* Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139; Division of Nephrology and Newborn Medicine, Children's Hospital and
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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Previously we found that 3
1 integrin-deficient neonatal mice develop micro-blisters at the epidermal-dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of
3
1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show
that the absence of
3
1 results in an increase in stress
fiber formation in keratinocytes grown in culture and at
the basal face of the basal keratinocytes of
3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and
-actinin at focal contact sites in the
3-deficient keratinocytes. These changes in focal contact composition
were not due to a change in steady-state levels of these
proteins, but rather to reorganization due to
3
1 deficiency. Apart from the loss of
3
1 there is no change
in expression of the other integrins expressed by the
3-null keratinocytes. However, in functional assays,
3
1 deficiency allows an increase in fibronectin and
collagen type IV receptor activities. Thus, our findings
provide evidence for a role of
3
1 in regulating stress
fiber formation and as a trans-dominant inhibitor of the
functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during
wound healing.
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Introduction |
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THE epidermis is composed of multiple layers of cells
known as keratinocytes. In normal skin, keratinocyte proliferation is restricted to the basal layer
of the epidermis and cells undergo differentiation as they
migrate up through the suprabasal layers. Basal keratinocytes adhere to the underlying basement membrane via
the extracellular matrix receptors known as integrins, a
family of heterodimeric glycoproteins each composed of
an - and a
-subunit. Heterodimer composition confers
extracellular matrix ligand specificity (Hynes, 1992
). Keratinocytes express a subset of integrins. These include:
2
1, a receptor for collagen;
3
1, a receptor for laminin 5;
5
1, a fibronectin receptor;
v
5, known to be a receptor for various substrates including vitronectin and fibronectin;
6
4, another laminin-5 receptor known to be
important in hemidesmosome formation (for review see
Watt and Hertle, 1994
); and
9
1, a tenascin receptor
(Palmer et al., 1993
).
Cultured keratinocytes adhere to substrates via at least
two structures: focal contacts and stable anchoring contacts (SACs),1 the in vitro "equivalent" of hemidesmosomes (Carter et al., 1990b; Stepp et al., 1990
). Focal
contacts are defined as clusters of integrins whose extracellular domains adhere to the extracellular matrix and
whose cytoplasmic tails are linked to a concentration of
actin-associated proteins that in turn link to actin filament bundles (Burridge et al., 1988
). The
1 and
v integrins
link the extracellular matrix outside the cell with the actin
cytoskeleton inside the cell via a network of focal contact-associated proteins including talin, vinculin, and
-actinin
(Otey et al., 1993
; Clark and Brugge, 1995
; Johnson and
Craig, 1995
). On the other hand, hemidesmosomes can be
defined as electron-dense units containing a high concentration of
6
4 integrins whose cytoplasmic tails associate
with cytoplasmic plaque proteins that in turn link to keratin filaments (Carter et al., 1990b
; Borradori and Sonnenberg, 1996
). So, although focal contacts and SACs are distinct structures on the keratinocyte surface both are
essential for effective adhesion to a common ligand, laminin 5.
In addition to being a receptor for laminin 5, 3
1 has
been implicated as a weak receptor for fibronectin (FN),
collagen type IV (Coll IV), and laminin 1 (Gelson et al.,
1988; Elices et al., 1991
) and is recruited to focal contacts
in cells cultured on these proteins (Grenz et al., 1993
;
DiPersio et al., 1995
). Adhesion-blocking assays using antibodies specific for
3
1 have suggested a variety of functions for
3
1 (Wayner and Carter, 1987
; Wayner et al.,
1988
; Carter et al., 1990a
). For example, it has been reported that function-blocking antibodies to
3
1 can inhibit long-term keratinocyte-substrate adhesion and cell- cell contact (Carter et al., 1990a
), and that blocking
3
1
function induces keratinocyte differentiation (Symington
and Carter, 1995
). It has also been shown that antibodies
to
3
1 may increase migration on FN and Coll IV (Kim
et al., 1992
). Studies in vivo have indicated changes in
3
1 integrin expression patterns during wound healing (Hertle et al., 1992
; Cavani et al., 1993
), psoriasis (Hertle et al., 1992
; Pellegrini et al., 1992
), and tumorigenesis
(Bartolazzi et al., 1994
). Taken together, these data suggest multiple roles for
3
1.
In this report we have used 3 integrin-deficient mice to
address directly the roles of
3
1 in keratinocytes.
3-deficient mice suffer kidney and lung abnormalities with
basement defects in the kidney (Kreidberg et al., 1996
).
We have shown that the
3-deficient mice form epidermal-dermal blisters that are associated with basement
membrane defects (DiPersio et al., 1997
). Mice deficient in
6 (Georges-Labouesse et al., 1996
) or
4 integrin subunits (Dowling et al., 1996
; van der Neut et al., 1996
) also
display blistering at the epidermal-dermal junction and
are models for the human blistering disease Junctional
Epidermolysis Bullosa with pyloric atresia (Niessen et al.,
1996
; Ruzzi et al., 1997
). These mice have normal basement membranes, but hemidesmosomes are absent with
alterations in associated keratin cytoskeletal morphology.
Since
3
1 links to the actin cytoskeleton, we hypothesized that there might be alterations in the actin filament
morphology in the
3-deficient mice.
We have analyzed the skin and isolated keratinocytes
from the 3-deficient mice to explore further the roles of
3
1 in keratinocytes. Here we show that the absence of
3
1 results in a change in filamentous actin (F-actin)
bundling both in keratinocytes grown in culture and in
basal keratinocytes of
3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins at
focal contact sites in the
3-deficient keratinocytes. In addition, there is an increase in FN and Coll IV receptor activity in the
3-null keratinocytes. Our results suggest a
role for
3
1 as a regulator of the actin cytoskeleton, focal
contact composition, and as a trans-dominant inhibitor of
fibronectin and collagen type IV integrin receptors in keratinocytes.
