| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Boehringer Ingelheim Pharma KG, Ingelheim, Germany;
Boehringer Ingelheim, Research and Development, Vienna, Austria; and
Research Institute of Molecular Pathology, Vienna, Austria
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The CD44 family of transmembrane glycoproteins is encoded by a single gene consisting of 21 exons, of which 12 can be differentially spliced (4, 5, 6). At least 17 different CD44 proteins generated through this great variability in splicing can be identified, including the predominant isoform, sCD44, which is a product of standard exons 1–5, 16–18, and 20 (7, 8).
The region encoded by the variant exons is very hydrophilic and is associated with several potential protein modification sites. Through these sites, CD44 has been proposed to bind multiple ligands including collagens, fibronectin (9), heparin-binding growth factors (10), osteopontin (11), and chondroitin sulfate (12). The major physiological interaction partner of CD44, hyaluronic acid (HA)3 (13, 14), has been described to bind to regions within standard exons s2 and s5, which are suggested to form a common domain stabilized through disulfide bridges (15, 16). Hyaluronate is a major component of the extracellular matrix surrounding proliferating and migrating cells in embryonic tissues. In the earliest embryonic developmental stages, cell proliferation and hyaluronate synthesis have been shown to be simultaneous events (17). CD44 as a mediator of HA uptake and degradation has been indicated by several lines of evidence (18).
CD44 has been shown to be functionally significant in hematopoiesis, lymphocyte homing, and formation of tumor metastasis (19, 20, 21, 22, 23). However, its expression is strongly increased during differentiation of T cells to memory cells (24). In addition, CD44 expression is enhanced during proliferation and differentiation of B cells (25). Mice depleted of thymic cells by irradiation display CD44 expression on the bone marrow-derived cells repopulating the thymus, an effect that can be inhibited by anti-CD44 Abs, suggesting CD44 as an essential player in this repopulation process (26, 27).
Tumor cell and leukocyte migration are thought to utilize similar mechanisms in the detachment of the cells from their primary locus; their travel through the lymph or blood stream; and their attachment to, invasion into, and settlement at secondary organs (28). The CD44 rat isoform p-Meta1, containing v4–v7, is not detected in normal tissues but is involved in the metastatic spread of tumors (29). The isoform p-Meta1 is closely related to a CD44 variant containing v6, which is required for the lymphatic spread of tumor cells and is believed to be involved in the process of lymphocyte activation (30). This observation supports the paradigm that metastasizing tumor cells mimic lymphocyte behavior.
In addition to its various functions within the immune system, a role for CD44 has been implicated in development of the mouse (31), as well as in a wide range of adult tissues (32). A detailed study on CD44 expression during wild-type mouse embryogenesis has been performed (33, 34), demonstrating that during embryogenesis, CD44 is strongly expressed in the forming heart, somites, and condensing limb bud mesenchyme during morphogenesis. This suggests an essential role of CD44 in cellular interactions during early development and pattern formation, an observation that has been confirmed independently (35).
To study the biological role of CD44 in vivo, we generated mice lacking all isoforms of CD44 by an in-frame insertion of the lacZ/neo (ß-galactoside and neomycin resistance genes) reporter cassette into the CD44 leader peptide, thus abolishing CD44 expression. We analyzed the role of CD44 in development, differentiation, and pattern formation of specific cell types and tissues during embryogenesis, especially where, based on previous results, lack of CD44 was likely to lead to irregularities. CD44 homozygous mutant mice are viable and fertile and do not exhibit any overt phenotype. Although the development of the lymphoid system is not affected in the absence of CD44, mutant lymphoid cells exhibit an impaired homing to the thymus and lymph nodes in the adult animals.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The targeting construct was generated as follows. A phage clone was isolated from a mouse genomic library (strain 129/Sv) containing 5' sequences of the CD44 gene. A fragment including sequences of about 2 kb 5' of the first standard exon to 5 kb of the first intron was subcloned into the EcoRV and XbaI sites of pBluescript II KS(+) (Stratagene, La Jolla, CA). The thymidine kinase gene of pMC1/TK (36) was inserted into the XhoI/SalI sites of the construct. A linker was introduced into the NcoI site connected with the CD44 start codon of exon s1, and another linker was introduced into the XbaI/SacII sites of pGNA (37); both linkers contained an SgfI restriction site. Using SgfI restriction, the lacZ/neo cassette of pGNA was inserted in frame directly 3' of the ATG start codon of exon s1. After linearization with NotI, the targeting construct was electroporated into 1.2 x 107 R1 embryonic stem cells (38).
