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Animal Models |
2 Integrin Subunit-Deficient Mouse
From the Departments of Pathology and Immunology*and Pediatrics,Washington University School of Medicine, St. Louis, Missouri
Abstract
The 
2ß1 integrin is a collagen/laminin receptor expressed on platelets, endothelial cells, fibroblasts, and epithelial cells. To define the role of the 2ß1 integrin in vivo, we created a genetically engineered mouse in which expression of the 2ß1 integrin was completely eliminated. Mice deficient in the 2ß1 integrin are viable, fertile, and develop normally with no excess lethality of homozygotes. Both 2ß1-integrin protein and 2 mRNA were undetectable in the 2-null mice. Gross and histological evaluation of the heart, lungs, kidneys, gastrointestinal tract, pancreas, skin, and reproductive tracts revealed no abnormalities. However, quantitative analysis of mammary gland branching morphogenesis demonstrated that branching complexity is markedly diminished in the 2-deficient animals. Studies in the 2-deficient animals do not support the proposed roles for the 2ß1 integrin on fibroblasts and keratinocytes in wound healing. When compared to platelets from wild-type littermates, platelets from 2-null mice failed to adhere to type I collagen under either static or shear-stress conditions. Although platelets from 2-deficient animals aggregated in response to collagen, they did so with prolonged lag time and lessened intensity. The 2ß1 integrin-null mouse thus exhibits diverse, sometimes subtle, phenotypes consistent with the widespread pattern of 2ß1 integrin expression.
2ß1 integrin functions as a collagen receptor on platelets and fibroblasts and as both a collagen and laminin receptor on endothelial cells and epithelial cell types.1-3
The 2ß1 integrin has been implicated in a variety of complex biological processes. An abundant literature supports an important role for the 2ß1 integrin in platelet function and hemostasis.4
Extensive in vitro studies of renal, pulmonary, gastrointestinal, and breast epithelia suggested that signals downstream of 2ß1-integrin ligation contribute to branching morphogenesis.5-8
Experiments using explant cultures and three-dimensional collagen gels have implicated the 2ß1 integrin in keratinocyte migration and fibroblast contraction during wound healing.9-11
To define better the role of the 2ß1 integrin in vivo, we created a genetically engineered mouse in which expression of the 2ß1 integrin was eliminated. We now show that mice deficient in the 2ß1 integrin are viable and fertile. However, 2ß1-integrin-deficient mice show defective hemostasis and abnormalities of branching morphogenesis, but apparently normal healing of skin wounds.
Materials and Methods
The Targeting Construct
Screening of a P1 129/Svj DNA genomic library (Genomic Systems, St. Louis, MO) by polymerase chain reaction (PCR) using primers directed against bp -15 to +6 and 165 to 186 of exon 1 of the murine 2-subunit cDNA sequence12
yielded a 100-kb P1 clone. The targeting vector [loxP neo/TK vector (a gift from Dr. Robert Schreiber, Washington University, St. Louis, MO)] was prepared from an 8.0-kb EcoRI restriction enzyme fragment from the P1 genomic clone containing exon 1 (Figure 1A)
. A 2.0-kb BamHI fragment consisting of 5' untranslated region of the 2 integrin gene was inserted upstream of the 5' loxP site. The contiguous 600-bp BamHI fragment containing exon 1 and flanking sequence was placed between the two downstream loxP sites. A 2.4-kb Aval/EcoRI fragment containing intronic sequence was placed between the 3' loxP site and the PGK-TK cassette, as shown in Figure 1A
.
