The CD44 family of cell adhesion molecules: Functional aspects

THE CD44 FAMILY OF CELL ADHESION MOLECULES: FUNCTIONAL ASPECTS

Charles B. Underhill

I. Introduction II. Structure of CD44 A. Functional Domains B. Multiple Isoforms of CD44 III. Lymphocyte Homing and Activation IV. Macrophages A. Distribution B. Hyaluronan Induced Aggregation C. Degradation of Hyaluronan V. Hemopoietic Tissue VI. Hair Follicle Development VII. Epithelial Cells A. Distribution B. CD44 is Associated with Proliferation C. Possible Functions of CD44 VIII. TumorCells A. Association with Metastatic Cancers B. Possible Mechanisms of Action IX. Conclusion Acknowledgments References Biomembranes Volume 3, pages 205-218. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-660-6.

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I. INTRODUCTION The realization that the hyaluronan receptor was identical to CD44 came as a surprise to many of us who had been working with this molecule (Aruffo et al., 1990; Culty et al., 1990; Miyake et al., 1990a). Indeed, there was no obvious reason to suspect that these molecules might be related, since they had been investigated in completely different functional contexts. The hyaluronan receptor was originally examined with regard to its ability to bind hyaluronan, while CD44 was characterized for its role in lymphocyte homing. As it turns out, both of these distinct functions were carried out by CD44 and, more recently, this same molecule has been implicated in other phenomena, including morphogenesis, inflammation, lymphocyte maturation, cell migration, and tumor metastasis. In the present review, I will concentrate on the functional aspects of this molecule, with particular emphasis on its interaction with hyaluronan. At the same time, I will speculate on some of the mechanisms by which CD44 mediates these diverse functions.

IL STRUCTURE OF CD44 A. Functional Domains

As illustrated in Figure 1, CD44 is a highly elongated molecule which can be divided into three distinct regions; the N-terminal, middle, and C-terminal domains. The N-terminal domain is responsible for binding hyaluronan and is structurally similar to the link protein and core protein of cartilage, which can also bind hyaluronan (Culty et al, 1990; He et al, 1992). However, CD44 recognizes a six-sugar sequence of hyaluronan while the cartilage core protein recognizes a 10-sugar sequence (Hascall and Heinegard, 1974; Underbill and Toole, 1979; Underbill etal., 1983). The middle domain of CD44 is responsible for the binding of lymphocytes to high endothelial cells of mucosal lymphoid tissue, a phenomenon which is blocked by an antibody (termed Hermes-3) directed against this region (Jalkanen et al., 1987; Berg et al., 1989). This central domain is the site at which most of the structural variations occur between the different isoforms of CD44. Not surprisingly, this region has also undergone a higher rate of evolutionary change than either the N- or C-terminal domains (Goldstein and Butcher, 1990). The C-terminal domain contains the cytoplasmic region, which can be associated with actin filaments of the cytoskeleton through an ankyin-like molecule, as illustrated in Figure 1 (Lacy and Underbill 1987; Kalomiris and Bourguignon 1988). Furthermore, Bourguignon and her associates have shown that the interaction between CD44 and the ankyrin-like molecule is modified by the attachment of phosphate and fatty acid residues and by the binding of guanine nucleotides to the cytoplasmic domain of CD44 (Kalomiris and Bourguignon, 1989; Bourguignon

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1 Membrane Ankyrin Actin Figure h Schematic view of CD44 and its interaction with hyaluronan and the cytoskeieton.

et al, 1991; Lokeshwar and Bourguignon, 1991, 1992). This interaction between CD44 and the cytoskeieton may be important in regulating the hyaluronan binding activity on the cell surface. If the cytoskeieton induces several molecules of CD44 to form clusters, then they v^ould be able to interact simultaneously with a single molecule of hyaluronan (see Figure 1). This cooperative binding would have a higher affinity for hyaluronan than that of a single molecule of CD44 interacting with hyaluronan (Underbill and Toole, 1980; Underbill, 1989). Thus, by controlling the distribution of CD44 on the cell surface, the cytoskeieton may indirectly regulate the binding affinity for hyaluronan on the cell surface. Consistent with this possibility is that cells transfected with forms of CD44 lacking the cytoplasmic domain have a greatly reduced binding affinity for hyaluronan (Hyman et al., 1991; Lesley etal, 1992). B. Multiple Isofornns of CD44

CD44 comes in a wide variety of different molecular weight forms, ranging from 80 to well over 200 kDa. Part of this variation is due to alterative splicing of the mRNA (Brown etal, 1991; Dougherty etal., 1991; Cooper etal, 1992). In humans, the single gene which codes for CD44 contains 19 exons which can give rise to 15