Electroporation of the vector, embryonic stem cell culture, G418 and gancyclovir selection, injection, and transfer of blastocysts were performed as described previously (39). Using a 5' flanking probe, an endogenous fragment of 15 kb and a recombinant 5-kb fragment were detected. Homozygous recombinant embryonic stem (ES) cells and germline transmission of the CD44 mutation were screened by Southern blot analysis of EcoRI-digested genomic DNA.
DNA and RNA analysis
Preparation of genomic DNA and Southern blot analysis to identify targeted clones and to genotype mice was performed as described previously (40). Embryos of E13.5 of gestation were homogenized and total RNA preparation and Northern blot analysis was performed as described (40).
Flow cytometry analysis
mAbs against the following structures were used (all from
PharMingen, San Diego, CA, unless specified otherwise): CD3
(145-2C11-PE), CD4 (RM4-5-FITC; GK1.5; American Type Culture Collection
(ATCC), Manasass, VA), CD8 (53-6.7-FITC,PE; 2.43; ATCC), CD25
(7D4-biotin), CD16/32 (FcR), CD44 (IM7-CyChrome; KM201; ATCC),
CD45R/B220-PE, CD62 (MEL14-FITC), CD69-PE, CD122 (TM-ß1-PE),
DX5-FITC, TCR-
ß-biotin, TCR-
(GL3-PE), I-Ab,d,-FITC.
Goat-anti-rat-FITC (Vector Labs., Burlingame, CA) and
streptavidin-PE or streptavidin-CyChrome were used as second-step
reagents where applicable. Cell suspensions from the respective organs
were prepared from mice 3 mo of age and stained using standard
procedures; data were collected using CellQuest on a FACSort cytometer
(Becton Dickinson, Mountain View, CA) and analyzed with WinMDI (J.
Trotter, Scripps Institute, La Jolla, CA).
Homing assay
Single-cell suspensions were prepared from spleen and thymus of CD44-deficient and wild-type mice and stained with PKH26 (Sigma, St. Louis, MO) or FITC (Molecular Probes, Eugene, OR) as described previously (41). Stained cells were introduced into CD44 homozygous and heterozygous mutant and wild-type recipients via injection into the tail vein. Donor and recipient mice were 5 wk old and originated from inbreds between sibling parents. Mice were sacrificed 2 or 24 h later, and the spleen, thymus, and peripheral lymph nodes removed and analyzed by flow cytometry.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To inactivate the CD44 gene through homologous recombination, a
targeting vector was constructed in which the leader peptide of CD44
was disrupted by an in-frame fusion of the lacZ/neo cassette
directly behind the ATG start codon, resulting in total loss of CD44
transcription and function. Transcription and translation of the
targeted allele subsequently lead to the synthesis of the
lacZ protein under control of the 5' regulatory elements of
the CD44 locus in all cells and tissues normally expressing one or
several of the CD44 isoforms. The targeting construct (Fig. 1
A) was introduced into R1
embryonic stem cells (38) of passage 14 by
electroporation. Colonies resistant to G418 and gancyclovir were
screened for homologous recombination by Southern blot analysis using a
0.75-kb EcoRI/EcoRV probe (Fig. 1A)
located 5' of the targeting vector. In addition to the endogenous 15-kb
EcoRI fragment, the 5' probe detects a 5-kb fragment in
targeted ES clones. Three different CD44 heterozygous mutant ES cell
clones could be identified, resulting in a gene-targeting frequency by
homologous recombination of 1 in 150.
|
B and Table I).
|
C). A
cDNA probe consisting of CD44 exons s1–s5 detected a 4.0-kb transcript
in wild-type mice corresponding to the sCD44 isoform, which makes up
the major part of isoforms expressed in this stage of embryonic
development. It was reduced in heterozygous mutant mice to about half
of the wild-type level, and was undetectable in homozygous mutant CD44
embryos, demonstrating that no RNA products are synthesized by
inappropriate splicing events. Furthermore, using flow cytometry
analysis, no CD44 expression was detected in several organs, e.g.,
thymus, spleen, and lymph nodes, of CD44 homozygous mutant mice,
whereas heterozygotes showed normal levels of CD44 surface expression,
comparable with wild-type animals (Fig. 2, data for wild-type not shown).