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Materials and Methods |
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Antibodies
Rabbit sera against the cytoplasmic domains of human 5,
1, and
chicken
3A integrin subunits were prepared as described (Marcantonio
and Hynes, 1988
; Hynes et al., 1989
; DiPersio et al., 1995
). Rabbit antiserum against
9 was a donation from Dr. D. Sheppard (University of California, San Francisco, CA) and the rabbit polyclonal antibodies to the
cytoplasmic domain of
2 and
6 were gifts from Drs. M. Hemler (Dana-Farber, Boston, MA) and V. Quaranta (Scripps Research Institute, La
Jolla, CA), respectively. Rabbit antiserum against the
v integrin subunit cytoplasmic domain was purchased from Chemicon International (Temecula, CA). Rat mAbs against mouse
5 and
1 extracellular domains were kind donations from Dr. B. Chan (University of Western Ontario, Canada), and rat mAb against mouse
4 was donated by S. Kennel (Oak
Ridge National Laboratory, Oak Ridge, Tennessee). The rat anti-mouse
6 extracellular domain-specific mAb GoH3 was purchased from Immunotech (Westbrook, ME), and the mouse anti-human
3 mAb P1B5 was
purchased from Gibco Laboratories, (Grand Island, NY).
The anti-mouse keratin 14 antibody was a donation from Dr. E. Fuchs
(University of Chicago, Chicago, IL). The anti-actin rabbit antibody
(751.1) was a generous donation from P. Matsudaira (Whitehead Institute,
Massachusetts Institute of Technology, Cambridge, MA). Anti-vinculin
and talin antibodies were obtained from Sigma Chemical Co. (St. Louis,
MO) and the anti--actinin antibody was obtained from ICN Biochemicals (Costa Mesa, CA). All fluorescently conjugated secondary antibodies
were obtained from TAGO BioSource (Camarillo, CA). The FITC-conjugated phalloidin was obtained from Sigma Chemical Co. (St. Louis, MO).
Extracellular Matrix Components
Purified human laminin 5 was a generous donation from Dr. R. Burgeson (Harvard/Massachusetts General Hospital, Charlestown, MA). Rat fibronectin was obtained from Gibco Laboratories and human collagen type IV and mouse laminin 1 were obtained from Collaborative Biomedical Products (Bedford, MA).
Preparation of Tissue for Histology
Animals were killed by CO2 narcosis and limbs removed. Tissue for paraffin embedding was fixed in 4% formaldehyde solution for 24 h at room temperature. Tissue for cryosectioning was snap frozen in OCT. Microscopy was carried out on a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY).
Isolation and Culture of Primary Mouse Keratinocytes
Epidermal keratinocytes were prepared from neonatal mice essentially as
described previously (Dlugosz et al., 1995). In brief, newborn mice were
killed by CO2 narcosis, washed in 0.01 N iodine in PBS for 10 min, rinsed
with PBS, washed in 70% ethanol for 10 min, and then rinsed in PBS.
Tails were used for genotyping by PCR (as described in DiPersio et al.,
1997
), and limbs were prepared for frozen skin sections (see above). Skins
were removed from the torso and head, and then floated on 0.25% trypsin
solution (Gibco Laboratories) overnight at 4°C, with the epidermis facing upward. Skins were then transferred to a dry, sterile surface with the epidermis facing down, and the dermis was separated from the epidermis.
The epidermis was minced, suspended in growth medium (see below), and
then agitated to release keratinocytes. Suspensions were passed through a
sterile, 70-µm nylon filter (Becton Dickinson, Franklin Lakes, NJ) to remove cornified sheets. Keratinocytes were seeded onto tissue culture
plates coated with 30 µg/ml denatured rat tail collagen (Collagen Corp.,
Palo Alto, CA) at a density of ~2-4 × 105 cells/cm2. To prevent differentiation of keratinocytes, cultures were grown in low calcium medium consisting of MEM (BioWhittaker, Walkersville, MD) supplemented with
4% FBS (Intergen Co., Purchase, NY) from which Ca2+ had been removed by chelation (Brennen et al., 1982
), 0.05 mM CaCl2, "HICE" mix
(5 µg/ml insulin [Sigma Chemical Co.], 0.5 µg/ml hydrocortisone [Calbiochem-Novabiochem Corp., La Jolla, CA], 10
10 M cholera toxin [ICN
Biomedicals, Costa Mesa, CA], 10 ng/ml EGF [Upstate Biotechnology
Inc., Lake Placid, NY], 2 × 10
9 M T3 [Sigma Chemical Co.]), 100 units/
ml penicillin, and 100 µg/ml streptomycin (Gibco Laboratories). Mouse
keratinocytes were cultured at 33°C, 7.5% CO2.
Immunofluorescence Microscopy
Unfixed neonatal mouse limbs were embedded in OCT and snap-frozen
in chilled isopentane in liquid nitrogen. The following procedure was used
for the immunostaining of 8-µm cryosections. Sections were fixed either in
4% formaldehyde in PBS for 20 min at room temperature when staining
for F-actin, 3,
6, or
4 integrin subunits or in 100% acetone for 10 min
at
20°C when staining for
1,
5, or
9 integrin subunits. When staining
for
v integrin subunits, sections were treated with 0.1% NP-40 in PBS for
10 min at room temperature, and then fixed in 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. All sections were blocked
with 0.1% BSA, 0.2% Triton X-100 in PBS for 1 h before 1-h incubations
with primary antibodies. Sections were washed in PBS and incubated with
secondary antibodies at a 1:200 dilution in block solution. Finally, sections
were washed in PBS, and then distilled water and mounted in Gelvatol
containing the anti-bleaching agent, DABCO.
