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Molecular and Cellular Biology, February 2000, p. 755-759, Vol. 20, No. 3
Lung Biology Center,1
Gladstone Institute of Cardiovascular
Disease,3 and Department of
Medicine,2 University of California, San
Francisco, San Francisco, California 94143-0854
Received 19 October 1999/Accepted 27 October 1999
Integrins have been shown to play important roles in embryonic
development, wound healing, metastasis, and other biological processes.
Integrins are heterodimeric
receptors for extracellular matrix and cell surface counterreceptors,
which play important roles in diverse biological processes including
embryonic development, inflammation, and wound healing (16,
24). In addition to recognizing distinct subsets of RGD-containing ligands,
Generation of Northern blotting.
Total RNA was isolated from primary
cultured mouse keratinocytes with TRIzol solution (Gibco/BRL) as
instructed by the manufacturer. Thirty micrograms of total RNA was
separated on an agarose gel containing formaldehyde and transferred to
nylon membranes. The membranes were blotted with a cDNA probe specific
for mouse Cells and cell culture.
Murine keratinocytes were obtained
and grown in keratinocyte growth medium (KGM; Clonectics) as previously
described (14). Briefly, mouse skin was kept in 0.1%
bacterial protease (P8811; Sigma) overnight at 4°C. The following
day, the epithelial layer was scraped off and incubated in 0.05%
trypsin for 40 min at 37°C. Then the cells were disaggregated by
pipetting and washed twice with phosphate-buffered saline (PBS).
Finally, the cells were resuspended in KGM and plated onto dishes
coated with type I collagen (10 µg/ml; Sigma, St. Louis, Mo.).
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Normal Development, Wound Healing, and Adenovirus
Susceptibility in
5-Deficient Mice
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
v
5 is a receptor for RGD-containing extracellular matrix proteins
that has been suggested to be important in cutaneous wound healing and
adenovirus infection. To examine the in vivo function of this receptor,
we have generated mice lacking 5 expression, using homologous
recombination in embryonic stem cells. Mice homozygous for a null
mutation of the 5 subunit gene develop, grow, and reproduce
normally. Keratinocytes harvested from
5
/ mice demonstrate impaired migration
on and adhesion to the v5 ligand, vitronectin. However, the rate
of healing of cutaneous wounds is not different in
5/ and 5+/+
mice. Furthermore, keratinocytes and airway epithelial cells obtained
from null mice show adenovirus infection efficiency equal to that from
wild-type mice. These data suggest that v5 is not essential for
normal development, reproduction, adenovirus infection, or the healing
of cutaneous wounds.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
v integrins (v1, v3, v5, v6, and
v8) mediate cell adhesion to various matrix proteins including
fibronectin, vitronectin, tenascin, osteopontin, and fibrinogen at
sites containing the tripeptide sequence arginine-glycine-aspartic acid
(RGD). Although the first-described member of this subfamily, v3,
appears to bind to virtually all of these proteins, the other v
integrins have been reported to be more restrictive in their
interactions with ligands. For example, v5 (25) and v8 (21) have been reported to be principally
vitronectin receptors, and v6 has been reported to principally
bind fibronectin and, to a lesser extent, vitronectin and tenascin
(4, 15, 22, 26, 29).
v integrins have been reported to exert distinct effects on cell
behavior. v5 is widely expressed on epithelial cells including
keratinocytes, airway epithelial cells, fibroblasts, osteoclasts, and
monocytes. v5 has been suggested to play important roles in
activation-dependent cell migration (18, 19), in promoting
adenovirus-mediated gene delivery (11, 27), and in cutaneous
wound healing. To investigate the role of v5 in vivo, we have
generated mice lacking 5 expression, using homologous recombination
in embryonic stem (ES) cells. Our results demonstrate an important role
for 5 in keratinocyte adhesion and migration on vitronectin but
suggest that functions of v5 in cutaneous wound healing or
adenovirus infection can be either developmentally or functionally
replaced by other receptors.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
v5-deficient mice.
A genomic clone
containing one exon of the mouse 5 gene (corresponding to
amino acids 114 to 230 in the reported human sequence [23]) was isolated from a genomic library derived from
mouse strain 129/sv. We used an 8.2-kb XbaI/SalI
fragment of this clone to construct a replacement vector that contained
a neomycin resistance gene replacing part of the exon and an adjacent
intron in our clone, and a thymidine kinase gene at one end for
negative selection. This vector was introduced into mouse RF8 ES cells
(14), and targeted clones were identified by Southern
blotting with both external and internal probes. Chimeric mice were
established and mated with C57BL/6J females to obtain mice carrying the
mutated 5 allele.