|
To test for potential phenotypic and functional deviations of the immune system such as lymphocyte homing and T cell activation of CD44 homozygous mutant mice, the following issues were investigated: 1) the cellular composition of various lymphoid organs (thymus, peripheral lymph nodes, and spleen) in adult mice to detect potential irregularities in the subset distribution; 2) the phenotype of cells in fetal thymus at E16.5–17.5, when CD44 expression was reported to be highest in this organ; and 3) trafficking of isolated and labeled lymphoid cells in wild-type and CD44 heterozygous and homozygous mutant mice.
No subset deviation in adult mice in the absence of CD44 protein
Thymus, spleen, and peripheral lymph nodes were removed from adult
CD44 wild-type, homozygous, and heterozygous mutant animals, and
single-cell suspensions were stained for CD44 expression and analyzed
using flow cytometry. T cell subsets appeared to be normal both in
numbers and in CD4/CD8 expression patterns in various lymphoid organs
of CD44 homozygous mutant mice and comparable with those of CD44
heterozygous mutant mice (Fig. 3),
indicating that T cells of CD44-deficient mice develop normally. Abs
against other markers, which are generally used to further characterize
these subsets, such as TCRß and CD25, also did not reveal any
deviations (data not shown).
|
Fetal thymus was removed from E16.5–17.5 embryos originating from
(CD44-/- x CD44+/-)
matings. Single-cell suspensions were prepared and analyzed using a
broad panel of mAbs. Staining with anti-CD44 Abs was performed to
determine the genetic background of the samples. Again, no major
difference was detected between homozygous and heterozygous mutant (and
wild-type) mice either in cell yield or in cell subset distribution
(data not shown). However, we repeatedly encountered an interesting
phenomenon in CD44-deficient mice compared with wild-type mice:
both CD4+ and CD8+ cells
had lost a CD25low+ population (Fig. 4).
|
CD44 has been described to play a role in thymus colonization of T cells. It is expressed on bone marrow cells repopulating the thymus of irradiated mice and in long-term bone marrow reconstitution. Anti-CD44 Abs inhibit thymus repopulation by bone marrow progenitors (26, 27). A similar effect would be expected for CD44-deficient lymphocytes. This expectation was indeed confirmed for the homing of CD44 homozygous mutant lymphocytes to the thymus and peripheral lymph nodes after tail vein injection of equal numbers of mixed fluorescent-labeled CD44-deficient and wild-type lymphocytes.
The ratio of CD44-deficient and wild-type fluorescent-stained donor
cells from various recipients recovered 2 or 24 h after
reintroduction via the tail vein should be 
1 in an ideal situation
in which no homing difference exists. The ratio would be <1 in the
case of a selective disadvantage of the homozygous mutant lymphocytes
compared with wild-type cells. A ratio of 1 is reasonably approximated
in spleens at all times in all recipients, irrespective of the
recipients’ genetic background (Fig. 5).
Peripheral lymph node ratios are generally lower at the early time
point (2 h) but are then (over)compensated later on: this suggests a
delayed entry of CD44 homozygous mutant cells into peripheral lymph
nodes, supporting the proposed functional role of CD44 in lymph node
homing.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This work demonstrates that complete loss of CD44 from the very beginning has no overt effect on mouse development, viability, and differentiation of tissues and organs. These results are unexpected, because it was suggested from previous studies (33, 32, 35, 42) that the loss of CD44 would interfere with normal embryonic and adult development. In addition, former reports display evidence for a role of CD44 during tumor development (43, 44, 45, 46), whereas our data indicate that total loss of CD44 information in mice has no effect on tumorigenicity over an observation period of more than 2 yr. Mice were checked frequently by autopsy and by occasional histological examination of specific organs. Despite the lack of any overt phenotype, the CD44 homozygous mutant mice described in this report show various changes in the lymphoid system.