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2-Deficient Mice
R1 embryonic stem cells derived from a 129/Sv X 129/Sv-CP F1 3.5-day blastocysts from the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada, were electroporated with 25 µg of linearized targeting construct and selected in both G418 (26 µmol/L) and gancyclovir (0.2 µmol/L). Genomic DNA from resistant clones was digested with Xhol, and analyzed by Southern blot hybridization with the 1.7-kb BamHI/Xhol genomic fragment (probe a in Figure 1A
). In a second step embryonic stem cell clones that had undergone homologous recombination were transfected with 25 µg of Cre-recombinase-expressing plasmid and selected for G418. Clones in which both the neo-cassette and exon 1 were deleted were identified by Southern blot analysis and PCR and injected into C57BL/6 blastocysts. PCR was performed using oligonucleotides directed against the 5' UTR [primer 1 (5'-GCGTTGGGGGGTGGAGGGAACTGCC-3')] and intron 1 [primer 2 (5'-CTGGCTCCACGAA-GCCTGG)]. Male chimeric mice were bred to C57BL/6 females to obtained heterozygous 2-deficient mice. Heterozygous mice were intercrossed to obtain wild-type, heterozygous, and 2-deficient animals.
Morphological and Histological Analyses
Mice at ages 9 to 11 weeks were euthanized by CO2 asphyxiation. Tissues were fixed in 4% formaldehyde and paraffin-embedded. Sections (4-µm) were stained with Harris’ hematoxylin and eosin.
Whole mounts of the mammary gland were prepared from the no. 4 inguinal gland by spreading the fatpad onto a precleaned glass slide, air-drying for 10 minutes, and fixing in acetic acid:ethanol (1:3) for 1 hour at room temperature. The tissue was dehydrated in 70% ethanol and ductal structures were stained with carmine solution (0.2% carmine and 0.5% aluminum potassium sulfate). The gland was dehydrated through graded ethanol, defatted in acetone, clarified in toluene, and mounted.
Reverse Transcriptase (RT)-PCR Analysis
Total RNA from whole mammary glands was isolated by the guanidine isothiocyanate extraction technique. First-strand cDNA synthesis of total RNA was performed using an oligonucleotide primer complementary to bp 3141 to 3161 of the murine 2 cDNA. RT-PCR was performed with the same PCR primer and one complimentary to bp 2320 to 2341 using standard PCR protocol.
Immunoblot Analysis
Purified platelet protein preparations or mammary glands were lysed in sodium dodecyl sulfate sample buffer [10% (v/v) glycerol/62.5 mmol/L Tris (pH 6.8)/2% sodium dodecyl sulfate/2% 2-mercaptoethanol], subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and electroblotted onto Immobilon P transfer membrane (Millipore, Bedford, MA). Immunoblots were incubated with the appropriate dilution of each primary antibody and secondary horseradish peroxidase-conjugated donkey/anti-rabbit IgG antibody (Amersham, Arlington Heights, IL). The enhanced chemiluminescence system (Amersham) was used for visualization. The polyclonal antiserum against a portion of the carboxy terminus of the murine 2 integrin has been previously described.12
The goat polyclonal antiserum against actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Platelet Preparation, Adhesion, and Aggregation Assays
Murine platelets were purified from citrated whole blood and resuspended in Ca2+-free solution to a final concentration of 1 x 108 platelets per ml, as previously described.13 When fixation was required, an equal volume of 2% paraformaldehyde in platelet buffer was added to the platelet suspensions, and the mixture was incubated at 37°C for 1 hour. Platelets were quantitated on a Hemavet 850 (CDC Technologies, Inc., Oxford, CT) and showed <0.1% contamination with leukocytes or erythrocytes.
Static adhesion assays were performed as previously described.14
Platelets purified from wild-type, heterozygous, and 2-integrin-deficient animals were prepared as described above and aliquotted into flat-bottom cells of a 96-well plate coated with either type I collagen or bovine serum albumin. The platelets were allowed to adhere for 1 hour at 37°C. Nonadherent platelets were removed by washing and the remaining attached platelets quantitated, as described.14
Platelet adhesion to substrates prepared with 100 µg/ml of equine tendon collagen (Helena Laboratories, Beaumont, TX) was assessed in a parallel-plate flow chamber apparatus as previously described.15 Platelets (1 x 108/ml) were perfused through the chamber at a shear rate of 150 seconds-1 for 5 minutes. The surface area covered by adherent platelets at the end of each experiment was determined (Image Pro Plus software) and expressed as a percentage of wild-type platelet adhesion. All experiments were performed in duplicate on different days.
