of Cx43 into Triton-insoluble Gap Junctions
We determined if adhesion to collagen and laminin 5 had
different effects on expression of mRNA encoding Cx43.
Total RNA was purified from cells incubated on each substrate for 2.5 h, quantitated, and electrophoresed. After
checking for equal loading with ethidium bromide staining, the gel was blotted and probed with a 32P-labeled
cDNA for Cx43. No significant difference between cells plated on collagen or laminin 5 was observed (Fig. 4 A,
Cx43 mRNA is ~3 kb). Reprobing the same blot for
changes in mRNA encoding the 3 chain of laminin 5 also
showed essentially no difference (not shown).
|
The quantities of total Cx43 protein found in whole cell
extracts did not vary when the HFKs were plated on collagen or laminin 5 for 2.5 h as determined by immunoblot
analysis (Fig. 4 B). Brefeldin A (BFA), an inhibitor of intracellular protein trafficking, also did not affect the whole
cell level of Cx43 on either ligand. Cx43 can be fractionated into Triton X-100-soluble and -insoluble fractions.
The Triton-insoluble fraction has been shown to be enriched for connexin subunits assembled into gap junctional
structures (Musil and Goodenough, 1991). The assembled
gap junctions contain phosphorylated forms of Cx43 which
migrate more slowly in SDS-PAGE as multiple bands
(Musil and Goodenough, 1991
). In HFKs, the nonphosphorylated form of Cx43 is the predominant detectable
species present in the whole cell extracts. However, Triton
extraction enriches for slower migrating, presumably phosphorylated species, present in the Triton-insoluble gap
junctional structures. The fact that the site of gap junction
formation, the cell-cell interface, is Triton resistant indicates that relative comparisons of the Triton insolubility of
Cx43 should yield relative levels of gap junction formation. HFKs plated on laminin 5 had fourfold more Triton-insoluble Cx43 than cells plated on collagen (Fig. 4 B,
compare the fifth and seventh lanes) and this increase in
Triton insolubility appeared to be dependent upon protein
trafficking since BFA eliminated more than half of the increase (Fig. 4 B, eighth lane).
Integrin 3
1 Is Sufficient to Mediate Laminin 5 Induction of GJIC
We investigated which integrins mediate signals from laminin 5 that increase assembly and communication of gap
junctions. To this end, antibodies to different integrins and
control proteins were immobilized on culture dishes. Soluble integrin antibodies (Symington et al., 1993) and beads
coated with antibodies to specific integrins have been
shown to elicit cellular responses (e.g., focal adhesion kinase phosphorylation) normally initiated by ligand-integrin interaction (Miyamoto et al., 1995
). A mixture of calcein- and DiI-loaded HFKs was plated on the dishes and
dye transfer was recorded after 2-3 h of incubation. Cells
readily adhered to the antibody-coated dishes if the antibody was to an extracellular portion of an integrin receptor and a confluent monolayer was produced. The best dye
transfer was observed when cells were plated on immobilized mAbs against the integrin
3 (P1B5, P1F2; Fig. 5 A).
Antibodies specific for
2 integrin (P4B4, P1H5), a collagen adhesion receptor, did not enhance dye transfer. An
antibody to
6 (G0H3), a receptor for laminin 5, supported dye transfer at about the same level as antibodies
to
2. In controls, if nuclear protein-specific antibodies or
antibodies that do not react with the cells were used, very
little cell attachment and hence no dye transfer was observed (not shown).
|
Integrin function in dye transfer was also investigated
using CHO cell lines that express either human 2 (collagen receptor),
3 (laminin 5 receptor), or
6
4 (laminin
5 receptor) (Symington et al., 1993
; Kamata et al., 1994
).
GJIC in each of the transfectants or parental cells was investigated using the calcein/DiI dye transfer method with
cells plated on either laminin 5 or collagen for 1.5 h. When
the
3 transfectant was plated on laminin 5, essentially all
cells showed dye transfer (Fig. 5 B). However, when the
2 and
3 transfectants were plated on collagen or the
2
transfectant was plated on laminin 5, they transferred dye
at a much lower level (Fig. 5 C, range 25-32%). The parental CHO cells have only low levels of endogenous
2
and
3 (Symington et al., 1993
) and are deficient in the
ECM signaling required to stimulate gap junctional communication. CHO cells transfected with
6
4 bound to
laminin 5 and transferred dye at an intermediate level
compared with
2
1 and
3
1, but they did not bind well
to collagen so dye transfer was not measured.