Lymphoid organs deficient in CD44 develop normally but lymphocyte trafficking is impaired
No deviations of T cell subsets both in number and phenotype were detected in any lymphoid organs of adult CD44-deficient mice. In addition, fetal CD44-deficient thymus displayed no gross abnormalities in cell yield or cell subset distribution. However, an interesting phenomenon was the loss of a small, transient, CD25low+ population in both CD4+ and CD8+ cells in the thymii of CD44 homozygous mutant embryos. Light scatter properties suggested that these CD4/8+CD25low+ cells, which are absent in deficient mice, may undergo apoptosis, a well-characterized, fundamental process in T cell homeostasis. The fact that this subset difference was specifically detected only by the anti-IL-2R (CD25) mAbs may also favor this possibility, because IL-2 and IL-2R are thought to be associated with the deletion of lymphoid cells (47). We thus speculate that CD44 influences the apoptotic deletion of immature T cells. Along this line, it is also quite intriguing that in IL-2R-deficient (CD25 homozygous mutant) mice a consistent up-regulation of CD44 has been documented, accompanied with enlargement of various lymphoid compartments (48).
Although CD44-deficient mice develop no deviations of T cell subsets in comparison with heterozygous or wild-type mice, lymphocyte trafficking of reintroduced CD44-deficient T cells into the thymus is impaired, irrespective of the recipients’ genetic background. In CD44 homozygous mutant animals, T cell precursors may have no problems in colonizing the thymus during embryonal development, but upon reinjection into adult mice, CD44-/- cells encounter a selective disadvantage in comparison with wild-type cells. These results support the notion of CD44 as a receptor for lymphocyte homing, a process that is orchestrated by cooperation with several other molecules, for example L-selectin, ICAM-1, and LFA-1 (49, 50). In wild-type T cells, both CD44 and the integrin LFA-1 have been reported to function as adhesion molecules and costimulators of resting T cells. Because Abs against LFA-1 reduce the migration capability of lymphocytes to lymph nodes by 40–60%, and CD44-mediated lymphocyte adhesion has been shown to be triggered by interaction with LFA-1 (50, 51), we propose a mechanism in which CD44 and LFA-1 cooperate for efficient homing of lymphocytes, although each molecule alone is sufficient to conduct the process at a reduced level in the absence of the other. Accordingly, we attribute the reduction of homing capability of CD44-deficient T cells to the suspended interaction between CD44 and LFA-1.
Putative substitute molecules for CD44 are required to bind to HA and fibronectin
Because CD44 seems to act through different binding interactions,
different mechanisms for CD44 substitution are conceivable. First, in
either cell migration or pattern forming and organ differentiation
processes in embryonic development, CD44 has been demonstrated to
interact through hyaluronate binding. Second, lymphocytes have been
described as using CD44 to bind to fibronectin but not to HA
(52). Furthermore, in addition to CD44,
4ß1 integrin-mediated
adhesion of colony-forming cells to a heparin-binding domain of
fibronectin can be blocked by anti-CD44 Abs, suggesting that
4ß1 integrin and CD44
cooperate in establishing progenitor cell adhesion (53).
Thus, compensation of the homing function of the CD44 molecule could be
achieved in its absence by the described adhesion molecules. However,
our lymphocyte homing experiments demonstrate that the proposed
compensation does not occur to a full extent: in a competitive
situation between homozygous mutant and wild-type CD44 cells, the
wild-type cells possess a clear advantage in homing capability over the
mutant cells, at least when the targets are the thymus or the
peripheral lymph nodes.
Putative CD44 substitutes are not only presumed to interact with fibronectin, but also with HA, as, for example, lectin, aggrecan, selectin, and link protein do (54, 16), supposedly via their B(X7)B motif (16). This motif is predicted to be a minimal binding requirement for HA and occurs in all HA binding proteins described to date. Whether candidate substitutes for the CD44 function contain both kinds of binding specificities (the one to HA and the one to fibronectin) or whether different individual molecules would overtake the respective functions remains to be determined. Detection of the identity of the putative proteins substituting for CD44 function and comparison of their expression pattern and role in developmental and differentiation processes with that of CD44 would be interesting to provide important new insights into the action of CD44 and its participation in interaction networks.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Frank Hilberg, Boehringer Ingelheim, R&D Vienna, 1121 Vienna, Austria. E-mail address:
3 Abbreviations used in this paper: HA, hyaluronic acid; ES cells, embryonic stem cells; neo, neomycin resistence gene; lacZ, ß-galactosidase gene.
Received for publication December 28, 1998. Accepted for publication August 11, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
4ß1 and the CD44 adhesion receptor. Blood 15:1802.