When staining for integrins at focal contacts, keratinocyte cultures
were first cross-linked in 0.4 mM BS3 (Pierce Chemical Co., Rockford, IL)
10 min at room temperature, washed in 10 mM Tris-HCl, pH 7.4, in PBS,
extracted in 0.5% NP-40 in PBS for 10 min and fixed in 4% PFA in PBS
for 10 min before blocking and antibody incubations as described above
(Enomoto-Iwamoto et al., 1993; DiPersio et al., 1995
). To stain for filamentous actin, keratinocyte cultures were first permeabilized with 0.5% Triton X-100 in PBS for 5 min followed by 20 min in 4% formaldehyde in
PBS before incubating in a 1:1,000 dilution of rhodamine-conjugated phalloidin. To stain keratinocyte cultures for cytoplasmic focal contact proteins, cells were fixed in 4% PFA for 20 min, permeabilized with 0.5%
NP-40 for 10 min, and then blocked for 1 h before incubation with the appropriate antibodies.
Interference Reflection Microscopy
Keratinocytes were cultured on glass coverslips coated with denatured rat tail collagen (Vitrogen, Palo Alto, CA) and were photographed for interference reflection microscopy on a Zeiss Photo Microscope III (Carl Zeiss, Inc.).
Transfection of Primary Keratinocytes
An 3 expression plasmid was constructed by inserting an XbaI restriction
fragment encompassing the cDNA for the human
3 integrin subunit
(provided by M. Hemler, Dana Farber Cancer Institute, Boston, MA) into
the XbaI site of the plasmid pcDNA3.1/Zeo(+) (Invitrogen Corporation,
Carlsbad, CA). Primary keratinocytes were transfected using LipofectACE reagent according to the manufacturer's directions (Gibco Laboratories). In brief, 2 µg of the
3 expression plasmid was mixed with 12 µl of
LipofectACE reagent (Gibco Laboratories) in 1 ml of Optimem and
added to subconfluent cultures of keratinocytes on 35-mm plates. After a
6-h incubation at 33°C, the cells were fed with normal culture medium and
grown for an additional 48 h, and then prepared for immunofluorescence
as described above.
Adhesion Assays
96-well bacteriological plates (Nunc Inc., Naperville, IL) were coated with 10 µg/ml of fibronectin, collagen type IV, EHS laminin (laminin 1), or laminin 5 in PBS overnight at 4°C. The plates were washed briefly and then blocked in 10 mg/ml BSA in PBS for 2 h at 37°C. Cells were trypsinized, washed in MEM medium containing 10% serum, and then resuspended at 3 × 104 cells/100 µl of serum-free medium containing 25 µM cycloheximide. 3 × 104 cells were plated per well for 1 h at 37°C. Non-adherent cells were washed off in PBS and the remaining adherent cells were fixed and stained in 0.1% methylene blue in H2O and counted.
Phagokinetic Assays
Phagokinetic assays were carried out as described by Albrecht-Buehler
(1977). In brief, 8-chamber Nunc slides were coated in BSA and colloidal
gold (prepared from gold chloride from Sigma Chemical Co.). The colloidal gold was then coated in 50 µg/ml of either fibronectin, collagen type
IV, EHS laminin, or laminin 5 overnight at 4°C. 103 cells were plated in serum-free medium overnight at 33°C (the temperature of mouse keratinocyte culture), fixed in 4% formaldehyde, and then viewed under dark
field on an inverted Zeiss IM35 microscope. Using a Hamamatsu CCD
camera linked to a Hamamatsu Argus 10 image processor (Hamamatsu
Phototonics, Bridgewater, NJ) the area translocated per cell per hour was
calculated.
FACS® Analysis of Surface Integrins
Keratinocytes were trypsinized, washed in medium containing serum, and then resuspended in PBS containing a 1:100 dilution of antibody for 30 min at 4°C. Cells were then washed three times in PBS and incubated with a suitable FITC-conjugated secondary antibody for 30 min at 4°C, washed three times in PBS and resuspended in PBS-containing propidium iodide, and then analyzed on a FACScan® from Becton Dickinson.
Surface Iodination and Immunoprecipitation of Integrins
Monolayers of mouse keratinocytes that had been passaged once were
surface labeled with 1 mCi/10 cm plate of Na-[125I] (New England Nuclear, Boston, MA) using the lactoperoxidase-glucose oxidase method
(Hynes, 1973). Cells were washed four times with 50 mM NaI in PBS/
Ca2+/Mg2+ and lysed for 15 min on ice in 1 ml of a detergent buffer containing 200 mM octyl-
-D-glucopyranoside (Calbiochem-Novabiochem
Corp.), 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM PMSF (Sigma Chemical Co.), 0.02 mg/ml aprotinin (Sigma Chemical Co.),
and 0.0125 mg/ml leupeptin (Calbiochem-Novabiochem Corp.). Lysates
were sedimented for 10 min at 10,000 g. Supernatants were preincubated
with 100 µl of protein A-Sepharose (1:1 slurry; Pharmacia Biotech, Inc.,
Piscataway, NJ) for 1 h and the beads sedimented for 2 min at 10,000 g.
Protein concentrations of supernatants were determined using a Bio-Rad
kit. To compare the relative amounts of each integrin between wild-type
and
3-null cells an equal amount of lysate was immunoprecipitated with
each anti-integrin antibody as described (Marcantonio and Hynes, 1988
).
In brief, BSA was added to lysates (60-100 µg total protein) to a final concentration of ~5 mg/ml, followed by 5-10 µl of antiserum. After incubation at 4°C for 1 h, 50 µl of protein A-Sepharose (1:1 slurry preabsorbed
with 10 mg/ml BSA in lysis buffer) were added, and reactions were incubated overnight at 4°C. Samples were washed extensively with cold lysis
buffer plus protease inhibitors, and then suspended in sample buffer (5%
SDS, 80 mM Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol and bromophenol blue) and boiled for 5 min. Non-reducing SDS-PAGE was performed by the method of Laemmli (1970)
using 5% acrylamide and a 3%
stacking gel.