5 (corresponding to nucleotides 744 to 1095 in
the reported human sequence [23]).
Immunoprecipitation.
Murine keratinocytes were labeled with
0.5 mCi of [35S]methionine overnight in methionine-free
Dulbecco modified Eagle medium (DMEM) supplemented with 1% fetal
bovine serum, 2% KGM, and penicillin-streptomycin and were lysed in
immunoprecipitation buffer (20 mM Tris-HCl [pH 7.5], 1% Triton
X-100, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2).
The lysates were immunoprecipitated with antiserum raised against the
human 5 cytoplasmic domain (kindly provided by Martin Hemler, Dana-Farber Cancer Institute, Boston, Mass.) or with antiserum 4377 against the human 5 cytoplasmic domain (kindly provided by Louis
Reichardt, University of California, San Francisco). Samples were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) on 7.5% acrylamide gels. Gels were impregnated with
2,5-diphenyloxazole (PPO; Fisher Scientific) and exposed to film at
80°C.
Migration assay. Cell migration assays were performed with matrix-coated transwell plates (8-µm pores; Costar, Cambridge, Mass.). The undersurface of the membrane was coated with collagen (10 µg/ml) or vitronectin (10 µg/ml) in PBS for 1 h at 37°C and blocked with 1% bovine serum albumin (BSA). Primary cultured keratinocytes were harvested with trypsin-EDTA, and trypsin was inactivated with soybean trypsin inhibitor. Cells were suspended in serum-free KGM and plated in the upper chamber at a density of 3.6 × 104 per well in 100 µl of medium in the presence or absence of phorbol myristate acetate (PMA; 10 ng/ml). After a 6-h incubation, cells were fixed with 2% paraformaldehyde and stained with 0.5% crystal violet in 1% formaldehyde. Cells in the upper chamber were removed, and cells on the lower surface were counted with a 10× grid at high-power magnification (×40). Multiple fields were counted and averaged for each condition studied.
Cell adhesion assay. Ninety-six-well non-tissue culture-treated polystyrene multiwell microtiter plates (Linbro/Titertek; Flow Laboratories, McLean, Va.) were coated with vitronectin or collagen. A 100-µl solution containing various concentrations of each protein was added to the wells and incubated at 37°C for 1 h. After incubation, wells were washed with PBS and then blocked with 1% BSA in serum-free DMEM at 37°C for 30 min. Control wells were filled with 1% BSA in DMEM. Cells were harvested in the same way as for the migration assay, resuspended in serum-free KGM, and then added to each protein-coated well. The plates were centrifuged (top side up) at 10 × g for 5 min before incubation for 1 h at 34°C in humidified 7% CO2. Nonadherent cells were removed by centrifugation top side down at 48 × g for 5 min. The attached cells were fixed with 1% formaldehyde and stained with 0.5% crystal violet, and then the wells were washed with PBS. The relative number of cells in each well was evaluated by measuring the absorbance at 595 nm in a Microplate Reader (Bio-Rad).
In vivo wounding. Animals were anesthetized with Metofane solution (Pitman-Moore Inc., Mundelein, Ill.). A small area of the animal's back was shaved, and 2- and 4-mm full-thickness punch biopsy specimens were taken. At day 2, 4, 6, 12, and 24 after wounding, the maximum diameter of the wounds was measured to evaluate the rate of wound closure.
Adenovirus-mediated gene delivery.
Adenovirus
(H5.010CMVlacZ; a gift from Alan Davies, University of Pennsylvania
Medical Center) expressing the lacZ gene under the control
of the cytomegalovirus promoter was used at log dilutions from 2 × 1011 particles per ml to infect keratinocytes and airway
epithelial cells from 5/ and wild-type
controls. We used this adenovirus construct because it has been
reported to utilize integrin v5 to infect human airway cells
(11). Cells were incubated with infection medium (DMEM
supplemented with 2% fetal bovine serum) for 90 min. The cells were
then washed and returned to their normal growth medium. Forty hours
after infection, the cells were fixed with 2% formaldehyde and 0.2%
glutaraldehyde in PBS and stained for 2 h at 37°C with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal;
1 mg/ml; Promega, Madison, Wis.) dissolved in potassium ferrocyanide (5 mM) and magnesium chloride (2 mM) in PBS. The blue-stained cells expressing viral lacZ were counted under an inverted
microscope, and the total number was expressed as a percentage of the
number of positive-staining wild-type cells per well incubated with the highest concentration of virus particles in the infection medium.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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5/ mice develop and grow
normally.
