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Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Japan; and
Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, 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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On the other hand, CD44 can interact with ligands apparently unrelated to ECM, such as serglycin (13, 14), osteopontin (15), and the MHC class II invariant chain (16). Of interest, serglycin enhances CD3-dependent granzyme A release of CTL clones (13), suggesting that non-HA ligands can also signal through CD44.
The CD44-mediated signaling from cell membrane to the nucleus was
initially shown experimentally using anti-CD44 mAbs. Engagement of
CD44 with specific mAbs induces proliferation of T cells when
coincubated with anti-CD2 or anti-CD3 mAbs
(17, 18, 19). Similarly, stimulation of CD44 with mAbs
enhances the cytotoxic activity of NK cells or CTL (20, 21) and induces the release of TNF-
and IL-1
in monocytes
(22). However, various anti-CD44 mAbs that recognize
different epitopes can apparently stimulate CD44-positive cells,
although it is not clear at present which domain(s) of CD44 should be
stimulated and what exactly leads to signal delivery through this
interesting multifunctional molecule.
To further understand the precise requirements for CD44-mediated signaling, we expressed on a lymphoid cell line, the chimeric CD44 proteins consisting of the extracellular (EC) domain of CD44 and the intracellular (IC) domain of a death-inducing molecule, Fas, which transduce apoptosis signals into cells upon appropriate cross-linking of the extracellular region. We then investigated the important sites for signal delivery in the CD44 EC domain using apoptosis as a parameter of CD44-mediated signaling, subsequent to stimulation with a variety of mAbs against different epitopes of the CD44 EC domain. We have previously used a similar strategy to demonstrate that stimulation through the lectin domain is crucial to signal generation in a leukocyte adhesion molecule, L-selectin (23). The results of the present study indicate that not only the HA binding domain but also another non-HA binding domain play a critical role in signal generation in CD44.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Full-length murine CD44H cDNA (24) was inserted into a mammalian expression vector pEF-BOS (25) to construct a plasmid encoding wild-type CD44. A recombinant plasmid, 44/F-4, encoding the EC domain, the transmembrane (TM) domain, and most of the IC domain of murine CD44 (nucleotides 1–1092) and the IC domain of human Fas, was constructed as follows. First, we introduced an NheI site in the multicloning site of the pUC119 vector. Then a DNA fragment encoding the full-length CD44 or Fas (26) was inserted into the modified pUC119 to construct pUCcd44 or pUCfas, respectively. The fragment encoding the IC domain of Fas (nucleotides 756-1427) was amplified by PCR by using pUCfas as the template. The PCR primers used were 5'-TGGCTGCAGGTTTGGGTGAAGAGAAAG-3' containing a PstI site at the 5' terminus and 5'-TACTTAGCATGCCACTGCATT-3' containing an SphI site. The resultant PstI-SphI fragment was ligated with pUC119 (pUCfasPCR). A KpnI-PstI fragment containing the nucleotide sequence 1–1092 of CD44 was isolated from pUCcd44 and transferred to pUCfasPCR at KpnI-PstI sites. Then a chimeric fragment consisting of nt 1–1092 of CD44 and 756-1427 of Fas was isolated by digestion with NheI and XbaI, and inserted into pEF-BOS.
The 44/F-4RA plasmid was prepared by modifying 44/F-4. For this purpose, a fragment encoding the EC domain of CD44 (nt 1–827) containing the Arg23Ala mutation was amplified by two-step PCR (amino acid numbers used here are those of mature murine protein; thus, Arg23 corresponds to Arg41 in man) (27). First, two sets of reaction were performed using pUC44/F-4 as a template. The following primers were used (1): 5'-GCGCGGTACCCCGAATTC-3' containing a KpnI site at the 5' terminus and 5'-TTTTTACCGCGGATGTCATAG-3' (2), 5'-AAAAATGGCGCCTACAGTATC-3', and 5'-CACTGAGTACCTAGGCTT-3' containing a BamHI site at the 3' terminus (mutated nucleotides are underlined). In the next reaction, products of the first PCR were used as primers, and pUC44/F-4 was used as the template. The amplified KpnI-BamHI fragment with the Arg23Ala mutation was then exchanged with corresponding KpnI-BamHI fragment in 44/F-4. All constructs were verified by DNA sequencing.