Analysis of the Levels of F-Actin in Keratinocyte Cultures
Keratinocytes from wild-type or 3-null mice were grown to 75% confluency in 60-mm dishes as described above. Cells were stained for F-actin as
described in "Immunofluorescence Microscopy." The adherent cells were
then washed in PBS and extracted in 600 µl of 100% methanol for 2 h at
room temperature in a humidified box. The extract was then read on a fluorescent plate reader (Titertek Fluoroskan II). Unstained, equivalent
plates were fixed in 4% formaldehyde, stained in 0.1% crystal violet, extracted in 10% acetic acid, and then read at 600 nm on a Titertek Multiscan Plus plate reader. These readings were used to determine protein
concentrations of the samples and indicate relative cell numbers. Results
were calculated as fluorescent units for equal cell numbers from wild-type or
3-null cells.
Western Blotting
Keratinocytes were grown in 10-cm dishes, extracted in 1 ml of radioimmunoprecipitation assay buffer (2% Triton X-100, 2% NaDOC, 0.2%
SDS, 316 mM NaCl, 20 mM Tris-HCl, pH 7.3, 2 mM EDTA with protease
inhibitors (as for surface iodinations) on ice for 10 min, extracts were centrifuged and protein concentration assessed by Bio-Rad "Dc" Protein Assay. 5 µg of each sample mixed with non-reducing sample buffer were
boiled and run on a 7.5 or 5% acrylamide gel (Laemmli, 1970). Samples
were then transferred onto nitrocellulose (Schleicher & Schuell, Keene,
NH), blocked (5% "Marvel," 0.1% Tween-20 in PBS) overnight at 4°C,
incubated with primary antibody in block solution for 2 h at room temperature, washed in 0.1% Tween-20 in PBS, incubated with appropriate
HRP-conjugated secondary antibody, washed again, and then bands detected by chemiluminescence (Renaissance NEN, Life Science Products).
For Western blotting of integrin immunoprecipitations, unlabeled lysates from wild-type or 3-null keratinocyte cultures were prepared in 200 mM octyl-
-D-glucopyranoside buffer, preabsorbed, and then immunoprecipitated as described above. For each immunoprecipitation, 565 µg of
lysate were incubated with 5-10 µl of rat mAb against the
1 (PharMingen, San Diego, CA),
6 (GoH3), or
4 integrin subunit, followed by 100 µl
of goat anti-rat IgG conjugated to Sepharose-4B (ICN Biomedicals, Costa
Mesa, CA); 1:1 slurry preabsorbed with 20 mg/ml BSA in lysis buffer; control reactions contained Sepharose beads only. Immunoprecipitates were
resolved on non-reducing, 7% polyacrylamide gels, and assayed by Western blotting, as described above, with an antiserum against the cytoplasmic domain of the
6 integrin subunit.
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Results |
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3 Integrin Deficiency Is Associated with Changes in
F-Actin Localization in Basal Keratinocytes
Previous work showed that deficiency in 3
1 integrin resulted in areas of disorganized basement membrane and
microblister development at the epidermal-dermal junction (DiPersio et al., 1997
). Here, we examine the nature
of the skin defects in further detail with a focus on the expression and function of integrins, cytoskeleton, and focal
contact proteins in the
3-null keratinocytes.
Newborn mice from 3+/
crosses were examined immediately after birth for skin defects. At the microscopic
level micro-blisters were observed in 80% of
3-null neonate feet and ~15% of these were blood-filled blisters
(Fig. 1, A-F). From previous work it was not clear whether
the blisters in the
3-null mice were caused by handling
the pups during examination. The presence of blood-filled blisters on live newborn mice demonstrates that the blisters observed in the
3-null mice are not due to post-mortem trauma (Fig. 1, A and B). Wild-type and heterozygous
3-knockout mice had indistinguishable and normal skin
morphology (Fig. 1, C and D). However, in the
3-knockout mice, microblisters were evident at the epidermal-dermal interface (Fig. 1, E and F). Note the presence of blood
in the blister shown in Fig. 1 F. We did not observe any abnormalities in epidermal stratification or proliferation in
the
3-deficient mice.
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The cytoplasmic tails of 6
4 integrins are linked via
hemidesmosomal proteins to the keratin cytoskeleton.
Mice deficient in
6 and
4 integrins develop blisters that
are associated with altered keratin expression (Dowling et al.,
1996
; Georges-Labouesse et al., 1996
; van der Neut et al.,
1996
). Since the cytoplasmic tail of
3
1 is associated with
focal contact-associated proteins such as talin, vinculin,
and
-actinin, which link integrins with the actin cytoskeleton, we explored the possibility that
3 deficiency would
be associated with changes in actin cytoskeletal morphology. In Fig. 1, G-K we show that there are no changes in
keratin 14 localization in the basal keratinocytes, but that
there are changes in the actin cytoskeleton.
Immunostaining of cryosections from skin showed that
expression of keratin 14 (Fig. 1, G and H) and keratin 5 (data not shown) were restricted to the basal keratinocytes
and appeared normal in both non-blistering and blistering
regions of the skin of 3-null mice. Since keratin 14 or 5 were not observed in suprabasal layers, or split between
the roof and the floor of the blisters, the results show that
neither hyperproliferation nor basal keratinocyte lysis occurred in the
3-null blisters and that cleft formation was truly at the epidermal-dermal interface.
Using rhodamine-conjugated phalloidin, F-actin had a
pericellular distribution in all layers of the epidermis of
both wild-type (Fig. 1 I) and 3-deficient skin (Fig. 1, J and
K). In addition, F-actin staining was also concentrated at
the basal face of the basal keratinocytes in the
3-null skin
but not in the wild type. Increased concentration of F-actin
in the basal keratinocytes was observed not only over blistering regions of the
3-nulls (Fig. 1 J), but also in intact
areas of skin (Fig. 1 K, arrowheads). These results suggested that
3
1 integrin regulates the expression pattern
of F-actin in the epidermis.