The murine v5 gene was inactivated by homologous
recombination in ES cells (5), using the strategy depicted
in Fig. 1A. Mice bearing the desired
mutant genotype were identified by Southern blot analysis (Fig. 1B). To
test whether the mutation leads to a loss of 5 mRNA, we
performed Northern blotting with a murine 5 cDNA probe.
5 mRNA was detected from RNA of
5+/+ keratinocytes, but no band was
detectable from RNA of 5/ cells (Fig.
2A). To confirm that
5/ mice were not able to make 5
protein, we performed 5 immunoprecipitation from metabolically
labeled lysates of cultured keratinocytes with antiserum raised against
the cytoplasmic domain of human 5. The anti-5 antiserum
immunoprecipitated two bands of the appropriate molecular masses to be
v and 5 from 5+/+ keratinocytes;
however, no bands were immunoprecipitated from 5/ keratinocytes (Fig. 2B). Similar
results were obtained with another antiserum, 4377, against the human
5 cytoplasmic domain (data not shown).
|
|
5/ mice were born at the expected
Mendelian frequency from heterozygous intercrosses (30% +/+, 45%
+/, and 25% / of 98 offspring analyzed), demonstrating that the
5/ subunit is not required for mouse
embryonic development. There were no gross abnormalities of the lungs,
heart, skin, liver, kidney, spleen, or intestine of any of the
5/ mice analyzed at up to 3 months of age.
Impaired adhesion and migration of
5/ keratinocytes on vitronectin.
To
determine whether the adhesive properties of cells lacking 5 were
altered, cell adhesion assays were performed with keratinocytes harvested from 5/ and wild-type mice.
Cells were plated in wells coated with vitronectin or collagen. In
comparison to wild-type cells, 5/ cells
showed decreased adhesion to vitronectin but no obvious alteration of
adhesion to collagen (Fig. 3A), an
extracellular matrix protein that is not a ligand for v5.
5/ keratinocytes did not completely lose
the ability to adhere to vitronectin, presumably because this function
is also mediated by the related integrin, v6, as we have
previously described (15).
|
v5 antibodies have previously implicated
v5 in cell migration on vitronectin of human keratinocytes and
epithelial tumor cells. To determine whether genetic deletion of
v5 impaired cell migration, we used modified Boyden chambers as
described previously (15). As for cell adhesion, the loss of
v5 had no effect on baseline or phorbol ester-stimulated migration of collagen but significantly inhibited stimulated migration on vitronectin in the presence of PMA (Fig. 3B).
Cutaneous wound healing was not altered in
5/ mice.
Expression of v5 has
been previously reported to be upregulated on keratinocytes at the
migrating edge of healing wounds. To investigate whether cutaneous
wound healing is impaired in the absence of this integrin, 2- and 4-mm
full-thickness cutaneous wounds were made in wild-type and
5/ mice, and the rate of healing was
determined by measuring the wound size at days 2, 4, 6, 12, and 24 after wounding. A similar healing rate was observed in null and
wild-type mice (Table 1). In both groups,
the wounds were completely healed by day 6 for 2-mm wounds and by day
12 for 4-mm wounds.
|
Adenovirus infection of murine keratinocytes and airway epithelial
cells is independent of v5 integrin expression.
To test the
role of the integrin v5 in adenovirus infection, primary cultures
of murine keratinocytes and airway epithelial cells were incubated with
adenovirus expressing the -galactosidase gene. Approximately 75% of
murine keratinocytes and 30% of murine airway epithelial cells were
stained by X-Gal following incubation with the highest concentration of
viral particles in the infection medium. Cells from wild-type and
5/ mice were equally efficiently infected
by all concentrations of viral particles (Fig.
4). These data demonstrate that v5 is not essential for infection of primary cultures of murine
keratinocytes or airway epithelial cells with adenovirus.
|
| |
DISCUSSION |
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|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
We have generated mice with a null mutation of the integrin 5
subunit by homologous recombination in ES cells. Mice homozygous for a
null mutation of the 5 subunit gene develop, grow, and reproduce
normally. The mutant allele generated a true null as assayed by
Northern blotting and immunoprecipitation. Keratinocytes from mutant
animals have impaired attachment and migration on the principal
v5 ligand, vitronectin. However, the rate of healing of cutaneous
wounds does not differ between wild-type and
5/ mice. Also, keratinocytes and airway
epithelial cells obtained from 5/ mice
are as susceptible to adenovirus infection as those from wild-type
mice. Thus, we conclude that v5 is not essential for normal
development, reproduction, the healing of cutaneous wounds, or
productive infection with adenovirus.
