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Tumor Biology |
Institute for Protein Research [T. F., Y. M-H., K. S.] and Department of Orthopedic Surgery, Faculty of Medicine [M. S., K. K., N. Ya.], Osaka University, Suita, Osaka 565-0871; Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Osaka 594-1011 [T. F., N. Yo., Y. K., R. M., K. S.]; and Sekiguchi Biomatrix Signaling Project, Japan Science and Technology Corporation, Nagakute, Aichi 480-1195 [R. M., K. S.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FNs exhibit molecular heterogeneity arising from alternative splicing of a primary transcript at three distinct regions termed EDA, EDB, and IIICS (5, 6, 7, 8) . Two type III repeats, EDA and EDB, are either included or excluded from FN mRNA by exon skipping (8) . Alternative splicing at the EDA and EDB regions is regulated in a tissue-specific and developmental stage-dependent manner. FNs expressed in fetal tissues contain a greater percentage of the EDA and EDB segments than those expressed in adult tissues (9, 10, 11, 12, 13) . In mice, FNs lacking the EDA and EDB segments are expressed in many adult tissues, although FNs containing either EDA or EDB segments are expressed in restricted tissues such as blood vessels and lung interstitium (EDA-containing FNs) and cartilage and cornea (EDB-containing FNs; Ref. 14 ).
Despite accumulating evidence for the regulated expression of EDA- and/or EDB-containing FNs in vivo, the biological functions of these isoforms are poorly understood. Recently, we showed that the EDA segment regulates the binding affinity of FNs for integrin 
5ß1 and thereby stimulates integrin-mediated signal transduction and subsequent cell cycle progression (15
, 16) . The EDA segment was also shown to stimulate the conversion of hepatic lipocytes to myofibroblast-like cells (17)
and cytokine-dependent matrix metalloproteinase expression (18)
, although the molecular basis of these phenomena remain to be elucidated. Unlike the EDA segment, the EDB segment did not enhance FN binding to integrin 5ß1 (15
, 16)
or stimulate myofibroblast conversion of lipocytes (17)
, but it was more readily incorporated into the extracellular matrix (19)
. The amino acid sequence of the EDB segment is 100% identical for human, rat, and mouse. Even between human and chicken, the identity of this amino acid sequence is as high as 97%. Given that EDB is selectively expressed during embryonic development, wound healing, and malignant transformation, the exceptionally high sequence homology of EDB may imply that it has an important function in these biological processes.
One approach to elucidate the hitherto unknown functions of EDB, particularly those in vivo, is to generate mice in which the EDB exon has been deleted from the FN gene. To generate such mice, we used the Cre-loxP system of the bacteriophage P1 (20) to excise the EDB exon from the FN gene together with the selection marker cassette which had been introduced to isolate the homologously recombined ES cells. Surprisingly, the homozygous EDB-deficient mice were apparently normal and fertile, although the fibroblasts obtained from the homozygous mice exhibited reduced potential for cell growth and FN matrix assembly in vitro.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A gene cassette containing the herpes simplex virus thymidine kinase gene, which was also kindly provided by Dr. K. Rajewsky, was then introduced 5' to the mouse FN genomic DNA for negative selection.
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. These primers generate a 1.2 kb fragment from successfully targeted cells. Targeted cells were verified by Southern blot analysis of the BlnI digest of genomic DNA using a probe covering the 3' region of exon III8a and its 3' intronic sequence (designated ‘Bl’; Fig. 1
). The homologously recombined clones were then transfected with pIC-Cre (a gift from Dr. K. Rajewsky; Gu et al., Ref. 21
) and grown on irradiated mouse embryonic fibroblasts at a low cell density (1 x 103 cells/10-cm dish) to allow transfected cells to form colonies. The colonies were individually picked up and expanded in duplicate 24-well plates, one of which was fed with medium containing G418 to select transfected cells from which the neo cassette had been deleted by Cre recombinase. G418-sensitive clones were screened for Cre-mediated deletion of the EDB exon by Southern blot analysis of the EcoRI digest of genomic DNA using a probe covering the intron between the EDB exon and exon III8a and 5' region of exon III8a (designated ‘Ec’). The clones, yielding a 1.2-kb band, were expanded and injected into C57BL/6 blastocysts. Male chimeric mice were mated with 129/Svter females to obtain heterozygous (i.e., EDB+/-) mice. Mice were genotyped by PCR using the following pairs of primers: (a) 5'-CCAGATATCACTGGCTACAGAATAACTACT-3' (primer III7bF), which was positioned within exon III7b and the primer III8aR; and (b) 5'-GAGGTGCCCCAGCTCACTGACCTAAGC-3' (primer EDBF), which was positioned within the EDB exon and the primer III8aR. DNA from tail biopsies was isolated by proteinase K digestion followed by isopropanol precipitation.