3 Integrin Deficiency Results in Enhanced F-actin
Stress Fibers and Focal Contacts
Since F-actin localization was altered in 3-null skins, we
investigated the effect of
3 integrin deficiency on the
distribution of F-actin and focal contact proteins in keratinocytes grown in culture (Fig. 2). In wild-type cells,
phalloidin-stained actin bundles appeared thin and cobweb-like (Fig. 2 A). In contrast, F-actin in the
3-null keratinocytes was arranged into thick stress fibers especially at
the basal face of the cells (Fig. 2 B). This was reminiscent
of the altered F-actin staining seen in skin sections, where
it also appeared to be concentrated at the basal face of the
basal keratinocytes in the
3-null mice (see Fig. 1, J and K).
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F-actin stress fibers in cultured cells are known to terminate at focal contact sites. Therefore, we asked whether
the increase in stress fiber thickness in the 3-null keratinocytes was associated with any changes in focal contacts.
Interference reflection microscopy revealed that focal
contact sites in the
3-null keratinocytes (Fig. 2 D) were
darker and larger than those observed in the wild-type cells (Fig. 2 C). This result implies that
3
1 deficiency
leads to closer apposition of the ventral cell surface to the
substrate at focal contact sites and also suggests that the
focal contacts in the
3-null keratinocytes may be structurally different from wild-type focal contacts.
Consistent with the increased stress fiber formation and
focal contact apposition to substrate in the 3-null keratinocytes, all the focal contact-associated proteins tested,
including vinculin (Fig. 2, E and F), talin (Fig. 2, G and H),
and
-actinin (Fig. 2, I and J), were more concentrated in
the focal contacts of the
3-null keratinocytes than in the
wild-type cells. In addition, PY20 staining for phosphotyrosine in the focal contacts was stronger in the
3-null keratinocytes than in wild-type cells (Fig. 2, K and L). Thus,
in the
3-null keratinocytes there is a change in focal contact composition. The increase in phosphotyrosine expression in the focal contacts of the
3-null keratinocytes likely reflects a change in focal contact signaling activities.
To verify that the change in actin cytoskeletal organization observed in 3-null keratinocytes was due to a deficiency in
3
1 integrin,
3-null primary keratinocytes
were transiently transfected with a recombinant plasmid
expressing the
3 subunit. Double-label immunofluorescence revealed that ~80% of cells expressing the exogenous
3 (Fig. 3, A and C), no longer displayed the thick stress fibers typical of
3-null keratinocytes (Fig. 3, B and D). These
3-transfected keratinocytes also showed reduced vinculin staining in focal contacts (Fig. 3, E and F).
These results support a role for
3
1 integrin in regulating
the organization of the actin cytoskeleton.
|
The Enhancement in Stress Fibers and Focal Contacts
in the 3-null Keratinocytes Is Due to
Redistribution and Not Elevated Levels of Proteins
We wished to determine whether the altered F-actin and
focal contact protein staining patterns observed in the 3-null keratinocytes were due to changes in protein levels.
To compare the levels of F-actin in the
3-deficient and
wild-type keratinocytes, cultures were stained with fluorescently conjugated phalloidin, washed, and then the
bound phalloidin extracted and quantified. The results showed that for equal protein concentrations of wild-type
and
3-null keratinocytes the levels of F-actin were the
same (Fig. 4 A). Combined with the immunofluorescence
staining of F-actin (Fig. 2), this result indicated that the increased stress fiber formation in
3-null keratinocytes is
not due to an increase in F-actin assembly, but rather to a
redistribution of existing F-actin.
|
Western blot analyses of protein extracts from 3-deficient (
) and wild-type (+) keratinocyte cultures showed
that, although the
3-null cells were indeed
3 integrin deficient, the levels of total actin, vinculin (Vn), talin (Tn),
and
-actinin (
-act) were each the same in the
3-null
and wild-type keratinocytes (Fig. 4 B). Once again, these
results, in combination with the immunofluorescence studies of focal contact-associated proteins (Fig. 2), demonstrate that
3 integrin deficiency results in a redistribution
of existing proteins rather than increased steady state levels of these proteins.
Expression of Other Integrins Is Normal in 3-deficient
Skin and Keratinocytes in Culture
Since integrins are known to be critical in regulating focal
contact functions, one might predict that 3-deficiency
could result in changes in other integrins that may compensate for the absence of
3
1. The effect of
3 integrin
deficiency on the expression patterns of other integrins in
the skin was investigated by immunofluorescence microscopy. Cryosections from wild-type and
3-null mice were stained with antibodies to various integrin subunits including
3,
v,
1, and
4 (Fig. 5, A-H). As expected,
3 was
present on all surfaces of the basal keratinocytes in wild-type epidermis (Fig. 5 A) and was completely absent from
3-null skin (Fig. 5 E). There were no detectable differences in distributions of the other integrins tested in the
skins of the wild-type or
3-null mice.
v and
1 subunits
were confined to the basal keratinocytes in both wild-type
(Fig. 5, B and C, respectively) and
3-null skin (Fig. 5, F
and G).
4 integrins were concentrated at the basal face of
the basal keratinocytes in both wild-type (Fig. 5 D) and
3-null skin (Fig. 5 H). It should be noted that, although
6
4 expression was strongest on the basal face of the
basal keratinocytes, faint
6
4 staining was occasionally
also observed on the lateral faces of basal keratinocytes in
the
3-deficient mice.
|
The distributions of integrin subunits were also tested
on keratinocytes grown in culture (Fig. 5, I-P). 3 integrin
was present in the focal contacts of wild-type (Fig. 5 I), but
not
3-null cells (Fig. 5 M, nuclei showed background
staining). The expression of all the other integrins tested
was normal in the
3-null cells.
v and
1 subunits both localized to focal contacts (Fig. 5, J and N, and K and O, respectively).
6
4 distribution was also normal in the
3-null keratinocytes, localizing to structures known as SACs.
