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Proteins CD44 Requirements for Signal Delivery Through CD44: Analysis Using CD44-Fas Chimeric Proteins1 Haruko Ishiwatari-Hayasaka,2,3* Takashi Fujimoto,2* Tomoko Osawa,2* Toshiyasu Hirama,4* Noriko Toyama-Sorimachi,† and Masayuki Miyasaka5* CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemo- poiesis, and tumor metastasis. To examine the requirements of CD44 for signal delivery through the extracellular domain, we constructed a chimeric CD44 protein fused to the intracellular domain of Fas on its C-terminus. In cells expressing the CD44-Fas fusion protein, apoptosis could be induced by treatment with certain anti-CD44 mAbs alone, especially those recognizing the epitope group d, which has been previously shown to play a role in ligand binding, indicating that ligation of a specific region of the CD44 extracellular domain results in signal delivery. Of note was that appropriate ligation of the epitope h also resulted in the generation of apoptotic signal, although this region was not thought to be involved in ligand binding. In contrast, the so-called blocking anti-CD44 mAbs (epitope group f) that can abrogate the binding of hyaluronate (HA) failed to induce apoptosis even after further cross-linking with the secondary Ab, indicating that a mere mAb-induced oligomerization of the chimeric proteins is insufficient for signal generation. However, these blocking mAbs were instead capable of inhibiting apoptosis induced by non- blocking mAb (epitope group h). Furthermore, a chimeric protein bearing a mutation in the HA binding domain and hence lacking the ability to recognize HA was incapable of mediating the mAb-induced apoptosis, suggesting that the functional integrity of the HA binding domain is crucial to the signal generation in CD44. The Journal of Immunology, 1999, 163: 1258–1264. D44 is a versatile adhesion molecule implicated in var- extracellular matrix (ECM) components such as collagen or fi- ious cell traffic events, such as migration of activated T bronectin (11, 12). Thus, it is suggested that one of the important C cells into sites of inflammation and distant metastasis of biological functions of CD44 is acting as a matrix receptor that tumor cells (1–3). CD44 has an N-terminal link module involved mediates cell adhesion to ECM (1). in the binding to hyaluronic acid (HA)6 (4, 5), and multiple lines On the other hand, CD44 can interact with ligands apparently of evidence indicate that recognition of HA is important in various unrelated to ECM, such as serglycin (13, 14), osteopontin (15), and CD44-mediated cellular events. For instance, early lymphopoiesis the MHC class II invariant chain (16). Of interest, serglycin en- is inhibited by anti-CD44 mAbs and also by treatment with hyal- hances CD3-dependent granzyme A release of CTL clones (13), by guest on October 1, 2021. Copyright 1999 Pageant Media Ltd. uronidase (6, 7). CD44-negative cell lines transfected with CD44 suggesting that non-HA ligands can also signal through CD44. cDNA become adherent to high endothelial cells of lymph nodes The CD44-mediated signaling from cell membrane to the nu- or an endothelial cell line, of which binding is sensitive to hyal- cleus was initially shown experimentally using anti-CD44 mAbs. uronidase (8, 9). Soluble CD44-Ig fusion protein binds to high Engagement of CD44 with specific mAbs induces proliferation of endothelial cells of lymph nodes, which is sensitive to hyaluron- T cells when coincubated with anti-CD2 or anti-CD3 mAbs (17– idase and blocked by soluble HA (10). Activation of T cells in- 19). Similarly, stimulation of CD44 with mAbs enhances the cy- creases their binding to HA and enables CD44-mediated lympho- totoxic activity of NK cells or CTL (20, 21) and induces the release cyte rolling (3). In addition to HA, CD44 can interact with other of TNF-a and IL-1b in monocytes (22). However, various anti- http://classic.jimmunol.org CD44 mAbs that recognize different epitopes can apparently stim- ulate CD44-positive cells, although it is not clear at present which *Department of Bioregulation, Biomedical Research Center, Osaka University Grad- domain(s) of CD44 should be stimulated and what exactly leads to uate School of Medicine, Suita, Japan; and †Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan signal delivery through this interesting multifunctional molecule. Received for publication April 20, 1998. Accepted for publication May 20, 1999. To further understand the precise requirements for CD44-medi- The costs of publication of this article were defrayed in part by the payment of page ated signaling, we expressed on a lymphoid cell line, the chimeric charges. This article must therefore be hereby marked advertisement in accordance CD44 proteins consisting of the extracellular (EC) domain of Downloaded from with 18 U.S.C. Section 1734 solely to indicate this fact. CD44 and the intracellular (IC) domain of a death-inducing mol- 1 This work was supported by a grant-in-aid from the Ministry of Education, Science, ecule, Fas, which transduce apoptosis signals into cells upon ap- and Culture, Japan, and a grant from the Science and Technology Agency, Japan. propriate cross-linking of the extracellular region. We then inves- 2 H.I.