This article has been cited by other articles:
|
|
 |
|
  M. Rajasagi, M. Vitacolonna, B. Benjak, R. Marhaba, and M. Zoller CD44 promotes progenitor homing into the thymus and T cell maturation J. Leukoc. Biol., February 1, 2009; 85(2): 251 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kawana, H. Karaki, M. Higashi, M. Miyazaki, F. Hilberg, M. Kitagawa, and K. Harigaya CD44 Suppresses TLR-Mediated Inflammation J. Immunol., March 15, 2008; 180(6): 4235 - 4245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Huebener, T. Abou-Khamis, P. Zymek, M. Bujak, X. Ying, K. Chatila, S. Haudek, G. Thakker, and N. G. Frangogiannis CD44 Is Critically Involved in Infarct Healing by Regulating the Inflammatory and Fibrotic Response J. Immunol., February 15, 2008; 180(4): 2625 - 2633. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Matzke, V. Sargsyan, B. Holtmann, G. Aramuni, E. Asan, M. Sendtner, G. Pace, N. Howells, W. Zhang, H. Ponta, et al. Haploinsufficiency of c-Met in cd44 / Mice Identifies a Collaboration of CD44 and c-Met In Vivo Mol. Cell. Biol., December 15, 2007; 27(24): 8797 - 8806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Scimone, I. Aifantis, I. Apostolou, H. von Boehmer, and U. H. von Andrian A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus PNAS, May 2, 2006; 103(18): 7006 - 7011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ori, M. Nardini, P. Casini, R. Perris, and I. Nardi XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development Development, February 15, 2006; 133(4): 631 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Walter, J. S. Park, R. E. Ries, S. K. M. Lau, M. McLellan, S. Jaeger, R. K. Wilson, E. R. Mardis, and T. J. Ley Reduced PU.1 expression causes myeloid progenitor expansion and increased leukemia penetrance in mice expressing PML-RAR{alpha} PNAS, August 30, 2005; 102(35): 12513 - 12518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Mochizuki, K. Ojima, A. Uezumi, S. Masuda, K. Yoshimura, and S. Takeda Participation of Bone Marrow-Derived Cells in Fibrotic Changes in Denervated Skeletal Muscle Am. J. Pathol., June 1, 2005; 166(6): 1721 - 1732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Katayama, A. Hidalgo, J. Chang, A. Peired, and P. S. Frenette CD44 is a physiological E-selectin ligand on neutrophils J. Exp. Med., April 18, 2005; 201(8): 1183 - 1189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hayer, G. Steiner, B. Gortz, E. Reiter, M. Tohidast-Akrad, M. Amling, O. Hoffmann, K. Redlich, J. Zwerina, K. Skriner, et al. CD44 is a determinant of inflammatory bone loss J. Exp. Med., March 21, 2005; 201(6): 903 - 914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Bohlson, M. Zhang, C. E. Ortiz, and A. J. Tenner CD93 interacts with the PDZ domain-containing adaptor protein GIPC: implications in the modulation of phagocytosis J. Leukoc. Biol., January 1, 2005; 77(1): 80 - 89. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nedvetzki, E. Gonen, N. Assayag, R. Reich, R. O. Williams, R. L. Thurmond, J.-F. Huang, B. A. Neudecker, F.-S. Wang, E. A. Turley, et al. RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: A different interpretation of redundancy PNAS, December 28, 2004; 101(52): 18081 - 18086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mukhopadhyay, A. Asari, M. S. Rugg, A. J. Day, and C. Fulop Specificity of the Tumor Necrosis Factor-induced Protein 6-mediated Heavy Chain Transfer from Inter-{alpha}-trypsin Inhibitor to Hyaluronan: IMPLICATIONS FOR THE ASSEMBLY OF THE CUMULUS EXTRACELLULAR MATRIX J. Biol. Chem., March 19, 2004; 279(12): 11119 - 11128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sumoza-Toledo and L. Santos-Argumedo The spreading of B lymphocytes induced by CD44 cross-linking requires actin, tubulin, and vimentin rearrangements J. Leukoc. Biol., February 1, 2004; 75(2): 233 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Fieber, P. Baumann, R. Vallon, C. Termeer, J. C. Simon, M. Hofmann, P. Angel, P. Herrlich, and J. P. Sleeman Hyaluronan-oligosaccharide-induced transcription of metalloproteases J. Cell Sci., January 15, 2004; 117(2): 359 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Donskoy, D. Foss, and I. Goldschneider Gated Importation of Prothymocytes by Adult Mouse Thymus Is Coordinated with Their Periodic Mobilization from Bone Marrow J. Immunol., October 1, 2003; 171(7): 3568 - 3575. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Louis, G. Dulude, S. Corneau, S. Brochu, C. Boileau, C. Meunier, C. Cote, N. Labrecque, and C. Perreault Changes in the lymph node microenvironment induced by oncostatin M Blood, August 15, 2003; 102(4): 1397 - 1404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Weng, D. R. Van Bockstaele, J. Wauters, E. Van Marck, J. Plum, Z. N. Berneman, and J. Merregaert A Novel Alternative Spliced Chondrolectin Isoform Lacking the Transmembrane Domain Is Expressed during T Cell Maturation J. Biol. Chem., May 23, 2003; 278(21): 19164 - 19170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Orian-Rousseau, L. Chen, J. P. Sleeman, P. Herrlich, and H. Ponta CD44 is required for two consecutive steps in HGF/c-Met signaling Genes & Dev., December 1, 2002; 16(23): 3074 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mu, S. Cambier, L. Fjellbirkeland, J. L. Baron, J. S. Munger, H. Kawakatsu, D. Sheppard, V. C. Broaddus, and S. L. Nishimura The integrin {alpha}v{beta}8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-{beta}1 J. Cell Biol., April 29, 2002; 157(3): 493 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Tammi, A. J. Day, and E. A. Turley Hyaluronan and Homeostasis: A Balancing Act J. Biol. Chem., February 8, 2002; 277(7): 4581 - 4584. [Full Text] [PDF]
|
|
|
|
|
|
O. Christ, U. Gunthert, D.-S. Schmidt, and M. Zoller Allogeneic reconstitution after nonmyeloablative conditioning: mitigation of graft-versus-host and host-versus-graft reactivity by anti-CD44v6 J. Leukoc. Biol., January 1, 2002; 71(1): 33 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Christ, U. Günthert, R. Haas, and M. Zöller Importance of CD44v7 isoforms for homing and seeding of hematopoietic progenitor cells J. Leukoc. Biol., March 1, 2001; 69(3): 343 - 352. [Abstract] [Full Text]
|
|
|
|
|
|
L. S. Sherman, T. A. Rizvi, S. Karyala, and N. Ratner Cd44 Enhances Neuregulin Signaling by Schwann Cells J. Cell Biol., September 4, 2000; 150(5): 1071 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Prevo, S. Banerji, D. J. P. Ferguson, S. Clasper, and D. G. Jackson Mouse LYVE-1 Is an Endocytic Receptor for Hyaluronan in Lymphatic Endothelium J. Biol. Chem., May 25, 2001; 276(22): 19420 - 19430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Mu, S. Cambier, L. Fjellbirkeland, J. L. Baron, J. S. Munger, H. Kawakatsu, D. Sheppard, V. C. Broaddus, and S. L. Nishimura The integrin {alpha}v{beta}8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-{beta}1 J. Cell Biol., April 29, 2002; 157(3): 493 - 507. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) was inserted into the
NcoI site, located at the translation initiation start
site in exon 1 (
). 
, Genomic sequences of CD44 integrated into
the targeting vector. The probe used to detect homologous recombination
at this locus is the indicated 5' 0.75-kb
EcoRI/EcoRV fragment (
). pBS,
pBluescript II KS(+); TK, thymidinkinase gene of vector pMC1/TK.
Restriction sites: RI, EcoRI; RV, EcoRV;
N, NcoI; X, XbaI; S, SacI.
B, Southern blot analysis of genomic DNA of CD44
wild-type, heterozygous, and homozygous mutant mice digested with
EcoRI and hybridized with the 5' 0.75-kb
EcoRI/EcoRV probe. In CD44 wild-type
mice, a 15-kb fragment is detected; the correct targeting of the gene
is represented by the 5-kb EcoRI fragment.
C, Northern blot analysis of CD44 wild-type,
heterozygous, and homozygous mutant embryos of E13.5 of gestation,
using a 580-bp s1 to s5 standard cDNA probe. CD44 mRNA of 4.0 kb is
observed in wild-type embryos and, at a reduced level, in heterozygous
mutant embryos but not in homozygous mutant embryos. GAPDH
hybridization is shown as a measure for the amount of RNA in the
samples.