Cells were also stained for 2,
5, and
9.
2 and
5
showed very low or undetectable levels of expression with
no differences between wild-type and
3-null cells in vivo
and in vitro (data not shown).
9 staining was confined to
the basal layer of keratinocytes and was normal in wild-type and
3-null skin (data not shown). However,
9 was
not detectable in wild-type or
3-null cells in culture. The
distribution of the
6-subunits was identical with the
4
staining patterns both in vivo and in vitro.
3 Integrin Deficiency Does Not Result in Changes in
the Surface Expression of Other Integrins
To investigate whether there were any changes in processing and surface expression levels of other integrins, primary and secondary passage mouse keratinocyte cultures
from wild-type and 3-null mice were either stained with
anti-integrin antibodies and used in flow cytometric assays
(Fig. 6 A) or were surface iodinated and immunoprecipitated with anti-integrin antibodies (Fig. 6, B and C).
|
In FACS® analysis (Fig. 6 A) pools of either wild-type or
3-null cultured keratinocytes were labeled with anti-integrin extracellular domain-specific antibodies to
1,
4, or
v, or with IgG2a as a negative control. The levels of
1
expression were considerably reduced in the
3-null cells.
This reflects the fact that
3
1 constitutes a large proportion of the
1 integrins on the keratinocyte cell surface.
4
and
v showed no consistent changes in levels of surface
expression (Fig. 6 A).
Surface iodination and immunoprecipitation with antibodies to integrin subunits 3,
1,
2,
5,
v, or
6 was
carried out on wild-type (+) and
3-null (
) keratinocytes (Fig. 6 B). Immunoprecipitations with anti-
3
and anti-
1 antibodies confirmed the absence of
3
1 in
the
3-null keratinocytes and the expected reduction of
1. There were no detectable differences in surface expression of
2,
5,
v, or
6
4 between the wild-type and
3-null cells.
Thus, the surface iodination and immunoprecipitation
data confirm the absence of detectable differences between wild-type and 3-null cells in the surface expression
of the different integrin subunits. The
6 integrin subunit
can also pair with the
1 subunit in some cell types, and
6
1 can function as a weak receptor for laminin 5. Western blot analysis demonstrated that the loss of
3
1 from
the surfaces of
3-null keratinocytes resulted in an increase in the intracellular pool of
1 (data not shown);
therefore, it seemed possible that some of the excess
1
might dimerize with endogenous
6 (normally dimerized
with
4) and cause an upregulation of
6
1 on the cell surface. Although the immunoprecipitation data in Fig. 6 B
do not support this idea, we addressed this possibility directly by immunoprecipitation of
1 integrins followed by
immunoblotting with antiserum against the
6 subunit.
For both wild-type and
3-null keratinocytes
6 was
present in immunoprecipitates of either
6 or
4 integrins,
but was not detected in immunoprecipitates of
1 integrins or in control immunoprecipitations lacking antibody (Fig. 6 C). Similar immunoblots with antiserum against the
1 subunit confirmed the presence of
1 in immunoprecipitates of
1 integrins, and the absence of
1 from immunoprecipitates of
6 integrins (data not shown). These results confirm the absence of detectable levels of
6
1 in
both wild-type and
3-null keratinocytes. Thus, although
3 deficiency affects F-actin and focal contact protein recruitment to focal contact sites, it has no effect on the expression patterns or levels of the other integrins in keratinocytes.
The Presence of 3
1 Inhibits Fibronectin and
Collagen Type IV Receptor Functions
We have demonstrated that 3 integrin deficiency enhances accumulation of actin-associated proteins to focal
contacts, but does not change other integrin expression
patterns. To examine the effect of
3 integrin deficiency
on keratinocyte behavior, secondary passage wild-type
and
3-null keratinocytes were tested in adhesion and migration assays. The numbers of
3-null or wild-type keratinocytes able to adhere to laminin 1 (LM-1) or laminin 5 (LM-5) were the same. This result implied that, in the absence of
3 integrins,
6
4 (another receptor for LM-5)
was sufficient to allow cell attachment to LM-5. Saturating
concentrations of GoH3 (an anti-
6 integrin function-blocking antibody; Sonnenberg et al., 1987
) had no effect
on the number of wild-type cells adherent to LM-5 since
3
1 is sufficient for adhesion (Delwel et al., 1994
; DiPersio et al., 1997
). In the presence of GoH3,
3-null keratinocytes were completely inhibited from adhering to LM-5 (Fig. 7 A) demonstrating that either
3
1 or
6
4 is sufficient for adhesion to LM-5. Surprisingly, the numbers of
3-null cells able to adhere to FN and Coll IV were significantly higher than the numbers of wild-type cells able to
adhere (P = 0.01 and 0.05, respectively) (Fig. 7 A). That is,
the fibronectin and collagen receptors are more active in
the
3-null cells.
|
To compare the migration of wild-type and 3-null keratinocytes, phagokinetic assays were performed (Fig. 7
B). The phagokinetic assays involved plating cells on an
even field of matrix-coated, colloidal gold particles and allowing the cells to attach and migrate over the substrate.
As the cells migrate they phagocytose the gold particles;
the area of the particle-free traces was measured and
quantitated as described in Materials and Methods. The
3-null keratinocytes migrated significantly further on FN
and Coll IV than did the wild-type cells (P = 0.005 and
0.005, respectively). In contrast to the migration on FN the
average area cleared by the
3-null keratinocytes on LM-1
was not significantly different between the
3-deficient
and wild-type keratinocytes. The
3-null keratinocytes
showed a significantly reduced level of migration on LM-5
when compared with wild-type cells (P = 0.005). This was consistent with our previous observation that
3-null keratinocytes do not spread well on LM-5, but the wild-type
cells do (DiPersio et al., 1997
). Thus, although the
3-null
cells can adhere as well as the wild-type cells to LM-5, they
do not migrate as well.