Isolation of Extracellular Matrix from Cultured Fibroblasts.
Mouse embryonic fibroblasts were grown in DMEM supplemented with 10% FN-depleted FBS in 10-cm dishes. FN-depleted FBS was prepared by passing the FBS through a gelatin affinity column twice. The FN-containing extracellular matrix was prepared from cells that had been kept in culture for 3 days after they had reached confluency (22)
with a slight modification. Briefly, the cell layers were rinsed three times with PBS at room temperature and lysed twice 10 min each in 50 mM Tris-HCl (pH 8.0) containing 0.5% sodium deoxycholate, 1 mM PMSF, 1 mM EDTA, and 145 mM NaCl at 4°C on a slowly moving four-way shaker. The resulting insoluble matrices were washed twice with low ionic strength buffer [2 mM Tris-HCl (pH 8.0), containing 1 mM PMSF, and 1 mM EDTA], twice with high ionic strength buffer [20 mM Tris-HCl (pH 8.0), containing 1 mM PMSF, 1 mM EDTA, 1 M NaCl], and finally twice with low ionic strength buffer again at 4°C. The remaining FN-containing matrices were recovered in 1 ml of 10 mM Tris-HCl (pH 6.8) containing 0.1% SDS, 50 mM NaCl, and 0.5 mM EDTA, lyophilized, and analyzed by immunoblotting as described below.
SDS-PAGE and Immunoblot Analysis.
SDS-PAGE of purified FNs or isolated extracellular matrices were performed as described by Laemmli (23)
using 6% polyacrylamide gels under reducing conditions. The separated proteins were transferred to nitrocellulose membranes and probed with polyclonal antibodies against human plasma FN (24)
or against the EDB segment of mouse FN. The anti-EDB antibody was raised in rabbits by immunization with a mixture of two synthetic peptides, EGIIFEDFVDSSVGY and YTVTGLEPGIDYDIS, after conjugation to keyhole limpet hemocyanin by the glutaraldehyde method (25)
. The specificity of the anti-EDB antibody was verified by immunoblot analysis using recombinant human FN containing the EDB segment (15)
. The bound antibodies were visualized with peroxidase-linked goat antibodies against rabbit IgG using the enhanced chemiluminescence system (Amersham Corp., Arlington Heights, IL). Recombinant human FNs containing both EDA and EDB as well as plasma FN were purified as described previously (15)
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Histological Examination.
Knee joint tissues from newborn, 5-week- and 6-month-old mice were fixed in 4% paraformaldehyde, decalcified in EDTA, dehydrated with ethanol, and embedded in paraffin. Sections (4-µm thick) were stained with H&E. For the experimental model of fracture healing, the right eighth ribs of 5-week-old mice were fractured by surgery under anesthesia as described previously (26)
. The mice were sacrificed on the seventh day after fracture, and the process of fracture healing was examined histologically. Sections of other tissues were processed as described above, except that the decalcification step was omitted.
Quantitation of FNs in Plasma and Culture Medium.
Blood (0.5 ml) was drawn from the mouse tail vein into a syringe containing a 25-µl aliquot of 0.2 M EDTA (pH 7.5). Plasma was separated from blood cells by centrifugation at 20,000 x g for 30 s and snap-frozen in small aliquots. All assays were performed on freshly thawed aliquots. The concentration of FNs in plasma was measured by ELISA, using collagen type I-coated 96-well polystyrene plates (Iwaki Glass, Tokyo, Japan) to capture the FNs, and a polyclonal antimouse FN antibody (Biogenesis, Poole, United Kingdom) to quantify captured FNs. The FN concentration in the conditioned medium of cultured fibroblasts was also measured by ELISA as described above.
Immunofluorescence Staining of FN Matrix.