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or more different splicing variations (Goodfellow et al., 1982; Goldstein et al., 1989; Dougherty et al, 1991; He et al., 1992; Screaton et al., 1992). As indicated above, most of these variants result in changes in the middle domain of the molecule (for review see Haynes et al, 1991). The two most common isoforms are CD44H (approximately 85 kDa) which is associated with hemopoietic tissue and CD44E (approximately 150 kDa) which is associated with epithelial cells. The different isoforms differ both structurally and functionally. For example, cells transfected with the CD44H isoform can mediate the binding of lymphocytes to high endothelial cells, whereas cells transfected with the CD44E isoform lack this activity (Berg et al, 1989; Picker et al., 1989). Presumably, a structural change in the middle domain of CD44 alters its ability to interact with mucosal addressin (see following section). The various isoforms of CD44 may also differ with respect to their ability to bind hyaluronan, however, the studies examining this issue are not totally consistent. Stamenkovic and his associates (1991) have shown that cells transfected with a human CD44E isoform do not readily bind to a hyaluronan-coated substrate. On the other hand, He et al. (1992) found that cells transfected with most of the higher molecular weight isoforms of CD44 can bind soluble hyaluronan. Perhaps, this discrepancy is due to differences in the techniques used to measure hyaluronan binding. Clearly, the binding of hyaluronan by the different isoforms of CD44 is an area that deserves further investigation. Another source of structural variation in CD44 is due to differences in the degree of glycosylation. All of the different isoforms of CD44 are highly glycosylated with both N- and 0-linked carbohydrate side-chains which can account for more than half of the molecular mass of the molecule (Carter and Wayner, 1988; Jalkanen et al., 1988; Brown et al, 1991). In the case of macrophages, the extent of glycosylation of CD44 can vary depending upon the activation state of the cell (Camp et al., 1991). It is possible that these carbohydrates modify the ability of CD44 to bind hyaluronan or mediate lymphocyte homing. Heparan and chondroitin sulfate can also be attached to some isoforms of CD44 present in the epidermis and other tissues (Jalkanen et al, 1988; Brown et al, 1991; Kugelman et al, 1992). The chondroitin sulfate side-chains have been shown to be responsible for the binding of CD44 to Type I collagen and fibronectin (Carter and Wayner, 1988; Jalkanen and Jalkanen, 1992), and are required for the migration of melanocytes on collagen (Faassen et al, 1992).

III. LYMPHOCYTE HOMING AND ACTIVATION The connection between CD44 and lymphocyte homing was first suggested by the work of Dr. Eugene Butcher and his collaborators (Berg et al, 1989). These researchers were studying the mechanism of lymphocyte homing by examining the adhesion of lymphocytes to cryostat sections of lymphoid tissue. In the course of these studies, they found that a monoclonal antibody, directed against CD44

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(termed Hermes-3), could block the binding of lymphocytes to the high endothelial cells of mucosal lymphoid tissue (i.e., Peyer's patches), but not to endothelial cells of peripheral lymph nodes (Jalkanen et al., 1987; Streeter et al., 1988). Subsequent research revealed that the CD44 was interacting with a protein termed mucosal addressin, which was specifically present on the endothelial cells of mucosal lymphoid tissue. In addition to its involvement in lymphocyte homing, CD44 may also participate in the activation of lymphocytes. While antibodies against CD44 do not directly stimulate lymphocytes, they do enhance the stimulatory effect of other agents such as antibodies to CD2 and interleukin-2 (Demming et al, 1990; Chong et al, 1992; Conrad et al., 1992). Presumably, this activation occurs through the cross-linking of the cell surface proteins. The question still remains as to whether hyaluronan itself has a similar effect on lymphocytes as the antibodies to CD44. It is possible that hyaluronan helps to prime the lymphocytes so that they are more susceptible to activation with other agents. In this manner, lymphocytes in connective tissue, which is rich in hyaluronan, may be more readily activated than those in lymphoid tissue, which is relatively deficient in hyaluronan.

IV. MACROPHAGES A.

Distribution

While CD44 is particularly abundant on most mature macrophages, it is not uniformly expressed throughout the family of mononuclear phagocytes. In rodents, CD44 is very prominent on mature macrophages of the lungs, peritoneal cavity, and dermis, but is greatly reduced or absent from the Kuppfer cells of the liver and microglia of the brain (Green et al., 1988a). Likewise, peripheral blood monocytes, which give rise to the mononuclear phagocytic cells, generally express lower levels of CD44 than mature macrophages of the lungs or peritoneal cavity. B. Hyaluronan Induced Aggregation

The ability of pulmonary and peritoneal macrophages to interact with hyaluronan has been recognized for some time. More than a decade ago, Mervik and his associates showed that the addition of hyaluronan to a suspension of macrophages induces them to aggregate (Love et al., 1979). Subsequently, Green et al. (1988b) showed that this aggregation was inhibited by blocking antibodies against CD44. Presumably, individual molecules of hyaluronan interact with CD44 on the surfaces of adjacent cells, linking them together. Hyaluronan-induced aggregation of macrophages may occur in both the lungs and peritoneal cavity under certain conditions. In response to inflammatory cytokines, both the fibroblasts of the lungs and the mesothelial cells of the peritoneal cavity secrete hyaluronan which, in turn, causes the aggregation of macrophages (Love et al,