Taken together, these data suggest that the changes in
focal contact protein composition observed in the 3-null
keratinocytes may be associated with the functional upregulation of fibronectin and collagen type IV receptors in
these cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
3
1 is an abundant integrin on the surfaces of keratinocytes. However, to date, the precise roles of
3
1 in
skin biology have been unclear. The generation of
3 integrin knockout mice has permitted us to isolate
3-deficient keratinocytes, which we have used to investigate the
roles of
3
1. Comparison of the characteristics and functional behaviors of these cells with those of wild-type keratinocytes has shed light on novel functions of
3
1 as a
regulator of cytoskeletal organization and as a trans-dominant inhibitor of other integrins. Our major findings are as
follows: (a)
3
1-deficient mice show an enhanced concentration of F-actin at the basal face of the basal keratinocytes; (b) in
3-null keratinocytes there is an increase in
stress fiber formation and actin-associated protein localization to focal contacts, and this is associated with increased levels of phosphotyrosine in focal contacts, and (c)
in functional assays the absence of
3
1 in the
3-null keratinocytes permits increased function of fibronectin and
collagen receptors when compared with wild-type cells,
implying a novel role of
3
1 as a trans-dominant inhibitor of other integrins.
The blisters observed in the 3-null mice are small
compared with the extensive blistering found in the
6- (Georges-Labouesse et al., 1996
) or
4-knockout mice
(van der Neut et al., 1996
; Dowling et al., 1996
) and are
particularly evident on the foot pads where they are sometimes filled with blood (Fig. 1). Mutations in
6
4 and
laminin 5 have been shown to be causes of severe human blistering disorders such as Junctional Epidermolysis
Bullosa with pyloric atresia (for review see Burgeson and
Christiano, 1997
). However, the etiologies of many other
human blistering diseases are still unknown. It will be of
interest to determine whether mutations in
3
1 may also
contribute to human blistering disorders.
3
1 Deficiency Results in F-Actin Reorganization
That Is Reminiscent of Wound Healing
Although 6
4 and
3
1 integrins are both receptors for
laminin 5, they differ in that the cytoplasmic tails of
6
4
interact with the keratin cytoskeleton, whereas the
3
1
cytoplasmic tails interact with the F-actin cytoskeleton. At
the ultrastructural level, the
6- and
4-knockout skins
have altered keratin filament organization (Dowling et al.,
1996
; Georges-Labouesse et al., 1996
; van der Neut et
al., 1996
). Our immunofluorescence studies show that,
although keratin 14 expression patterns are similar in
3-null and wild-type skin, the pattern of F-actin changes. In the
3-null epidermis F-actin was concentrated at the
basal face of the basal keratinocytes in both blistering and
non-blistering areas (Fig. 1, I-J). These observations were
reflected in cultured keratinocytes where thin fibers of
F-actin became concentrated into thick bundles in the absence of
3
1 integrins (Fig. 2, A and B). Reconstitution
of
3
1-expression in
3-null keratinocytes reduced the
thick actin bundles typically presented in these cells (Fig. 3).
Our findings both in vivo and in vitro show a striking
resemblance to the patterns of reorganized F-actin seen
during epithelial and corneal endothelial wound healing
(Gabbiani et al., 1978; Brock et al., 1996
; Gordon and
Buxar, 1997
) and suggest that the basal keratinocytes in
the
3-null skin may be mimicking some kind of wound
healing process. We postulate that deficiency for
3
1 results in a wound healing-like response, which in turn is manifested by the repeated deposition of basement membrane proteins during epidermal development, thus giving
rise to the "layered" appearance of the basement membrane in the
3-null skin (DiPersio et al., 1997
).
3
1 Is a Trans-dominant Inhibitor of Fibronectin and
Collagen Type IV Receptor Function
In the 3-null keratinocytes the changes seen in F-actin organization are also associated with an increased recruitment of actin-associated proteins such as vinculin, talin
and
-actinin to focal contacts (Fig. 2). Immunochemical
data show that this increased recruitment is not due to an
increase in production of these proteins, but rather to
changes in localization (Fig. 4). A similar change in actin
organization was also observed in corneal epithelial cells
during wound healing with no change in actin synthesis
(Gordon and Buxar, 1997
). The phenomenon of enhanced focal contacts in the
3-null keratinocytes implies that,
when
3
1 is present, it prevents these events from occurring (Fig. 2, C-L). This is a novel function for
3
1, but
the mechanism is unclear.
Since 3
1 is such an abundant integrin on the surface
of keratinocytes and most of it is not concentrated in focal
contact sites (DiPersio et al., 1995
), it is possible that
3
1
competes for certain proteins thereby regulating focal contact composition. Hence, when
3
1 is absent, actin-associated proteins may be "free" to localize more readily to
focal contact sites. The increase in PY20 staining at the focal contacts in the
3-null keratinocytes demonstrates that
there is an increase in tyrosine phosphorylation at these
sites. Elevated tyrosine phosphorylation is associated with
activation of focal adhesion proteins and this in turn with integrin-mediated cell binding to the extracellular matrix
(Hynes, 1992
; Juliano and Haskill, 1993
). Thus, in the absence of
3
1 integrin, such an increase in phosphotyrosine at focal contacts may reflect a change in the signaling of other integrins.
Another way in which 3
1 might regulate stress fiber
and focal contact assembly is via effects on cell polarization and cadherin function. Antibodies to
3
1 inhibit
cell-cell adhesion (Carter et al., 1990a
), so loss of
3
1
could similarly affect cadherin function including recruitment of F-actin and certain focal contact proteins to cell-
cell adherens junctions. This in turn would release these
components for assembly at the basal surface in response
to other integrins. We have observed that E-cadherin is
poorly organized in
3-null epidermis that overlies "diffuse" areas of basement membrane (data not shown). This
preliminary evidence supports the idea that
3 deficiency
may affect keratinocyte polarization in skin and is in accordance with the disorganized cadherins observed in immortalized kidney epithelial cells (Wang, Z., J.M. Symons,
and J.A. Kreidberg, personal communication). Whatever
the precise mechanism, it is clear that the absence of
3
1
allows greater recruitment of cytoskeletal components by other integrins.