Cells
A murine thymoma cell line AKR1 (28) was maintained in DMEM containing 10% FCS (Dainippon Pharmaceutical, Osaka, Japan), 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% (v/v) 100x nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium). To prepare clones that express CD44 or CD44-Fas fusion protein, the recombinant plasmid together with pSV2bsr carrying blasticidin S resistance gene were introduced into AKR1 cells by electroporation. Transfectants were selected in complete medium containing 10 µg/ml blasticidin S (Funakoshi, Tokyo, Japan), and drug-resistant cells were cloned by limiting dilution. The surface expression of CD44 or CD44-Fas fusion protein in each clone was examined by indirect immunofluorescence staining with anti-mouse CD44 mAb IRAWB14.4 (9, 29) and FITC-conjugated goat anti-rat (IgG+IgM) (Southern Biotechnology Associates, Birmingham, AL), followed by flow cytometry on an EPICS XL flow cytometer (Coulter, Hialeah, FL).
Monoclonal Abs
Table I
summarizes the
anti-CD44 mAbs used in this study and their specificities. Rat
anti-mouse CD44 mAbs IRAWB14.4, KM201, KM703 (6), and
IM7.8.1 (30) and anti-mouse MAdCAM-1 mAb MECA-367
(IgG2a) (31) were purified by protein G-Sepharose
(Pharmacia Biotech, Uppsala, Sweden). RAMBM5.5.8 and R7 166.7
(32) were purified from hybridoma culture supernatant
using the E-Z-SEP Ab purification kit (Pharmacia Biotech). Purified
RAWB45.106.2 and KM114 were provided by Dr. Jayne Lesley (The Salk
Institute, La Jolla, CA) and Dr. Kensuke Miyake (Saga University
Medical School, Saga, Japan), respectively. Anti-CD3 mAb 145-2C11
(33) free of endotoxin was purchased from PharMingen (San
Diego, CA).
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For induction of apoptosis, 1 x 104 cells were seeded into each well of a 96-well microtiter plate in complete medium containing 5 µg/ml anti-CD44 mAb with or without 5 µg/ml goat anti-rat (IgG+IgM) (Southern Biotechnology Associates) and incubated for 5 h. As a control for Ab treatment, cells were incubated in complete medium containing 5 µg/ml MECA-367. To examine the effect of KM201 or KM114, cells were pretreated with 20 µg/ml of each Ab for 10 min. After pretreatment, cells were incubated for 5 h with 5 µg/ml R7 166.7 or overnight with KM703. To examine HA-induced cell death, cells were incubated for 17 h with 100 µg/ml human umbilical cord HA (ICN, Costa Mesa, CA) and 5 ng/ml IRAWB14.4. Cell death was detected by Cell Death Detection ELISAPLUS (Boehringer Mannheim, Mannheim, Germany), which detects mono- and oligonucleosomes in cell lysates using mouse mAbs directed against DNA and histones, respectively. Briefly, cells were lysed, and the lysates were placed into a streptavidin-coated plate. Subsequently, biotin-labeled anti-histone mAb and peroxidase (POD)-labeled anti-DNA mAb were added and incubated for 2 h. After washing the wells to remove unbound Abs, we quantified the amount of nucleosomes derived from apoptotic cells. This represented the amount of POD-anti-DNA mAb bound to the DNA component of the nucleosomes. POD was determined with its substrate ABTS (2, 2'-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] by photometric analysis measured at 405 nm.