Platelet aggregation was performed in siliconized glass cuvettes at 37°C with constant stirring (1200 rpm) using a dual-channel aggregometer (Payton Scientific, Buffalo, NY) using washed murine platelets (2 x 108/ml). Aggregation was initiated by either the addition of 2.5 or 5 µg/ml of soluble equine tendon collagen (Helena Laboratories) or 0.5 U/ml of murine thrombin (Sigma Immunochemicals, St. Louis, MO).
Flow Cytometry
Suspensions of paraformaldehyde-fixed platelets were incubated with the appropriate monoclonal antibody, washed with phosphate-buffered saline containing 1% bovine serum albumin and 5 mmol/L ethylenediaminetetraacetic acid, and subsequently incubated with fluorescein isothiocyanate-labeled secondary antibody (Zymed Laboratories, Inc., South San Francisco, CA) for 30 minutes at room temperature. The platelets were again washed and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The following previously described antibodies to murine antigens were obtained from BD PharMingen (San Diego, CA): HM2 (anti-CD49b, hamster IgG), MEC 13.3 (anti-CD31, rat IgG2a), and isotype-matched control (rat anti-IgG2a). NAD-1 (anti-CD41/61, mouse anti-IgG) and anti-glycoprotein VI (GPVI) were generated to murine GPIIbIIIa complex and murine GPVI, respectively, using standard hybridoma techniques.16
Cutaneous Wounds
2-deficient and wild-type mice were anesthetized with ketamine, dorsal hair was removed, and a 100-mm2 full-thickness wound was created by excising the skin. The wounds were photographed on day 0 and on every third day until they were completely healed. The size of the wound was digitally quantitated using Scion Image software.
Results
Targeted Disruption of the 2 Integrin Gene in Embryonic Stem Cells
To generate 2-integrin-null animals, we used a Cre-loxP targeting approach. The targeting vector was designed to flank exon 1 of the 2-integrin gene by two loxP sites and to place the PGK-neomycin resistance gene cassette upstream of exon 1 and the PGK-thymidine kinase gene downstream in the first intron. Embryonic stem cell clones with a targeted allele were transiently transfected with a Cre-recombinase encoding plasmid. Clones with deletion of both exon 1 and the neo-cassette were injected into host 129/Svj blastocysts. Chimeric animals with germline transmission of the targeted allele were obtained. Heterozygous mice were intercrossed to obtain mice homozygous for the 2 mutation. Genotypic analysis confirmed that mice lacking exon 1 of the 2-integrin gene were obtained (Figure 1, B and C)
.
2-Deficient Mice Are Viable and Fertile
Mice deficient in the 2ß1 integrin are viable and fertile and develop normally. Matings of heterozygous progeny yielded 22% wild-type, 58% heterozygous, and 21% homozygous mutant animals, consistent with typical Mendelian inheritance with no excess lethality of homozygotes. 2-null females produced viable litters of the expected sizes and nursed their young.
To verify that 2-integrin gene disruption generated a null allele and that the 2-null mice were deficient in expression of the 2ß1 integrin, platelets purified from the blood of wild-type, heterozygous, and homozygous mutant animals were evaluated by flow cytometric analysis using a fluorescein isothiocyanate-conjugated anti-murine 2 (CD49b) integrin subunit antibody. As shown in Figure 2A
, the 2ß1 integrin was highly expressed by platelets from wild-type animals, as expected. In contrast, there was no detectable expression of the 2ß1 integrin on the platelets of 2-deficient animals. Intermediate levels of the 2ß1 integrin were detected on the surface of platelets from heterozygous littermates. Expression of CD31 and CD41/61 (IIbß3, GPIIb/IIIa, the most abundant integrin on platelets) (Figure 2A)
and GPVI on 2-deficient platelets was comparable to that on the platelets of wild-type and heterozygous littermates.
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2-integrin cytoplasmic domain. Neither full-length nor truncated 2-integrin subunit protein was detected from platelets of 2-null animals (Figure 2B)
. The 2-integrin subunit was detected at intermediate level from platelets of heterozygous littermates and at high level from the wild-type littermates.