Mouse embryonic fibroblasts were cultured in DMEM containing 10% FBS and 2 mM glutamine and harvested with 0.025% trypsin/1 mM EDTA. Cells (2 x 105) were plated onto glass coverslips placed in 24-well culture plates and cultured for 24 h in DMEM containing 5% FN-depleted FBS and 2 mM glutamine. Cells were then fixed with 3.7% formaldehyde for 10 min at room temperature and stained with the polyclonal anti-mouse FN antibody for 1 h at room temperature, followed by incubation with a FITC-labeled secondary antibody for immunofluorescence detection. Stained cells were observed using a Zeiss Axioplan fluorescence microscope, and representative fields were photographed using Kodak Ektachrome 400x film. Densitometric quantitation of the fluorescence intensities of FN matrix was performed using the public domain NIH Image program developed at the United States National Institute of Health and available on-line.7
Cell Growth Assay.
Mouse fibroblasts prepared from the embryos of knockout mice were cultured in DMEM containing 10% FBS and 2 mM glutamine and harvested with 0.025% trypsin/1 mM EDTA. Cells (1.5 x 104) were plated on 35-mm culture dishes and cultured in DMEM containing 5% FN-depleted FBS and 2 mM glutamine at 37°C under a 5% CO2/95% air atmosphere. Cells were harvested with 0.025% trypsin/1 mM EDTA, and the cell number was determined using a Coulter counter model Z1.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and used to construct the targeting vector as follows. A neo gene flanked by two loxP sites was inserted into the intron between exons III7b and EDB for positive selection, and a third loxP site was inserted into the intron between exons EDB and III8a. A herpes simplex virus-thymidine kinase gene was placed 5' to exon III3a for negative selection (Fig. 1)
. The linearized targeting vector was transfected into E14.1 ES cells by electroporation, and the cells were subjected to positive selection with G418 and negative selection with Gancyclovir. Clones containing the homologously recombined allele were identified by PCR and Southern blot analysis. These clones produced 4.5-kb and 14.5-kb bands upon Southern blots of EcoRI and BlnI digests of genomic DNA (Fig. 2, A and B
; Lane 2). Approximately 5% of the selected clones were positive for homologous recombination.
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; Lanes 4 and 5), indicative of the presence of the EDB exon; and those yielding a 1.2-kb band with EcoRI digest and a 10-kb band with BlnI digest (Fig. 2, A and B
; Lanes 3 and 6), indicative of the deletion of the EDB exon. Eighty-five percent of the G418-sensitive clones scored positive for Cre-mediated recombination. Two independent clones containing the EDB-deleted allele were injected to C57BL/6 blastocysts and used to generate chimeric males, of which four males were found to be capable of transmitting the targeted allele to their progeny. Mice heterozygous for the deleted EDB exon were identified by PCR using the primer pair III7bF/III8aR (Fig. 2C)
; wild-type mice gave rise to a 3 kb-fragment, whereas heterozygous mice produced only a 1.7-kb fragment. Failure to detect the 3-kb fragment in the PCR products of heterozygous mice could be because of a bias toward smaller fragments in PCR amplification of multiple-sized fragments with a single primer pair, thus resulting in a preferential amplification of the 1.7-kb fragment with respect to the 3-kb fragment. In support of this possibility, PCR using another pair of primers, i.e., EDBF/III8aR, yielded a 1.5-kb fragment from both wild-type and heterozygous mice, demonstrating that the wild-type allele was present in heterozygous mice. Intercrossing of heterozygous mice produced offspring homozygous for the EDB-deleted allele, yielding a 1.7-kb fragment with the III7bF/III8aR primer pair but no 1.5-kb fragment with EDBF/III8aR (Fig. 2C
; Lanes 6 and 7).
EDB-deficient Mice Are Apparently Normal.
Mice heterozygous for the targeted FN allele appeared healthy and were approximately the same size as their wild-type littermates. Although the EDB-containing FNs are highly expressed throughout early embryonic development, intercrossing heterozygous mice produced homozygous EDB-/- offspring at an expected Mendelian frequency (i.e., the numbers of wild-type, heterozygous, and homozygous mice were 84, 167, 77, respectively, indicating that lack of the EDB exon does not cause embryonic lethality). These mice developed apparently normally and were fertile. No apparent abnormalities were observed in homozygous mice upon anatomical inspection of major organs. Despite the proposed role of EDB-containing FN as an angiogenesis marker (27
, 28)
, the lung and kidney, two major organs with extensive microvasculature, did not show any noticeable anatomical abnormality with respect to vasculogenesis (data not shown). The concentration of FN in blood plasma was 418 ± 92 µg/ml in wild-type mice, 441 ± 40 µg/ml in heterozygous mice and 472 ± 93 µg/ml in homozygous mice, demonstrating that the deletion of the EDB exon did not perturb the expression of the FN gene either at the transcription or translation level.