Recently, mice lacking the v subunit have been described
(2). These mice all die during embryonic development or
immediately after birth, principally due to defects in the development
of the blood vessels of the brain and gastrointestinal tract.
5/ mice were born viable without obvious
anatomic or histologic abnormalities. These results suggest that loss
of v5 is not, by itself, responsible for the developmental defect
in v/ mice. Reports of the phenotype of
mice expressing null mutations of the 6 (14) and 3
(13) subunits suggest that the developmental defect in v
null mice is not solely explained by the loss of v3 or v6
either. Thus, this phenotype must be due either to the loss of other
specific v integrins (e.g., v1 or v8) or to the combined
effects of loss of multiple members of the v integrin subfamily.
Since the principal ligand for v5 appears to be vitronectin, our
results are consistent with the report that vitronectin knockout mice
also develop normally (30).
One of the principal reasons for examining the in vitro migration of
keratinocytes is to generate hypotheses about the mechanisms of
processes that require keratinocyte migration in vivo. One of the most
important of these is cutaneous wound healing. Several previous reports
have suggested that v5 would be likely to play important roles in
the wound healing process (6, 10, 18), based largely on the
findings that this integrin is rapidly induced on the keratinocytes
along the wound edge, the cells that must migrate across the wound bed
in order to facilitate wound closure. However, the results of the
present study suggest that despite the important role that this
integrin plays in in vitro migration of keratinocytes, it is not
required for normal wound healing to occur in vivo. The
5/ mice were fully capable of healing
cutaneous wounds in the punch biopsy model. This result is not
particularly surprising given the large number of matrix proteins
present in wounds (8, 17), the large integrin repertoire
expressed on keratinocytes (1, 20), and the biological
importance of wound healing. We have reported similar findings for mice
lacking the integrin v6 (14), which is also highly
induced at the edge of cutaneous wounds (3, 7, 12), and we
have shown v6 plays an important role in keratinocyte migration
on defined ligands in vitro (15). Indeed, mice expressing
null mutations in both the 6 and 5 subunits also heal cutaneous
wounds normally (unpublished observations).
There is no impairment of adenovirus-mediated gene transfer and
expression in cells from 5/ mice,
suggesting that this integrin is not critical for adenovirus infection,
at least of keratinocytes and airway epithelial cells. Previous studies
that have suggested a predominant role for v5 in this process
have used transfected cells infected in suspension (27, 28).
The use of RGD peptide to antagonize adenovirus infection has been
cited as evidence for a role for this integrin in this process.
However, high concentrations of RGD (up to 4 mg/ml) have been required
to produce significant decrements of infection (11). Our
study, together with recent data implicating activated 51 in
adenovirus infection (9), suggests that adenoviruses can
readily infect epithelial cells by mechanisms independent of v5.
The results of this study demonstrate that despite the contribution of
v5 to cellular response to vitronectin and other ligands, any
role the integrin plays in murine development, wound healing, or
adenovirus infection can be compensated for by other v5-independent pathways. Identification of critical in vivo roles
for the integrin may depend on use of these mice in additional disease
models and/or the generation of mice lacking multiple -subunit
partners of v.
| |
ACKNOWLEDGMENTS |
|---|
We thank Eric Sande for technical assistance in generating the
5/ mice and Martin Hemler and Louis
Reichardt for generously providing anti-5 antiserum.
This work was supported by NIH grants HL/AI33259, HL47412, HL53949, and HL56385 (to Dean Sheppard) and the J. David Gladstone Institutes (to Robert V. Farese, Jr.).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Lung Biology Center, UCSF Box 0854, San Francisco, CA 94143-0854. Phone: (415) 206-5901. Fax: (415) 206-4123. E-mail: deans{at}itsa.ucsf.edu.
Present address: 76 Bolingbroke Grove, London SW11 6HB, United Kingdom.
| |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
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Molecular and Cellular Biology, February 2000, p. 755-759, Vol. 20, No. 3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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36: 377-386
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Carey, B., Staudt, M. K., Bonaminio, D., van der Loo, J. C. M., Trapnell, B. C.
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Wang, W., Kirsch, T.
(2006). Annexin V/beta5 Integrin Interactions Regulate Apoptosis of Growth Plate Chondrocytes. J. Biol. Chem.
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Lucas, M., Stuart, L. M., Zhang, A., Hodivala-Dilke, K., Febbraio, M., Silverstein, R., Savill, J., Lacy-Hulbert, A.
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del Zoppo, G. J., Milner, R.