Western blot analysis and RT-PCR of protein lysates and mRNA of virgin female mammary glands established that the 2 integrin subunit was also effectively deleted from epithelial tissues. As shown in Figure 2, B and C
, the wild-type mammary gland expressed the 2-integrin subunit protein and mRNA, as expected from previous studies.17
In contrast, the 2-integrin subunit was not detected in protein lysates (Figure 2B)
or total RNA (Figure 2C)
from the 2-null mammary gland.
2 Integrin Subunit-Deficient Mice Develop Normally
Analysis of in vitro models of pulmonary, mammary, gastrointestinal, and renal development suggested a role for the 2ß1 integrin in epithelial differentiation and branching morphogenesis.5-8,18
However, 2-deficient mice appear grossly normal, are fertile, and are able to nurse their young. Gross and histological evaluation of a number of organs including the heart, lung, gastrointestinal tract, liver, kidney, pancreas, and reproductive tract failed to detect differences between the wild-type, heterozygous, and 2-deficient animals. Although the organization of the lungs, gastrointestinal tract, kidney, and mammary gland was entirely normal by histological sections in the 2-null animals (Figure 3A
, top), whole-mount preparations of virgin female mammary gland revealed marked differences in branching complexity. As shown in Figure 3A
, middle and bottom panels, the branching was much more extensive throughout the gland as well as at the terminal ducts in the wild-type mammary gland than in the 2-null gland. The number of branch points per terminal duct was 4 ± 1.7 in the wild-type gland versus 1.6 ± 0.2 (P = 0.0008) in the 2-null mammary gland, as shown in Figure 3B
. These findings support a role for the 2ß1 integrin in branching morphogenesis in vivo.
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2 Integrin Deficiency on Platelet Function
Many studies have suggested that the 2ß1 integrin is a critical mediator of platelet adhesion to collagen within the vessel wall after vascular injury. Tail vein bleeding times in wild-type, heterozygous, and 2-deficient mice were similar (data not shown). Platelets purified from wild-type animals adhered under static conditions to collagen substrates in a Mg2+-dependent manner, as expected for 2ß1 integrin-mediated adhesion (Figure 4A)
. In contrast, platelets purified from 2-deficient mice failed to adhere to type I collagen. Platelets from heterozygous littermates exhibited intermediate adhesion. Platelets from all animals adhered to fibronectin in a comparable manner (data not shown), but failed to adhere to bovine serum albumin.
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, purified platelets from wild-type mice also effectively adhered to type I collagen at shear rates of 150 seconds-1, a flow rate in which exogenous Von Willebrand factor is not required. In contrast, platelets from 2-deficient mice failed to adhere under shear stress. Platelets from heterozygous mice again adhered to an intermediate extent, indicative of a gene dosage effect and suggesting that there are few, if any, spare 2ß1 collagen receptors. As also shown in Figure 4B
, platelets from both wild-type and heterozygous mice formed small platelet thrombi on the collagen substrates under conditions of flow.
Platelets from wild-type and heterozygous animals comparably aggregated in response to low concentrations of collagen (2.5 and 5 µg/ml) (Figure 4D)
. Although platelets from 2-null animals aggregated in response to type I collagen, they did so with a significantly prolonged lag phase and a decreased rate of aggregation.
2ß1 Integrin and Wound Healing
The 2ß1 integrin is highly expressed by the basal cell layer of the skin and moderately expressed by dermal fibroblasts. In vitro and explant studies of matrix contraction and wound healing suggested that the 2ß1 integrin may serve a role in the normal organization of the dermis and an obligatory role in wound healing.9,10
Histological evaluation of full-thickness skin biopsies of wild-type and 2-null animals identified no detectable differences (Figure 5A)
. In both cases the epidermis was composed of layers of stratified squamous epithelium attached to the basal cell layer. The dermis in the 2-null and wild-type animals contained comparable numbers of fibroblasts and well-organized collagen bundles.