Intermediate Processes in the Pathway Linking
3
1-Laminin 5 Interaction to GJIC
We wished to map cellular processes and signals that
may be required intermediates in the complex process
linking cell-substrate adhesion via laminin 5-3
1 to GJIC.
Therefore, we examined intracellular trafficking of proteins as a requirement for assembly of gap junctions and
the Rho family of GTP binding proteins in the signaling
pathway.
CHO cells were
treated with BFA to test whether the increased gap junctional assembly observed when cells were plated on laminin 5 was dependent on protein trafficking. Equal numbers of 2- and
3-transfected CHO cells were plated on
collagen or laminin 5 for 1 h in the presence or absence of
BFA. The level of total Cx43 protein did not vary when
the
2-transfected CHO cells were plated on collagen or
3-transfected CHO cells were plated on laminin 5 for 1 h
as determined by immunoblot analysis (Fig. 6 A, first and
third lanes). BFA treatment also did not significantly affect the level of total Cx43 protein (Fig. 6 A, second and
fourth lanes), but did apparently affect the phosphorylation/migration of Cx43 as has been reported previously
(Musil and Goodenough, 1991
; Laird et al., 1995
). In contrast, densitometry of the Western blots indicated that
the
3-transfected CHO cells plated on laminin 5 had 10-fold more Triton-insoluble Cx43 than
2-transfected CHO cells plated on collagen (Fig. 6 A, compare the fifth and
seventh lanes). Again, this increase in the Triton insolubility of Cx43 appeared to be at least partially dependent
upon protein trafficking since BFA eliminated 46% of the
increase. BFA also reduced the fraction of cells transferring dye from the control value of 0.94 to 0.35. To confirm
that the increase in Triton insolubility was due to increased gap junction assembly,
3-CHO cells were attached to laminin 5 or collagen, extracted with Triton, and
then processed for immunofluorescence with an anti-Cx43
antibody. Cells on both ligands showed some perinuclear
labeling. However, typical punctate gap junctional staining
was clearly evident when the cells were plated on laminin
5 whereas little was observed on collagen (Fig. 6 B). The
results in Figs. 5 and 6 indicate that interaction of integrin
3 with laminin 5 in transfected CHO cells is sufficient to
promote GJIC and assembly of Cx43 into Triton-insoluble
gap junctions. This implies that increased GJIC on laminin
5 may result from increased assembly of gap junctions.
However, increased GJIC could also result from changes
in the channel properties of the Cx43 complexes that are
already at the cell surface or increased trafficking of a protein other than Cx43 necessary for gap junction formation.
|
FEPE1L8 cells
are a human keratinocyte cell line that has been immortalized by transfection with human papilloma virus 16. FEPE1L8 cells retain their ability to differentiate in response to calcium, they stratify in organotypic cultures,
form focal adhesions, and express the same integrin profile
as HFKs, but produce little laminin 5 (Kaur and Carter,
1992). The fact that these cells produce little laminin 5 makes them dependent upon exogenous ECM ligands for
rapid adhesion. FEPE1L8 cells show a fivefold increase in
GJIC on exogenous laminin 5 versus collagen and a sixfold
increase versus fibronectin (Fig. 7 A). In addition, the increase in GJIC observed on laminin 5 could be largely inhibited if the Rho inhibitor Toxin B was included during
the incubation (Fig. 7 B).
|
Treatment of FEPE1L8 cells with lysophosphatidic acid
(LPA), a known activator of Rho, increased GJIC on both
collagen and laminin 5 (Fig. 7 B). Consistent with the
LPA-induced increase in GJIC observed in FEPE1L8
cells, treatment of HFKs with LPA increased GJIC (transfers/total interfaces) from 0.49 ± 0.08 to 0.79 ± 0.08 on
laminin 5 and from 0.25 ± 0.02 to 0.63 ± 0.09 on collagen.
The LPA-dependent increase observed in FEPE1L8
cells could be reduced by Toxin B treatment (Fig. 7 B).
In a similar manner, if integrin 3-transfected CHO cells
bound to laminin 5 were treated with Toxin B, we observed a 60% reduction in GJIC (results not shown).
To more clearly determine if Rho signaling could be
playing a role in integrin-based regulation of GJIC, we
transfected FEPE1L8 cells with a T17N mutant RhoA
(DN-Rho) that preferentially binds GDP and when overexpressed has been shown to function in a dominant negative manner (Qiu et al., 1995b). Western blotting of
DN-Rho-transfected and vector-transfected cells with
anti-RhoA antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) demonstrated a 105% increase in Rho expression in the DN-Rho cells. The DN-Rho cells also did not
spread well on either collagen or laminin 5, confirming a biological effect of the mutant Rho expression. However,
by plating the cells at confluence we were able to ensure
that cell-cell contacts occurred, an obvious prerequisite to
gap junction assembly. Interaction of these cells with laminin 5 failed to increase GJIC above that observed on collagen (Fig. 7 C, under DN-Rho). Taken together, these results indicate that Rho activity is necessary for the laminin
5-mediated upregulation of GJIC.
