[CANCER RESEARCH 59, 219 –226, January 1, 1999] CD44 Variants but not CD44s Cooperate with 1-containing Integrins to Permit Cells to Bind to Osteopontin Independently of Arginine-glycine-aspartic Acid, thereby Stimulating Cell Motility and Chemotaxis 1 Yohko U. Katagiri, Jonathan Sleeman, Hideki Fujii, Peter Herrlich, Hiroshi Hotta, Kumiko Tanaka, Shunsuke Chikuma, Hideo Yagita, Ko Okumura, Masaaki Murakami, Ikuo Saiki, Ann F. Chambers, and Toshimitsu Uede 2 Section of Immunopathogenesis, Institute of Immunological Science, Hokkaido University, Sapporo, 0600815, Japan [Y. U. K., H. H., K. T., S. C., M. M., T. U.]; Institute of Genetics, Forschungszentrum Karlsruhe, D-76021 Karlsruhe, Germany [J. S., P. H.]; Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, 9300194 Toyama, Japan [H. F., I. S.]; Department of Immunology, Juntendo University School of Medicine, 1138421 Tokyo, Japan [H. Y., K. O.]; and Division of Experimental Oncology, Department of Oncology, The London Regional Cancer Centre, Ontario, N6A 4L6, Canada [A. F. C.] ABSTRACT The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycine- aspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH- terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin 1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-1 antibodies. Finally, we show that OPN binding by CD44 variants/1-containing integrins promotes cell spreading, motil- ity, and chemotactic behavior. INTRODUCTION OPN 3 is a highly acidic calcium-binding glycosylated phosphopro- tein secreted by many cell types, including osteoblasts, osteocytes, kidney tubules, metrial gland cells of the decidium and placenta, macrophages, activated T cells, and vascular smooth muscle cells (1). OPN has been shown to support various functions including macro- phage chemotaxis, cell attachment, and cell migration (1–3). Re- cently, much interest has focused on the association of OPN with tumorigenesis and metastasis (4 – 6). OPN contains an RGD tripeptide sequence that can be bound by v3, v1, and v5 integrins on the cell surface (7, 8). The binding of integrins to OPN through the interaction with the RGD sequence results in distinct functional con- sequences. For example, in vascular smooth muscle cells, v3, v1 and v5 integrins mediate cell adhesion, whereas only the v3 integrin supports cell migration (9). The functional diversity of OPN can be also explained by the interaction of OPN with a cellular receptor(s) other than an v-containing integrin. For example, the 91 tenascin receptor is involved in the binding of human melanoma cells to the thrombin-cleaved amino-terminal half of human OPN (10). Additionally, we have previously shown that both human and murine OPN have domains that do not contain the RGD sequence, but which can be bound by cell surface receptors (11). Furthermore, it was recently demonstrated that OPN can interact with CD44 in an RGD- independent manner (12). CD44 has been implicated in a wide variety of cellular processes, including cell-cell and cell-matrix interactions, cell migration, metas- tasis, lymphocyte homing, and T cell activation (13–15). CD44 is encoded by a total of 20 exons, 7 of which form the invariant extracellular domain of the standard form (CD44s). The variant exons (v1-v10) are alternatively spliced within this invariant extracellular domain (16). The up-regulation of CD44 variant expression is asso- ciated with tumor progression in several tumor types (17). Functional differences between the isoforms are emerging. For example, certain CD44 variants have been shown to up-regulate binding to hyaluronic acid and other glycosaminoglycans, and to promote CD44 oligomer- ization (18 –20). In addition to binding to glycosaminoglycans, CD44 has been shown to bind to a range of other ligands (17). With regard to OPN binding, transfection of CD44v7-v10 into CD44-negative fibroblasts conferred on the transfected cells the ability to bind to OPN and HA, and this binding was clearly inhibited by exogenous OPN or HA (12). The transfected fibroblasts bound to OPN in an RGD-independent manner. Furthermore, OPN was able to induce chemotaxis in the transfected cells. This chemotaxis was inhibited by antibodies against OPN and CD44, suggesting that OPN is involved in CD44-mediated cell migration (12). However, the potential of differ- ent isoforms of CD44 to bind to OPN and induce chemotaxis has not been investigated. Integrins compose one of the major families of adhesion receptors and the heterodimeric association of different  and  subunits results in the formation of more than 20 integrin receptors. The different subunit combinations have remarkably different properties, being involved in processes as diverse as tissue development, angiogenesis, inflammation and the regulation of apoptosis (21). Furthermore, changes in integrin subunit composition are critically involved in tumorigenesis and metastasis (22, 23). Integrin activity is regulated by intracellular signaling, which can convert low affinity forms into high affinity forms. On the other hand, binding of integrin to ligand can result in signal transduction, which regulates other molecules includ- ing cytoskeletal organization (24, 25). We have previously reported that murine B16-F10 melanoma cells bound to OPN through interaction of the RGD tripeptide sequence within the OPN protein via v-containing integrin, as did L929 fibroblasts. In contrast, OPN domains lacking the RGD sequence were shown to be involved in the binding of OPN with murine B16-BL6 melanoma cells (11). However, the cellular receptors for the non- RGD-containing domains of OPN were not identified. In this study, we show that B16-BL6 but not B16-F10 or L929 cells express CD44 variants, and that CD44 variants but not CD44s can bind to OPN in an Received 6/23/98; accepted 10/28/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a Grant-in-Aid from Sagawa Cancer Research Foundation; the Ministry of Education, Science, and Culture of Japan Grant 10557024 (to T. U.); and by Grant 8426 from the National Cancer Institute of Canada (to A. F. C). J. P. S. was supported by a European Union Marie Curie Fellowship. 2 To whom requests for reprints should be addressed, at Institute of Immunological Science, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, 0600815, Japan. Phone and Fax: 85-11-736-9836; E-mail: toshi@imm.hokudai.ac.jp. 3 The abbreviations used are: OPN, osteopontin; CD44s, standard form of CD44; CD44v, variant form of CD44; GST, glutathione S-transferase, EC2.5.1.18; GRGDS, glycine-arginine-glycine-aspartic acid-cysteine; GRGES, glycine-arginine-glycine-gluta- mine-cysteine; HA, hyaluronic acid; RGD, arginine-glycine-aspartic acid; VN, vitronec- tin; PE, phycoerythrin. 219 on July 6, 2015. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from