Functional assays revealed that 3-null keratinocytes
adhere and migrate better on fibronectin and collagen
type IV than do wild-type controls (Fig. 7). Using P1B5, a
function-blocking anti-
3 antibody, Kim et al. (1992)
showed evidence that blocking
3 integrin function enhanced the migration of keratinocytes on FN. However, they and others (Carter et al., 1990a
) have suggested that
P1B5 blocked keratinocyte adhesion to FN. Our data instead support a role for
3
1 as a trans-dominant inhibitor
of FN and Coll IV receptor function.
Previous work suggests that occupancy of one integrin
can suppress the functions of other integrins in the same
cell. For example, anti-v
3 antibodies suppress
5
1-dependent phagocytosis (Blystone et al., 1994
) and ligation of
4
1 inhibits
5
1-dependent expression of metalloproteinases (Huhtala et al., 1995
). Diàz-Gonzàlez et al.
(1996) showed that ligand-specific ligation of a "suppressor" integrin such as
IIb
3 is associated with a blockade
in functioning of "target" integrins such as
5
1. Diàz-Gonzàlez's work also indicates that
llb
3 is dependent
on ligand interaction to inhibit
5
1 function. Our in vitro
results show that the trans-dominant role of
3
1 can be
independent of ligation with LM5 and relies on the presence of
3
1. It should be noted that unlike
llb
3,
3
1
appears to be constitutively active in cultured cells in that
focal contact localization may be ligand-independent in
some cases (DiPersio et al., 1995
). It is quite possible that
ligation of
3
1 with laminin 5 may enhance
3
1 activity
to send an inhibitory signal to the FN and Coll IV receptors on the surface of the cell and thus inhibit their functions further.
Potential Implications for Trans-dominant Inhibition During Wound Healing
Given the results reported here, one can make some hypotheses about the role of 3
1 in regulating keratinocyte
behavior in resting skin and during wound healing. In resting skin
3
1 is prevalent and is engaged with LM5 in the
basement membrane and the cells are sessile. This situation would be predicted to lead to
3
1-dependent suppression of the functions of other migratory integrins. At
the time of wounding,
5
1 and
v integrins are upregulated and are believed to promote keratinocyte migration on the provisional matrix, which is rich in ligands for these integrins (fibronectin, fibrin, and vitronectin).
2
1-collagen interactions could also play a role (Larjava et al.,
1993
). Our results suggest that migration would be enhanced if trans-dominant inhibition by
3
1 were relieved
(Fig. 8). There are several ways in which this could arise.
Metalloproteinases, which are upregulated on wounding
(for review see Clark, 1996
) could cleave constituents in
the basement membrane such as LM5 or
3
1 itself. This
might cause uncoupling of the
3
1-LM5 adhesion system, allowing efficient functioning of migratory integrins
(including
5
1,
2
1, and
v) and epithelial closure of
the wound. Relief of
3
1 trans-dominant inhibition could
also occur by some form of inactivation of
3
1 from inside the cell (i.e., inside-out signaling). Then, once the
wound is closed, reestablishment of a newly formed basement membrane and of functional
3
1-LM5 interactions,
could be hypothesized to restore trans-dominant inhibition of the other integrins, bringing migration to an end
and returning the keratinocytes to their sessile state.
|
In addition to the above hypothesis, 3
1-LM5 interactions may occur in two different modes. The first would be
as a trans-dominant inhibitor just as in the model above
and we would suggest that this is the role of this interaction in resting skin and after completion of healing. However, during migration,
3
1-LM5 interactions could also
play a role in migration. This change from a resting, adhesive, and trans-dominant inhibitory state of
3
1 to a migratory and non-inhibitory state could occur by alterations
in LM5 and/or in intracellular regulation of
3
1 function.
Such a model would accommodate a role for the high levels of LM5 during re-epithelialization (Larjava et al., 1993
;
Zhang and Kramer, 1996
; Lotz et al., 1997
).
Either model invokes 3
1 as a suppressor of keratinocyte migration in non-wounded skin and could explain
the apparent formation of multiple layers of basement
membrane in the
3-null skin (DiPersio et al., 1997
). The
models also lead to testable predictions about the behavior of
3-null skin before and after wounding; such predictions could be tested by transplanting neonatal skin (Medalie et al., 1996
) from
3-null pups to immuno-deficient
recipient mice. These hypotheses also raise questions
about
3
1 signaling processes which will be readily investigated using keratinocytes established from wild-type and
3-null mice and transfected with
3 cDNAs.
In conclusion, using 3-deficient mice and keratinocytes, we have uncovered a novel role for
3
1 in the
regulation of focal contact composition, F-actin organization and as a trans-dominant inhibitor of FN and Coll IV
receptor functions. This new role for
3
1 may be important in understanding more about the regulation of keratinocyte migration in wound healing physiology.
![]() |
Footnotes |
---|
Received for publication 11 March 1998 and in revised form 20 July 1998.
This work was sponsored in part by The Human Frontiers Science Programme and The Dystrophic Epidermolysis Bullosa Research Association, by a grant from the National Cancer Institute (RO1 CA7007), and by the Howard Hughes Medical Institute (HHMI). R.O. Hynes is an Investigator of the HHMI.The authors are grateful to K. Mercer for her excellent help with histology. Thanks also to all those who kindly provided antibodies and reagents. And finally, thanks to B. Burgeson, E. Clark, and M. Borowsky for their valuable comments while preparing this paper.
![]() |
Abbreviations used in this paper |
---|
F-actin, filamentous actin; FN, fibronectin; Coll IV, collagen type IV; LM1 laminin 1, LM5 laminin 5; SAC, stable achoring contact.
![]() |
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