Fluorescein-HA binding assay
Cells were incubated with or without 40 µg/ml KM201 for 20 min at 4°C. After incubation, cells were washed twice with PBS and incubated with 50 µg/ml fluorescein-conjugated HA (Seikagaku Kogyo, Tokyo, Japan) dissolved in PBS for 20 min at 4°C. Cells were then washed and analyzed by flow cytometry on an EPICS XL flow cytometer (Coulter).
T cell proliferation assays
Proliferation assays were conducted as previously described (34). Briefly, surface Ig- and Ia- T cells were obtained from lymph node of BALB/c mice by immunomagnetic negative selection. The purity of the resulting cell population as assessed by flow cytometry was >97% CD3+. Each anti-CD44 mAb (5 µg/ml) was mixed with the anti-CD3 Ab (2.5 µg/ml) and immobilized onto 96-well microculture plates. T cells were cultured in complete medium at 1 x 105 cells/well for 2 days, and then harvested after an 8-h pulse with 0.5 µCi of tritium-labeled thymidine.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first step we constructed plasmids encoding CD44 and the IC
domain of Fas (Fig. 1A). In
the 44/F-4 plasmid, the coding sequence of CD44 including EC, TM, and
nearly the entire IC domains (aa 1–332) was fused to that of the IC
domain of Fas. The 44/F-4RA plasmid was identical with the 44/F-4
plasmid, except that it had a single amino acid substitution of
arginine 23 (amino acid numbers used in this study refer to those of
mature protein) to alanine. This substitution corresponds to the
mutation at arginine 41 in human CD44, which destroys the B(X7)B motif
in the HA binding domain and thus abrogates the ability of CD44 to
recognize HA (27). These plasmids or plasmid encoding the
wild-type CD44 were transfected into CD44-negative murine thymoma AKR1
cells, and multiple clones expressing the corresponding proteins,
namely, wild-type CD44 (A-CD44WT), 44/F-4 (A44/F-4), or 44/F-4RA
(A44/F-4RA) at high levels were obtained. Representative results are
shown in Fig. 1B.
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We have previously reported that a chimeric adhesion molecule,
L-selectin-Fas generated as described above, transduces apoptotic
signals in cells upon ligation of a functional domain of the EC region
with mAb (23). Similarly, as shown in Fig. 2, ligation of the EC domain of CD44-Fas
with HA in conjunction with stimulation with a low dose (5 ng/ml) of
anti-CD44 mAb IRAWB14.4 that enhances HA binding (9, 29) induced apoptosis in cells, indicating that the CD44-Fas
protein can also transduce signals upon appropriate stimulation.
Therefore, we decided to use this strategy to investigate the
functional sites involved in signal transduction in the EC region of
CD44. To this end, AKR1 cells expressing the 44/F-4 chimeric protein
(A44/F-4) or the wild-type CD44 (A-CD44WT) were treated with various
anti-mouse CD44 mAbs with different epitope specificities
(29) and then examined to determine whether the cells
underwent apoptosis. The isotype and epitope specificity of each mAb
are shown in Table I
. Preliminary experiments indicated that these mAbs
bound to both the wild-type CD44 and CD44-Fas fusion proteins equally
well (data not shown) and thus were considered to provide a suitable
means to stimulate transfectant cells to induce apoptosis. As shown in
Fig. 3, six of the eight anti-CD44
mAbs tested induced apoptosis of A44/F-4 cells but not A-CD44WT cells,
indicating that the apoptotic effect was specifically mediated by the
chimeric CD44-Fas protein. Introduction of a stop codon in the coding
sequence of the death domain of Fas abrogated apoptosis (data not
shown), supporting the idea that the CD44-Fas chimeric protein
transduced apoptosis through Fas upon ligation of the CD44 EC
domain.