(2006). Integrin-Matrix Interactions in the Cerebral Microvasculature. Arterioscler. Thromb. Vasc. Bio.
26: 1966-1975
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Nandrot, E. F., Anand, M., Sircar, M., Finnemann, S. C.
(2006). Novel role for {alpha}vbeta5-integrin in retinal adhesion and its diurnal peak. Am. J. Physiol. Cell Physiol.
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Asano, Y., Ihn, H., Yamane, K., Jinnin, M., Tamaki, K.
(2006). Increased Expression of Integrin {alpha}v{beta}5 Induces the Myofibroblastic Differentiation of Dermal Fibroblasts. Am. J. Pathol.
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Hardman, M. J., Waite, A., Zeef, L., Burow, M., Nakayama, T., Ashcroft, G. S.
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Taverna, D., Crowley, D., Connolly, M., Bronson, R. T., Hynes, R. O.
(2005). A Direct Test of Potential Roles for {beta}3 and {beta}5 Integrins in Growth and Metastasis of Murine Mammary Carcinomas. Cancer Res.
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Zhang, Y., Bergelson, J. M.
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(2004). Loss of Synchronized Retinal Phagocytosis and Age-related Blindness in Mice Lacking {alpha}v{beta}5 Integrin. JEM
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Robinson, S. D., Reynolds, L. E., Wyder, L., Hicklin, D. J., Hodivala-Dilke, K. M.
(2004). {beta}3-Integrin Regulates Vascular Endothelial Growth Factor-A-Dependent Permeability. Arterioscler. Thromb. Vasc. Bio.
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Kaji, K., Kudo, A.
(2004). The mechanism of sperm-oocyte fusion in mammals. Reproduction
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Sengoku, K., Takuma, N., Miyamoto, T., Horikawa, M., Ishikawa, M.
(2004). Integrins are not involved in the process of human sperm-oolemmal fusion. Hum Reprod
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Taverna, D., Moher, H., Crowley, D., Borsig, L., Varki, A., Hynes, R. O.
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Hakkinen, L., Koivisto, L., Gardner, H., Saarialho-Kere, U., Carroll, J. M., Lakso, M., Rauvala, H., Laato, M., Heino, J., Larjava, H.
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Naik, M. U., Mousa, S. A., Parkos, C. A., Naik, U. P.
(2003). Signaling through JAM-1 and {alpha}v{beta}3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and {alpha}v{beta}3 complex. Blood
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SHEPPARD, D.
(2003). Functions of Pulmonary Epithelial Integrins: From Development to Disease. Physiol. Rev.
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Zhu, J., Motejlek, K., Wang, D., Zang, K., Schmidt, A., Reichardt, L. F.
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Schmid, R. S., Anton, E.S.
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McCarty, J. H., Monahan-Earley, R. A., Brown, L. F., Keller, M., Gerhardt, H., Rubin, K., Shani, M., Dvorak, H. F., Wolburg, H., Bader, B. L., Dvorak, A. M., Hynes, R. O.
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Burns, K. H., Owens, G. E., Fernandez, J. M., Nilson, J. H., Matzuk, M. M.
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Smyth, S. S., Patterson, C.
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Eliceiri, B. P., Puente, X. S., Hood, J. D., Stupack, D. G., Schlaepfer, D. D., Huang, X. Z., Sheppard, D., Cheresh, D. A.
(2002). Src-mediated coupling of focal adhesion kinase to integrin {alpha}v{beta}5 in vascular endothelial growth factor signaling. JCB
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HYNES, R.O., LIVELY, J.C., MCCARTY, J.H., TAVERNA, D., FRANCIS, S.E., HODIVALA-DILKE, K., XIAO, Q.
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Bouvard, D., Brakebusch, C., Gustafsson, E., Aszodi, A., Bengtsson, T., Berna, A., Fassler, R.
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Taverna, D., Hynes, R. O.
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Li, E., Brown, S. L., Stupack, D. G., Puente, X. S., Cheresh, D. A., Nemerow, G. R.
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Steffensen, B., Hakkinen, L., Larjava, H.
(2001). Proteolytic Events of Wound-Healing -- Coordinated Interactions Among Matrix Metalloproteinases (MMPs), Integrins, and Extracellular Matrix Molecules. CROBM
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Eliceiri, B. P., Puente, X. S., Hood, J. D., Stupack, D. G., Schlaepfer, D. D., Huang, X. Z., Sheppard, D., Cheresh, D. A.
(2002). Src-mediated coupling of focal adhesion kinase to integrin {alpha}v{beta}5 in vascular endothelial growth factor signaling. JCB
157: 149-160
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