To confirm the absence of EDB-containing FNs in homozygous mice, fibroblasts were isolated from embryos of wild-type, heterozygous, and homozygous mice and examined for the expression of EDB-containing FNs. Confluent monolayers of these fibroblasts were extracted with 0.5% sodium deoxycholate, and the resulting insoluble matrices were analyzed by immunoblotting with an antibody specifically recognizing the EDB segment (Fig. 3)
. Although FNs were deposited in the matrices of both wild-type and EDB-/- fibroblasts, EDB-containing FNs were only detectable in the matrix of the wild-type fibroblasts, confirming the inability of the homozygous mice to produce EDB-containing FNs. It was also noted that the intensity of the FN band (Fig. 3
, left panel) was less pronounced in the matrix from EDB-/- fibroblasts, suggesting that EDB-deficient FNs have reduced ability to assemble into the extracellular matrix.
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. In epiphysial growth plates, the chondrocytes of the distinct differentiation stages, i.e., resting, proliferating, and hypertrophic chondrocytes, are arranged in long parallel columns. Examination of the growth plates of the tibia and femur of 5-week-old homozygous mice did not show any anatomical aberration in the organization of the chondrocytes of the different differentiation stages. No clear differences were observed between wild-type and homozygous mice of different ages (e.g., neonates and 6-month-old mice; data not shown). Histological aberration was also not observed in the Descemet’s membrane of EDB-/- mice (data not shown). These data suggested that either EDB-containing FNs are not essential for the development of these tissues or that other protein(s) may compensate for the role of the EDB-containing FNs in the developmental processes.
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. In both EDB-deficient and wild-type mice, the fracture site was bridged by a typical cartilaginous external callus containing various stages of proliferating and hypertrophic chondrocytes. The bone cortex near the fracture end was surrounded by a periosteal bony callus with trabeculae. There were no significant differences in the histology of the fracture healing process between the two genetically different groups of mice. Normal condensation of prechondrogenic mesenchymal cells was observed in the EDB-deficient mice as well as in the wild-type mice at the periphery of the external callus. These results, taken together with the normal histology of the hyaline cartilage of wild-type and homozygous mice, indicate that EDB-containing FNs, although specifically expressed in chondrogenic tissues in adult mice, are dispensable for chondrogenic differentiation in both normal development and during the bone repair process.
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. The growth of EDB-/- fibroblasts was significantly slower than EDB+/+ fibroblasts, whereas EDB+/- fibroblasts exhibited an intermediate growth rate between EDB-/- and EDB+/+ cells, suggesting that the in vitro growth potential of embryonic fibroblasts might be modulated by the EDB segment of FNs.