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), indicating that the HA
binding-enhancing effect was not a major factor for induction of
apoptosis in CD44-Fas chimera-expressing cells. With RAWB45.106.2,
KM703, and RAMBM5.5.8, apoptosis was enhanced by further ligation with
a secondary Ab (goat anti-mouse Ig), suggesting that the efficiency
of apoptosis might be determined in part by the extent of cross-linking
of CD44-Fas by these mAbs. Interestingly, two groups of mAbs, one
recognizing the epitope group d (IRAWB14.4, RAWB45.106.2) and the other
recognizing the epitope group h (KM703, RAMBM 5.5.8, R7 166.7,
IM7.8.1), were uniformly capable of inducing apoptosis, and all mAbs
except IM7.8.1 in these groups induced apoptosis with the primary mAb
alone. IM7.8.1 also induced apoptosis once sufficient cross-linking was
established with the secondary Ab. Whereas the epitope group d is
thought to be involved in ligand binding, epitope group h is not
involved in HA binding (29). In contrast, both blocking
mAbs KM201 (IgG1) and KM114 (IgG1) that block HA binding failed to
induce apoptosis with the primary mAb alone and induced little
apoptosis, if any, with the secondary Ab. Both mAbs recognize a
particular region (epitope group f) of the CD44 link module and thus
block the binding to HA, although neither of them directly binds to the
B(X7)B motif critical for HA binding (29). Inhibition of mAb-induced apoptosis via CD44-Fas by blocking mAbs against CD44
Although neither KM201 nor KM114 was capable of inducing apoptosis
through the CD44-Fas chimeric protein despite the fact that these mAbs
are known to recognize the functional domain of CD44 (29),
it is of note that these mAbs were instead capable of inhibiting
apoptosis induced by nonblocking anti-CD44 mAbs. As shown in Fig. 4, KM201 inhibited KM703-induced
apoptosis of A44/F-4 cells by 
70%, while R7 166.7-induced apoptosis
by 80%, although KM201 did not inhibit binding of mAb KM703 or R7
166.7 to the chimeric CD44-Fas (data not shown). Similar inhibitory
effects were observed with another blocking mAb KM114.
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Inhibition of apoptosis by a HA-binding blocking mAb KM201 or
KM114 observed above (Fig. 4) suggests that the HA binding domain of
CD44 is involved in the signal generation. Therefore, we expressed in
AKR1 cells a mutant CD44/Fas protein, 44/F-4RA, which bears the
Arg23Ala mutation in the link domain. This
mutation has been reported previously to completely abrogate HA binding
of CD44 (27). As shown in Fig. 5A, 44/F-4RA-expressing cells
(A44/F-4RA) failed to show HA binding, whereas 44/F-4-expressing cells
(A44/F-4) bound HA, albeit at slightly lower levels than AKR1 cells
that expressed the wild-type CD44 (A-CD44WT). We then examined whether
the mutant 44/F-4RA protein could transduce apoptotic signal upon
cross-linking with mAb. As shown in Fig. 5B, the 44/F-4RA
chimeric protein failed to mediate mAb-induced apoptosis with any of
the mAbs tested, even in the presence of a secondary Ab (data not
shown), suggesting that the functional integrity of the HA binding
domain of CD44-Fas is crucial to the transduction of apoptotic signal
into cells.