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Discussion |
---|
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---|
This study establishes that cell interactions with laminin 5 via integrin 3
1 are sufficient to signal assembly of gap
junctions and promote GJIC. The pathway connecting
cell-substrate adhesion to intercellular communication
requires multiple intermediate or parallel signals from
RhoA and protein trafficking. Thus, adhesion of basal epithelium to laminin 5 in the BM instructs downstream cellular signaling and function distinct from interactions with exogenous connective tissue ligands.
Laminin 5 is identified as a promoter of gap junction assembly and intercellular communication based on structural (Triton insolubility) and functional (dye transfer) assays in three different cultured cell populations. We feel
this is a strong statement of the generality of the intracellular signaling between cell-substrate and cell-cell communication. Integrin 3
1 is sufficient to transmit signals
from laminin 5 to connexins based on studies with integrin-transfected CHO cells and immobilized integrin
antibodies. Immobilized integrin
3 antibodies were 2-3.5-
fold better at supporting GJIC between HFKs than antibodies to
2. CHO cell transfectants expressing integrin
3 showed approximately fourfold better GJIC on laminin 5 than
2 transfectants on collagen and ninefold better
than the parental cell line on collagen or laminin 5. For
the CHOs, we conclude that
3
1 interaction with laminin
5 is sufficient to enhance GJIC. However, these studies
have not eliminated the possible contribution of
6
4-
laminin 5 interactions to the promotion of GJIC in HFKs
or FEPE1L8 cells. The three different cell types (HFKs,
FEPE1L8s, and CHOs) used to investigate laminin 5-
3
1 promotion of GJIC did not vary in their qualitative responses to exogenous ECM ligands but did vary in the
quantitative responses: HFKs had a two- to threefold increase in GJIC on laminin 5 compared with collagen
whereas FEPE1L8s and CHOs were fivefold different. Another potentially important difference between these
cells is their ability to synthesize laminin 5: HFKs readily
express laminin 5, FEPE1L8 cells synthesize only limited
levels of laminin 5 (Kaur et al., 1992), and CHOs do not
synthesize laminin 5 that can be detected with any of our
antibody reagents. HFKs deposit endogenous laminin 5 on
exogenous collagen and fibronectin (Carter et al., 1990a
;
Carter et al., 1991
). The secreted laminin 5 likely generates a mixed signal from both the endogenous and exogenous
ligands. Thus, the production of laminin 5 by HFKs may
lead to a reduced level of enhancement on laminin 5 relative to collagen or fibronectin. Alternatively, collagen and
fibronectin may have a low but detectable ability to promote GJIC when compared with laminin 5.
Multiple intermediate functions are probably required
to link laminin 5-3
1 in cell substrate adhesion to gap
junctions in intercellular communication. Gap and adherens junctions appear to be interdependent possibly because adherens junctions are required to establish cell-cell
interphases. Antibodies to either Cx43 or N-cadherin inhibited both gap junction and adherens junction assembly
(Meyer et al., 1992
). Cell lines that were deficient in E-cadherin showed much less gap junctional communication
and transfection of these lines with E-cadherin increased
cell adhesion and GJIC (Musil et al., 1990
; Jongen et al.,
1991
; Miner et al., 1995
). It is also possible that
3
1-laminin 5 may promote assembly of adherens junctions before
assembly of gap junctions. Antibodies that inhibit cadherins block calcium-induced differentiation and prevent
loss of integrin mRNA, suggesting that the function of cadherins and integrins may be linked (Hodivala and Watt,
1994
). Consistently, antibodies that inhibit
3
1-laminin 5 interactions promote differentiation of keratinocytes (Symington and Carter, 1995
). Recently, it has been reported
that integrin and cadherin synergy regulates motility (Huttenlocher et al., 1998
). It is conceivable that
3
1-laminin
5 interactions could promote formation of adherens
junctions and thereby upregulate GJIC. In contrast,
2
1-
collagen interactions at the leading edge of the wound or in epibole cultures may not promote the formation of adherens junctions or GJIC.