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. Densitometric quantitation of the immunofluorescence intensities showed that EDB-/- cells deposited 
45% less FN matrix than EDB+/+ cells (Fig. 7B)
. FN fibrils assembled by EDB-/- fibroblasts were slightly thinner and shorter than those assembled by EDB+/+ fibroblasts. The decreased deposition of FN matrix in EDB-/- fibroblasts was not because of a reduction in the level of FN expression because the concentration of FNs in the conditioned medium of EDB-/- cells was higher than that of EDB+/+ cells (Fig. 7C)
. Furthermore, quantitation of the levels of FN mRNA by real-time PCR did not show any significant difference between EDB+/+ and EDB-/- cells (data not shown). These results, together with the normal level of plasma FN concentration in EDB-/- mice, indicate that the reduced FN matrix assembly of EDB-/- fibroblasts is because of the reduced potency of EDB-deficient FNs to assemble into the extracellular matrix.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another approach to elucidate the functions of the EDB segment is to produce animal models that cannot produce EDB+ FN isoforms via gene knockout technology. Because conventional knockout of the FN gene in mice results in embryonic lethality (2) , it is necessary to specifically eliminate the EDB exon without perturbing the level of expression of the FN gene. Georges-Labouesse et al. (3) constructed two targeting vectors focusing on the EDB exon, one for deletion of the EDB exon and the other for knockin of EDB. Unfortunately, both targeting constructs resulted in null mutation upon homologous recombination, failing to produce EDB exon-specific knockout or knockin mice. Abrogation of the FN expression in these mice could be because of the neo cassette introduced into the FN gene for selection of the homologously recombined ES cells or to partial deletion of intronic sequences downstream of the EDB exon that have been shown to be crucial for the recognition of EDB as an exon (29) . To overcome these potential problems, we used a gene targeting strategy using the Cre-loxP system of bacteriophage P1. We deleted the EDB exon by two-step recombination events, i.e., homologous recombination of the FN gene with the targeting vector, followed by Cre-mediated removal of the floxed EDB exon together with the neo cassette. One of the eight (T)GCATG repeats in the intron downstream of the EDB exon, the repeats proposed as the candidate motifs for correct splicing of EDB, was eliminated after Cre-mediated removal of the floxed EDB exon, but this did not cause any deleterious effects on the expression and/or splicing of the targeted allele, as evidenced by the normal levels of plasma FN in EDB-/- mice and by the comparable levels of FN expression by EDB+/+ and EDB-/- embryonic fibroblasts. It should also be noted that the single loxP site left behind between exons III7b and III8a after Cre-mediated recombination did not interfere with the transcription and/or splicing of the targeted FN gene either.
Mutant mice homozygous for the EDB-deficient allele were obtained at the predicted Mendelian frequencies with two independent targeted ES clones. Furthermore, the EDB-/- mice developed normally and were fertile. In normal adult mice, EDB+ FNs have been shown to be expressed only in restricted tissues such as hyaline cartilage and Descemet’s membrane of cornea (14) , but no histological abnormalities were detected in the articular cartilage of the femur and tibia or in cornea. No abnormalities were observed in the healing of bone fractures in EDB-/- mice either, supporting the conclusion that chondrocyte function during endochondral ossification was not impaired in EDB-/- mice. The failure to detect any developmental defects in EDB-/- mice, particularly in the process of ossification, does not seem to be because of variation in the genetic background because no abnormalities were detectable in homozygous mice either with the chimeric background of 129 and C57 strains or after repeated backcrossing with 129 strain. No apparent behavioral abnormalities were observed with EDB-/- mice either, although we cannot exclude the possibility that EDB-/- mice manifest aberrant behavior upon pathological intervention, as has been shown for the tenascin knockout mice (30) .
EDB+ FNs have been shown to be expressed under pathological conditions such as tissue repair and tumorigenesis (31) . In malignant tumors as well as tumor xenografts in nude mice, EDB+ FNs are deposited in the tumor stroma and more prominently in neovasculature within the tumor (11 , 28 , 32) . EDB+ FNs have therefore been proposed as a marker of angiogenesis because EDB+ FN is expressed not only in tumor neovasculature but also in the superficial layer of the endometrium where angiogenesis occurs physiologically (27 , 28) . Furthermore, injection of anti-EDB antibodies labeled with 125I or an infrared fluorophore into tumor-bearing mice resulted in selective accumulation at the tumor foci (33 , 34) , raising the possibility that EDB+ FNs could be used as a marker for tumor targeting. In support of this possibility, an anti-EDB antibody fused with a tissue factor, a coagulation-inducing protein, was successfully used to target the tissue factor to tumor xenografts in mice, thereby inducing selective infarction of different types of solid tumors followed by complete eradication of tumors in 30% of the mice treated (35) . Despite these observations, we could not find any defects in organogenesis in which extensive angiogenesis/vasculogenesis are closely associated. No anatomical abnormalities were observed in the lung and kidney, the organs with extensive microvasculature. Close association of EDB+ FN expression with various types of tumors (11 , 32 , 36) may imply a role of EDB+ FN in tumorigenesis, but no clear evidence supporting this possibility has ever been obtained. We have crossed EDB-/- mice with p53-null mice to generate double knockout mice lacking the EDB exon and a functional p53 gene. However, no clear difference in the length of survival periods was observed between EDB-/-p53-/- and EDB+/+p53-/- mice,8 arguing against the possibility that expression of EDB+ FNs potentiates tumor growth and/or tumor angiogenesis.