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The above results of the mapping of functional epitopes obtained
by the use of CD44-Fas chimeric proteins indicates that epitope h,
which has been thought to be uninvolved in ligand binding
(29), actually plays a role in signal generation. To
examine whether this observation can be extended to the intact CD44
molecule, we tested whether stimulation through this epitope leads to T
cell costimulation. Purified T cells were cultured for 2 days in
wells containing either of the anti-CD44 mAb coimmobilized with a
suboptimal dose of anti-CD3 mAb in the absence of APC and examined
for cell proliferation. As shown in Fig. 6, three of the four mAbs against
epitope group h (IM7, R7 166.7, RAMBM5.5.8) induced strong
proliferation of TCR-stimulated naive T cells, similar to
anti-CD28. KM703 recognizing the same epitope group induced much
lower, but significant, T cell costimulation. mAbs KM201 and KM114
against epitope group f also induced T cell costimulation in agreement
with the previous report (35). These results are in
agreement with those obtained with the chimeric CD44-Fas proteins, in
that epitope h plays a role in signal delivery.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The link domain of CD44, with a three-dimensional structure similar to the lectin domain of selectins (36), possesses at least five different epitope groups (d, e, f, g, and h) identified by the use of a panel of anti-CD44 mAbs (29), although the functional significance of these groups remains to be fully explored. In this study, we used CD44-Fas chimeric proteins to examine whether any EC domain(s) of CD44 is involved in signal generation. To stimulate a particular site of the CD44 EC domain, we used eight anti-CD44 mAbs recognizing three different epitope groups (d, f, and h). Our results showed that although these mAbs bound to CD44 and CD44-Fas equally well, ligation of the CD44-Fas chimeric receptor with mAbs that recognize epitope group d or h induced apoptosis, whereas mAbs against epitope group f did not, even in the presence of a secondary Ab. By contrast, ligation of wild-type CD44 with any of the mAbs tested did not induce apoptosis. These results suggest that stimulation through a specific site of CD44 is crucial for the generation of a signal and that at least two epitope groups (d and h) in the CD44 EC domain contribute to signal transduction.
The epitope group d is recognized by mAbs IRAWB14.4 and RAWB45.106.2 and spans eight amino acid residues (Asp46 to Ser53), among which Lys49 is apparently most important for Ag specificity (29). Because one of the mAbs belonging to this group (IRAWB14.4) has a potent enhancing effect on ligand binding by CD44, this epitope group is thought to be important in regulating the adhesive function of CD44 (29). Indeed, mutation of the lysine residue of human CD44, which is equivalent to Lys49 of the murine counterpart, has been shown to abrogate HA binding (37). Our observation that both IRAWB14.4 and RAWB45.106.2 generated apoptotic signal via CD44-Fas further indicates the importance of this epitope group. The finding that RAWB45.106.2, which does not enhance HA binding (29), also generated apoptotic signal suggests that the ligand binding-enhancing effect of mAbs does not necessarily correlate with their ability to generate a signal in CD44.
Epitope group h is dependent on two noncontiguous residues, namely
Pro108 and Thr113, which
are present in two neighboring regions interrupted by a disulfide bond
in the link domain of murine CD44 (29). Many of the mAbs
recognizing this region cross-react with human CD44, probably because
Pro108 is shared by mouse and human CD44
(29). Because most of these mAbs do not inhibit
hyaluronate recognition by CD44 expressed on the cell surface, it has
been suggested that epitope group h is not directly involved in ligand
binding (29). However, in the present study all four
mAbs in this group (R7 166.7, KM703, IM7.8.1, RAMBM5.5) generated
apoptotic signal in CD44-Fas-expressing cells in the presence of a
secondary Ab, and all but one (IM7.8.1) induced apoptosis by
themselves. Furthermore, stimulation of naive T cells through this
epitope induced potent costimulation (Fig. 6). These findings indicate
that this epitope actually plays a role in signal generation. In
agreement with this idea, IM7.8.1 has actually been shown to induce
shedding of CD44 (38, 39) and inhibit induction of murine
arthritis (39). In addition, preliminary results from our
laboratories have shown that at least three of the mAbs in this group
(R7 166.7, KM703, RAMBM5.5.8) can inhibit the binding of soluble
CD44-Ig chimera to hyaluronate (unpublished observations), confirming
the importance of this epitope group in ligand binding. That IM7.8.1
induced apoptosis only in the presence of a secondary Ab but not by
itself could mean that IM7.8.1 differs in fine specificity from other
mAbs of the same group. This conclusion was recently confirmed in a
series of preliminary studies in our laboratory showing that IM7.8.1
and KM703 cross-blocked each other only weakly (unpublished
observations). On the other hand, it is also possible that IM7.8.1 is
unable to induce a sufficient level of CD44 clustering unless further
cross-linked by a secondary Ab. Fas is known to exert its cytocidal
effect by forming a homotrimer (40), and it is expected
that sufficient oligomerization or cross-linkage of CD44-Fas is
necessary for generation of the apoptotic signal. Studies by others
indicate that receptor oligomerization can augment ligand recognition
in CD44 (1, 9, 41). Nevertheless, it is evident that a
mere Ab-induced receptor oligomerization is insufficient for signal
generation, because mAbs of epitope f (KM201, KM114) were completely
unable to induce apoptosis even in the presence of a secondary Ab
as discussed below. Clement and Stamenkovic (42) have
similarly observed, with CD40 fused to Fas or TNF receptor, that only
appropriate ligation via a functional site is critical for receptor
signaling.