Protein trafficking is also required for GJIC on laminin
5 since BFA inhibited accumulation of Cx43 in Triton insoluble gap junctions on laminin 5. We also examined
other possible pharmacological effectors of the signaling
pathway between laminin 5 and GJIC including wortmannin, genistein, orthovanadate, forskolin, LPA, and Clostridium difficule Toxin B. Only Toxin B inhibited the laminin 5-dependent increase in GJIC whereas LPA
and forskolin increased communication. Toxin B inhibits
members of the Rho family of GTPases: Rho, Rac, and
Cdc42. The Rho subgroup of the Ras superfamily are
small GTPase binding proteins that participate in different cell functions by regulating the actin cytoskeleton (Symons, 1996; Tapon and Hall, 1997
). Expression of a T19N
(dominant negative) form of RhoA in FEPE1L8 cells inhibited GJIC on laminin 5 indicating that RhoA is the Rho
family member that plays the key role in GJIC. Recently,
Rho and Rac were shown to be necessary for establishment of cadherin-dependent cell-cell contacts (Braga et
al., 1997
). Taken together, these results indicate that
RhoA activity is necessary for the laminin 5-mediated upregulation of GJIC. However, at this time it is not possible
to determine if Toxin B exerts its inhibitory function on
Rho and GJIC at the level of cell-substrate adhesion, cell-
cell interfaces, protein trafficking, or other functions in
the communication pathway between laminin 5-
3
1 and
GJIC.
The adhesive ligands, antibodies, and drugs used in
these studies have effects on cell shape that may affect
GJIC. For example, Toxin B disrupted cell-cell adhesion
in HFKs, consistent with the described role of RhoA in
E-cadherin-mediated intercellular adhesion (Hordijk et
al., 1997; Takaishi et al., 1997
). In contrast, Toxin B does
not have a major effect on cell-substrate adhesion of
HFKs because interactions with laminin 5 are mediated in
part by
6
4 and are insensitive to drugs that disrupt actin
filaments (Xia et al., 1996
). We attempted to reduce the effects of shape changes on GJIC by performing dye transfer
assays at confluent cell densities where changes in cell
shape will not limit intercellular contact and indirectly affect GJIC.
Differences in the kinetics of initial cell adhesion, spreading, and cell-cell contact on the different ligands could
contribute to differences in rates of assembly of gap junctions and GJIC. However, our results argue against a simple kinetic explanation. (a) We detect some differences in
the rates of attachment and spreading of HFKs on laminin
5 and collagen in the first 5-10 min of plating, but no differences are apparent after 30 min. However, we detect
significant differences in GJIC (Figs. 3 and 7) and gap
junction assembly (Figs. 4 and 6) 2-3 h after initial adhesion, long after any differences in adhesion or spreading can be detected. (b) In Fig. 3 C, ECM-coated beads were
added to the apical surface of HFKs that had already been
attached to poly-L-lysine and allowed to spread. Even with
this equivalent starting point, GJIC was threefold higher
in response to laminin 5-coated beads than beads coated
with collagen or fibronectin. (c) Although we have not detected differences in the kinetics of HFK attachment to
immobilized antibodies, attachment of HFKs to immobilized anti-3 mAbs, but not immobilized anti-
2 or anti-
6, increased GJIC (Fig. 5 A). (d) The differences in gap
junction assembly and GJIC reported for the epibole cultures (Fig. 2) in the leading cells contacting collagen verses
the following cells on laminin 5 were assayed after 7 d of
culture.
Published studies indicate that the function and expression of gap junctions are downregulated adjacent to the
wound bed within 2-6 h after injury but before keratinocytes begin to migrate into the wound (Goliger and
Paul, 1995). We also observed decreased expression of
Cx43 and upregulation of laminin 5 in basal keratinocytes adjacent to the wound only 8 h after injury. The rapid
changes in these two junctional components suggest that
they may be linked to each other and participate in the
subsequent migration into the wound bed. This possible
linkage is further supported by subsequent changes: 24 h
after wounding, connexin immunostaining was reported to
increase and then gradually return to prewound levels after several days (Gabbiani et al., 1978
; Goliger and Paul,
1995
; Saitoh et al., 1997
). Our results indicate that laminin
5 continues to be expressed at an elevated level in the
wound bed for 3-5 d and declines to baseline by 7 d (Ryan
et al., 1994
; Gil, S., E. Harper, and W.G. Carter, manuscript in preparation). This suggests that both the initial
activation of laminin 5 expression and the subsequent suppression may be linked to the changes in GJIC.