Although no clear function of EDB+ FNs emerged from the in vivo studies of EDB-null mice, embryonic fibroblasts prepared from EDB-/- mice proliferated more slowly than those from EDB+/+ mice in vitro. Furthermore, fibroblasts from EDB-/- mice produced FN fibrils that were shorter and thinner than those deposited by EDB+/+ fibroblasts. These differences were not dramatic but were reproducible in separate experiments. In support of these observations, recombinant rat FN containing either the EDA or EDB segment was more readily incorporated into existing FN matrices than those lacking these extra domains (19)
. In explant culture of articular cartilage, EDB+ FNs produced by cultured chondrocytes were preferentially retained in the cartilage matrix rather than exported to the culture medium (37)
, suggesting a role of the EDB segment in FN matrix assembly in cartilage. Consistent with this observation, immunohistochemical analysis of the articular cartilage showed that EDB-/- mice deposited significantly less FN in the cartilage matrix than EDB+/+ mice.9
These results, taken together, indicate that expression of EDB+ FNs may play a regulatory role in FN matrix assembly and proliferation of connective tissue cells. Given that matrix-assembled FNs transduce signals that stimulate cell cycle progression via integrin 5ß1 and some other Arg-Gly-Asp (RGD)-recognizing integrin(s) (38)
, decreased FN matrix assembly may well explain the reduced proliferative potential of EDB-/- fibroblasts in vitro. Nevertheless, neither growth retardation nor malformation of specific organs was detectable with EDB-/- mice, arguing against a regulatory role for EDB+ FNs in cell proliferation in vivo. Apparent normalcy of EDB-/- mice, however, does not exclude the possibility that EDB+ FNs may play a role in cell proliferation in some pathological processes. Although EDB+ FN deficiency does not have any discernible effects on bone fracture healing or in tumorigenesis associated with ablation of the p53 tumor suppressor gene, the search for distinctive phenotypes of EDB-/- mice needs to be continued to uncover the in vivo function of EDB+ FNs in normal and pathological processes.
In summary, we generated mice deficient in the EDB exon using the Cre-loxP recombination system. Unexpectedly, the homozygous EDB-/- mice grew normally and were fertile without showing any distinctive phenotypes, despite the restricted expression pattern of EDB+ FNs during embryogenesis and in normal adult tissues. Although in vivo function(s) of EDB+ FNs therefore remains to be elucidated, embryonic fibroblasts prepared from EDB-/- mice exhibited discernible differences from EDB+/+ fibroblasts in their ability to proliferate in vitro and to assemble FN matrices, indicating the possibility that the EDB segment may play a role in the regulation of FN matrix assembly and FN matrix-dependent cell growth. Given the exceptionally conserved amino acid sequence of the EDB segment between different species, i.e., 100% homology between human, rat, and mouse and 97% homology between human and chicken, it is important to continue the pursuit of the function(s) of EDB+ FNs by pathological intervention, as was successfully demonstrated with tenascin-C knockout mice. The EDB-/- mice developed should provide a valuable model to uncover hitherto unknown function(s) of the highly conserved EDB segment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Special Coordination Funds from Science and Technology Agencies of Japan (to K. S.) and Grants-in-Aid from Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. S., T. F.). 
2 Present address: Department of Pathology, University of Pittsburgh, 715 Scaife Hall, Pittsburgh, PA 15213.
3 Present address: The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
4 Present address: Department of Orthopedic Surgery, The University of Tokushima, Tokushima 770-8503, Japan.
5 To whom requests for reprints should be addressed, at Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8617; Fax: 81-6-6879-8619; E-mail: sekiguch{at}protein.osaka-u.ac.jp
6 The abbreviations used are: FN, fibronectin; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride; EDA, extra domain A; EDB, extra domain B; IIICS, type III-connecting segment.
7 Internet address: rsb.info.nih.gov/nih-image/.
8 T. Fukuda and K. Sekiguchi, unpublished observation.
9 Y. Mizuno-Horikawa and K. Sekiguchi, unpublished observation.
Received 4/25/02. Accepted 7/30/02.
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), EDB+/- (
), and EDB-/- (
) mice were plated at a density of 1.5 x 104 cells/35-mm dish in DMEM supplemented with 5% FN-depleted FBS and cultured for the indicated periods. Cell numbers were determined using a Coulter counter. Each bar represents the mean SD (n = 6).