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2ß1 integrin was required for wound healing, the ability to close a 100-mm2 full-thickness wound was evaluated. Compared to wild-type animals, 2-deficient mice demonstrated no defect in wound repair and all animals displayed a similar temporal pattern of wound healing (Figure 5B)
. In fact, wound healing on day 12 was slightly enhanced in the 2-null animals compared to wild-type littermates. The morphology of the wounds failed to demonstrate any difference in keratinocyte migration or angiogenesis into the wound site (data not shown). Discussion
The expression of the 2ß1 integrin by diverse cell types raised many questions regarding the role of the 2ß1 integrin in vivo. Indeed, the 2ß1-integrin-null mouse exhibits a multifaceted phenotype. In some cases, such as platelets and mammary epithelial cells, the null phenotype was consistent with predictions based on the expression pattern of the integrin and consistent with the in vitro data. In other instances, the in vivo findings suggest alternative mechanisms for biological processes such as wound healing, and reject the proposed obligatory role for the 2ß1 integrin.
Although the 2ß1 integrin is expressed at high level in the placenta and in many other tissues during embryogenesis,12
its expression is not required for normal murine development. Deficiency of the 2ß1 integrin had no effect on offspring viability or fertility of the 2-deficient animals.
The 2ß1 integrin is highly expressed at sites of terminal duct branching in the developing mammary gland and lung.12,17
The significant decrement in branching complexity observed in the mammary glands of 2ß1 integrin-deficient animals supports an important role for the integrin in branching morphogenesis in vivo.5-8
The results of the in vivo studies, although striking, were less dramatic than the complete absence of branching morphogenesis observed in the absence or inhibition of the 2ß1 integrin in earlier in vitro studies in three-dimensional collagen gels. The in vitro models were designed to study a single integrin interaction with a single matrix molecule. Mammary epithelial cells express a wide variety of integrin and nonintegrin adhesion receptors that interact with multiple components of the extracellular matrix in vivo and may mitigate the consequences of 2ß1 integrin deficiency.
The mechanism by which branching is impaired in the 2-deficient mouse is not yet understood. One possibility is defective proliferation of 2ß1-integrin-deficient mammary epithelial cells. Recent studies have established that the integrin is an important determinant of growth factor-dependent cell cycle progression of mammary epithelial cells.18,19
Whether the differences seen at weeks 9 to 11 are because of defective branching per se, or rather to a delay in development, is uncertain. Future studies will evaluate the mammary gland during all stages of maturation including puberty, gestation, and involution. However, the 2-deficient mammary gland is capable of lactation and the 2-deficient animals can adequately nurse their young despite the deficit in branching complexity. Litters of 2-deficient females do not show a growth defect. Our earlier studies established a strong correlation between diminished 2ß1 integrin expression and a less differentiated phenotype in breast cancer.20,21
The 2ß1-integrin-deficient mouse should afford a system to further assess the relationship between 2ß1 integrin expression and the development and phenotype of breast carcinomas.
The lack of obvious abnormalities in other organs such as the lung, gastrointestinal tract, and kidney may be because of the subtlety of lesions in these organs not detected by standard histopathological evaluation. In the mammary gland, the differences were most obvious in whole-mount preparations, but not readily apparent in cross sections of the mammary gland. More detailed examinations of other organs are required to exclude this possibility.
In vitro studies of human skin have suggested that the 2ß1 integrin is required for wound healing.9-10
Inhibitory antibodies against the 2ß1 integrin prevent keratinocyte migration on type I collagen and across wounded dermis and block fibroblast contraction of three-dimensional collagen gels.9-10
However, deficiency of the 2ß1 integrin failed to alter wound healing in vivo and had no effect on the migration of keratinocytes over exposed dermis (data not shown) suggesting that 2ß1 integrin does not play an obligatory role in wound healing. There are at least two possible explanations for the discrepancy. One arises from the use of two different organisms that may not be mechanistically equivalent as model systems. Second, on binding the integrin, inhibitory antibodies may stimulate signals downstream of the integrin that are responsible for the observed phenotype. Thus, antibodies could produce effects that differ from those because of lack of integrin expression.