The results presented here establish a communication
path from cell-substrate adhesion via laminin 5-3
1 to
GJIC. We suggest that gap junctions and laminin 5 may
participate in bidirectional communication to regulate pre-
and postmigratory responses in the wound as follows. (a)
Wounding of skin disrupts epidermal GJIC and cell-cell
adhesion in quiescent epidermis and may initiate wound
repair, including synthesis and deposition of laminin 5 before migration begins. (b) Keratinocytes at the leading
edge of the migrating tongue would contact dermal ECM
ligands (collagen, fibronectin) in the wound bed which do
not promote GJIC. (c) One possible scenario would include deposition of the precursor form of laminin 5 (recognized by mAb D2-1) into the provisional BM of the wound
bed by the leading edge cells that could partially mask the
exposed dermal ligands and facilitate
3
1-laminin 5 interactions that promote GJIC by the following cells. (d)
As the wound heals and gap junctional expression returns
to homeostatic levels (Gabbiani et al., 1978
; Goliger and
Paul, 1995
; Saitoh et al., 1997
), expression of laminin 5 would also reduce to prewound levels (Ryan et al., 1994
).
In one model, interaction of laminin 5 with
3
1 is replaced by interaction with
6
4 as the wound heals
(Carter et al., 1990a
; Carter et al., 1991
; Xia et al., 1996
;
Fuchs et al., 1997
) and
3
1 relocates from a polarized localization in the BM zone of the wound bed to the preactivation location at lateral cell-cell junctions. In an alternative model,
3
1 may function in conjunction with
6
4 to
mediate adhesion to laminin 5 in the normal BM. Consistent with the second model,
3
1 does codistribute with
laminin 5 at the ultrastructural level in the BM zone
(Carter et al., 1991
) particularly in the deep rete ridge of
epidermis in palms and feet (Symington et al., 1993
) suggesting that
3
1 may contribute to adhesion to the BM of
both normal epidermis as well as wounds. Also, in support
of the later model, targeted disruption of integrin
3 in
mice generates blisters in the legs and footpads (DiPersio
et al., 1997
). It is of related interest that laminin 5 is initially synthesized and deposited into the provisional BM
as a precursor protein recognized by mAb D2-1 (Fig. 1).
The precursor form of laminin 5 is subsequently proteolytically cleaved and converted to the mature
3 chain of laminin 5 present in the assembling BM (Fig. 1; Gil, S., E. Harper, and W.G. Carter, manuscript in preparation). Recent in vitro studies have suggested that proteolytic cleavage of the carboxy terminus of the
3 chain of laminin 5 with plasmin promotes downstream assembly of HDs
(Goldfinger et al., 1998
). Thus, modification of the precursor laminin 5 may be related to both changes in keratinocyte adhesion in the wound and to changes in GJIC.
In conclusion, we have shown that assembly of gap
junctions and intercellular communication are upregulated when integrin 3
1 interacts with laminin 5 in vitro
and that consistent correlative changes are observed in
wounds in vivo. Neither collagen nor fibronectin duplicates this communication path. The promotion of GJIC is
dependent on intracellular protein trafficking involved in
assembly of gap junctions, and Rho-mediated signaling.
We suggest that the intracellular signaling resulting from
3
1-laminin 5 interaction instructs assembly of gap junctions and increases GJIC in order to coordinate the functions of individual epithelial cells in the wound and in adjacent normal epidermis.
![]() |
Footnotes |
---|
Received for publication 23 July 1998 and in revised form 21 October 1998.
The authors would like to acknowledge financial support from National
Institutes of Health grants GM55632 (P.D. Lampe), GM47157 (Y. Takada), CA49259 (W.G. Carter), and AR-21557 (W.G. Carter).
Address correspondence related to ECM-integrin interaction and signaling to William Carter, Fred Hutchinson Cancer Research Center, A3-015,
1100 Fairview Avenue North, Seattle, WA 98109. Tel.: (206) 667-4478. Fax: (206) 667-3331. E-mail: wcarter{at}fhcrc.org Address correspondence
related to gap junctions to Paul Lampe, Fred Hutchinson Cancer Research Center, DE-320, 1100 Fairview Avenue North, Seattle, WA 98109. Tel.: (206) 667-4123. Fax: (206) 667-2537. E-mail: plampe{at}fhcrc.org
![]() |
Abbreviations used in this paper |
---|
BFA, Brefeldin A; BM, basement membrane; Cx43, connexin 43; DN, dominant negative; ECM, extracellular matrix; GJIC, gap junctional intercellular communication; HD, hemidesmosome; HFK, human foreskin keratinocyte; KGM, keratinocyte growth medium; LPA, lysophosphatidic acid.
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