Epitope group f is dependent on His83 or Val90/Thr91 (29) and recognized by hyaluronate blocking mAbs KM201 and KM114. Because this region is distantly located from the B(X7)B motifs known to be critical for HA recognition on the secondary structure (36), one possibility is that these blocking mAbs exert their effect not by blocking the ligand binding site but by altering conformation of CD44 so as to make hyaluronate recognition difficult, although further study is clearly required to verify this point. Substitution of a residue equivalent to His83 of mouse CD44 to alanine in human CD44 has been shown to abrogate HA binding (37). An unexpected observation in our study with mAbs recognizing this epitope group was that KM201 and KM114 blocking mAbs inhibited mAb-induced apoptosis by mAbs KM703 and R7 166.7 (both belong to group h), while they themselves failed to induce apoptosis. Because neither KM201 nor KM114 inhibited binding of KM703 to any extent (unpublished observations), this suppressive effect by KM201 or KM114 is certainly not attributable to inhibition of mAb binding. Rather, it may be that the ability to recognize hyaluronate has to be retained by CD44 so as to generate the signal. This is supported by our finding that the Arg23Ala CD44-Fas mutant without the ability to bind hyaluronate was completely unable to transduce apoptotic signal. Therefore, the functional integrity of the hyaluronate binding region may be critical for signal transduction by CD44. Clement and Stamenkovic (42) also prepared a chimeric human CD44-Fas molecule consisting of CD44 EC, Fas TM, and Fas IC domains, but were unsuccessful in generating apoptotic signal by mAb-induced cross-linking, although the specificity of the mAbs was not mentioned in their study. Failure of generation of the apoptotic signal in their study might have been due to the use of mAb blocking HA recognition.
Other than the three epitope groups examined in the present study, there is also another interesting site in CD44 that would merit functional analysis if appropriate mAb were available. It is an epitope recognized by mAb Hermes-3 (43) that can inhibit lymphocyte adhesion to high endothelial venules in mucosal lymphoid tissues in the absence of blocking ability to hyaluronate binding (44). However, no anti-mouse mAb against this epitope is available at present, and therefore, the functional importance of this epitope remains to be determined.
Finally, the present study reiterates the functional importance of the link domain of CD44 in receptor signaling and supports the idea that multiple regions in the link domain must function in concert for an effective ligand binding and signal generation. Our results also extend those of previous studies by showing that a site apparently uninvolved in ligand binding (epitope group h) is also important in signal generation. Elucidation of the role of this region will enhance our understanding of the complex regulatory mechanisms of ligand binding and signal transduction in CD44.
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Acknowledgments |
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Footnotes |
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2 H.I.-H., T.F., and T.O. contributed equally to the completion of this work.
3 Current address: Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Hon-Komagome, Bunkyo, Tokyo 113-8613, Japan.
4 Current address: National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba City 263-8555, Japan.
5 Address correspondence and reprint requests to Dr. Masayuki Miyasaka, Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail address:
6 Abbreviations used in this paper: HA, hyaluronic acid; ECM, extracellular matrix; EC, extracellular; IC, intracellular; TM, transmembrane; POD, peroxidase.
Received for publication April 20, 1998. Accepted for publication May 20, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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