The profound deficit in platelet adhesion to collagen observed with platelets from 2 integrin subunit-deficient animals is in accord with earlier studies addressing the role of the 2ß1 integrin as a collagen receptor on platelets by less direct approaches.4
The lack of 2ß1 integrin expression did not effect the expression of the other major platelet integrin, IIbß3 (CD41/61). The magnitude of the deficit under both static conditions and conditions of flow clearly indicates that the 2ß1 integrin is the dominant adhesive receptor for collagen on platelets.
Other putative receptors are unable to support significant adhesion in the absence of the 2ß1 integrin not only under conditions of flow, but also under the less rigorous static conditions. The dramatic reduction in adhesion of heterozygous platelets under both static and flow conditions is consistent with a gene dosage effect and suggests that the level of 2ß1 integrin expression profoundly influences the adhesive functions of platelets on collagenous substrates. This provides an animal model to evaluate the molecular mechanisms by which allelic differences in the 2 integrin gene and different levels of 2ß1 integrin expression are associated with increased risk of myocardial infarction, diabetic retinopathy, and stroke.22-25
The adhesive decrement in heterozygotes suggests that the receptor is not present in significant excess.
The results of the platelet studies are primarily consistent with the two-step, two-site model of collagen-induced platelet activation.26
In this model, adhesion is mediated by the 2ß1 integrin, whereas collagen-induced activation and aggregation is mediated by a second, low-affinity signal-transducing co-receptor. Recent studies suggest that GPVI serves the role of the signal-transducing co-receptor. Under the extremely low shear conditions of the aggregation assay, as opposed to the more stringent conditions of the adhesion assays, our data suggest that GPVI is sufficient to support activation and aggregation although at a somewhat reduced level. Collagen is only one of several physiologically relevant activators of platelets. The presence of other potent activators, such as thrombin, at the sites of vascular injury in vivo, likely accounts for the relatively normal tail vein bleeding times observed and the absence of an overt bleeding diathesis.
Some comparison with the recently published study of collagen-induced platelet aggregation and adhesion in platelets deficient in the ß1 integrin subunit is warranted.27
The findings regarding collagen-induced platelet aggregation and adhesion to substrates of monomeric collagen are primarily in accord. The studies of adhesion to fibrillar substrates described by Nieswandt and colleagues27
were likely confounded by the highly thrombogenic nature of fibrillar collagen substrates and the relatively intact aggregation response to collagen exhibited by platelets deficient in the 2ß1 integrin. It should also be noted that differences between 2ß1-deficient and control platelets were minimized by reconstituting washed platelets in media containing 1 mmol/L of Ca++ thereby suppressing 2ß1 integrin functions. The model presented above, but not that of Nieswandt and colleagues,27
is also consistent with the findings of studies with human platelets deficient in GPVI. GPVI-deficient platelets exhibit markedly impaired collagen-induced aggregation and activation, little or no defect in adhesion per se, and a modest decrement in total platelet deposition (adhesion plus aggregation) on collagen substrates measured under conditions of flow that was attributable to the loss of platelet-platelet interactions.28,29
In addition to profoundly deficient adhesion to collagen, the platelets deficient in the 2ß1 integrin of a patient described by Nieuwenhuis and colleagues30,31
also exhibited markedly impaired (essentially undetectable) collagen-induced platelet aggregation. This finding is at variance with both our study of the 2-integrin subunit null mouse and the study of Nieswandt and colleagues27
of the ß1-integrin subunit-deficient mouse. It seems likely that the platelets of the patient described by Nieuwenhuis and colleagues30,31
exhibit an additional defect resulting in the absence of collagen-induced platelet aggregation. Alternately, the relative contributions of the 2ß1 integrin, glycoprotein VI, and perhaps other receptors and signaling pathways differ in human and murine platelets.
Acknowledgements
We thank Bruce Linders and George Li for expert technical assistance and Mary Beth Flynn for excellent secretarial assistance.
Footnotes
Address reprint requests to Mary M. Zutter, Washington University School of Medicine, Department of Pathology & Immunology, 660 S. Euclid Ave., Campus Box 8118, St. Louis, MO 63110. E-mail: zutter{at}path.wustl.edu
Supported by the National Institutes of Health (grants CA70275, CA83690, and HL63446).
Accepted for publication April 5, 2002.
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