-H., T.F., and T.O. contributed equally to the completion of this work. tigated the important sites for signal delivery in the CD44 EC 3 Current address: Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Hon-Komagome, Bunkyo, Tokyo 113-8613, Japan. domain using apoptosis as a parameter of CD44-mediated signal- 4 Current address: National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage- ing, subsequent to stimulation with a variety of mAbs against dif- ku, Chiba City 263-8555, Japan. ferent epitopes of the CD44 EC domain. We have previously used 5 Address correspondence and reprint requests to Dr. Masayuki Miyasaka, Depart- a similar strategy to demonstrate that stimulation through the lectin ment of Bioregulation, Biomedical Research Center, Osaka University Graduate domain is crucial to signal generation in a leukocyte adhesion mol- School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail address: [email protected] ecule, L-selectin (23). The results of the present study indicate that 6 Abbreviations used in this paper: HA, hyaluronic acid; ECM, extracellular matrix; not only the HA binding domain but also another non-HA binding EC, extracellular; IC, intracellular; TM, transmembrane; POD, peroxidase. domain play a critical role in signal generation in CD44. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1259 Materials and Methods Table I. Isotypes and epitope specificities of anti-CD44 mAbs used in Construction of CD44-Fas fusion protein this study Full-length murine CD44H cDNA (24) was inserted into a mammalian Epitope Induction of Effect on HA expression vector pEF-BOS (25) to construct a plasmid encoding wild-type CD44 mAbs Isotypes Specificitiesa Apoptosis Bindinga CD44. A recombinant plasmid, 44/F-4, encoding the EC domain, the trans- membrane (TM) domain, and most of the IC domain of murine CD44 IRAWB14.4 IgG2a d 1 11 (nucleotides 1–1092) and the IC domain of human Fas, was constructed as RAWB45.106.2 IgM d 1 2 follows. First, we introduced an NheI site in the multicloning site of the KM201 IgG1 f 2 2 pUC119 vector. Then a DNA fragment encoding the full-length CD44 or KM114 IgG1 f 2 2 Fas (26) was inserted into the modified pUC119 to construct pUCcd44 IM7.8.1 IgG2b h 1b 3 or pUCfas, respectively. The fragment encoding the IC domain of Fas R7 166.7 IgM h 1 1 (nucleotides 756-1427) was amplified by PCR by using pUCfas as the RAMBM5.5.8 IgM h 1 3 template. The PCR primers used were 59-TGGCTGCAGGTTTGGGT KM703 IgG2a h 1 (2)c GAAGAGAAAG-39 containing a PstI site at the 59 terminus and 59- 9 a The data are based on the analysis presented in the article by Zheng et al. (29). TACTTAGCATGCCACTGCATT-3 containing an SphI site. The result- b ant PstI-SphI fragment was ligated with pUC119 (pUCfasPCR). A KpnI- Apoptosis was examined in the presence of a secondary Ab. c This effect depends on types of the cell expressing CD44. 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. treated with 20 mg/ml of each Ab for 10 min. After pretreatment, cells were The 44/F-4RA plasmid was prepared by modifying 44/F-4. For this incubated for 5 h with 5 mg/ml R7 166.7 or overnight with KM703. To purpose, a fragment encoding the EC domain of CD44 (nt 1–827) con- 23 examine HA-induced cell death, cells were incubated for 17 h with 100 taining the Arg Ala mutation was amplified by two-step PCR (amino acid m 23 g/ml human umbilical cord HA (ICN, Costa Mesa, CA) and 5 ng/ml numbers used here are those of mature murine protein; thus, Arg corre- PLUS 41 IRAWB14.4. Cell death was detected by Cell Death Detection ELISA sponds to Arg in man) (27). First, two sets of reaction were performed (Boehringer Mannheim, Mannheim, Germany), which detects mono- and using pUC44/F-4 as a template. The following primers were used (1): oligonucleosomes in cell lysates using mouse mAbs directed against DNA 59-GCGCGGTACCCCGAATTC-39 containing a KpnI site at the 59 ter- 9 9 9 and histones, respectively.
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