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1979; Sampson et al., 1992). This phenomenon is responsible for the "macrophage disappearance reaction," which is considered to be a form of delayed type hypersensitivity (Galindo et al., 1975; Shannon and Love, 1980; Shannon et al., 1980). This reaction occurs when mice are challenged with an immunogen and then the number of peritoneal macrophages is determined from peritoneal washes. In the case of naive mice, large numbers of macrophages are recovered, whereas in previously sensitized mice, the number of macrophages is greatly reduced. This phenomenon has been traced to a hyaluronan-induced aggregation of the macrophages, which prevents them from being removed during a peritoneal wash, resulting in their apparent disappearance. While the biological significance of this hyaluronan-induced aggregation is still an open question, one possibility is that aggregation of macrophages helps these cells to combat infection. Along these lines, hyaluronan has been shown to stimulate the rate of phagocytosis by monocytes (Ahlgren and Jarstrand, 1984). C. Degradation of Hyaluronan

Macrophages also play an important role in the degradation of hyaluronan, a process which depends upon CD44 (Culty et al., 1992). When fluorescein or isotopically tagged hyaluronan was added to cultures of alveolar macrophages, initially it was bound to the plasma membrane and then taken up inside the cells, where it appeared in small vesicles (Culty et al., 1992). Eventually this hyaluronan was degraded by acid hydrolases present in lysosomes. The process of hyaluronan binding, internalization and degradation was blocked by antibodies to CD44. This suggests that CD44 is required for the critical first step in the process of degradation, namely, the binding to the cell surface. The degradation of hyaluronan by macrophages appears to be an important factor during the morphogenesis of the lung tissue. During early stages of development, large amounts of hyaluronan are present in the interstitial spaces of the lungs, however, prior to birth most of this hyaluronan is lost and the interstitial space shrinks so that gas exchange can take place. The removal of the hyaluronan is accompanied by a general increase in the number of CD44 positive macrophages present in the lungs, many of which were found to contain hyaluronan in their cytoplasms (Underbill et al, 1993). Presumably, these cells had taken up hyaluronan from the lung tissue and were in the process of removing it, by either degrading it directly or by migrating out of the lungs, and taking the hyaluronan with them. Further evidence for the role of macrophages in removing hyaluronan from the lungs camefroman experiment in which newborn mice were injected with blocking antibodies to CD44. After a period of 10 days, the hyaluronan content of the lungs of the injected mice was approximately twice that of control mice (Underbill et al., 1993). Apparently, the antibody blocked the ability of the macrophages to degrade the hyaluronan. It is likely that macrophages play a similar role in the turnover of hyaluronan in other tissues, such as the skin and lamina propria of various tissues.

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V. HEMOPOIETIC TISSUE CD44 is present on a subset of the cells in both myeloid and lymphoid hemopoietic tissues. Indeed, the ability to interact with hyaluronan may be an important factor in the cell maturation process. Miyake et al. (1990b) have shown that antibodies directed against CD44 prevent the maturation of B cells when co-cultured with stromal cells from bone marrow. Presumably, the maturation process requires a hyaluronan-dependent adhesion between the lymphocytes and the stromal cells. Lymphoid cells expressing CD44 also play an important role in removing hyaluronan from the lymph so that the circulating levels are maintained at low levels. Fraser and coworkers (1988,1989) have shown that when [^H]-hyaluronan was injected either intravenously or into afferent lymphatic vessels, a large fraction of it was removed by the dendritic cells of the spleen and lymph nodes. Not surprisingly, dendritic cells also express relatively large amounts of CD44, which is presumably responsible for their ability to bind and degrade the hyaluronan.

VI. HAIR FOLLICLE DEVELOPMENT Histochemical staining of embryonic tissue has revealed an inverse correlation between the expression of CD44 and the presence of hyaluronan in a variety of tissues including the lungs, heart, liver, spleen, and other hemopoietic tissue. However, the most striking example of this occurs in the morphogenesis of the hair follicle, which involves an inductive interaction between the epidermal cells and a small cluster of mesenchymal cells termed the dermal condensation. The cells in this condensation specifically express CD44, while the surrounding mesenchymal cells do not (see Figure 2a). Furthermore, these mesenchymal cells expressed CD44 only during the inductive phases of follicle formation, and lost it once the mature hair follicle had formed. In contrast, hyaluronan was conspicuously absent from the region of the inductive mesenchymal cells as long as the receptor was expressed (see Figure 2b). However, in the mature hair follicle, when CD44 was absent, then hyaluronan reappeared. Thus, the presence of hyaluronan was inversely correlated with the expression of CD44 both temporally and spatially. This inverse correlation suggests, but does not prove, that the expression of CD44 allows the cells to degrade hyaluronan. The absence of hyaluronan from the tip of the developing hair follicle may account for the condensed nature of the inductive mesenchymal cells in this region. Indeed, several studies have demonstrated that treatment of embryonic tissue with hyaluronidase leads to a marked condensation of mesenchymal tissue (Solursh et al, 1979; Morriss-Kay et al., 1986). Apparently, hyaluronan present in these regions keeps the cells separated and its removal causes these tissues to collapse.

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a

Figure 2. Developing hair follicle from a hamster embryo. (A) The section is stained for CD44 and shows that it is present on the cells of the dermal condensation. (B) A similar section is stained for hyaluronan, which is present throughout most of the dermis except in the region of the dermal condensation.

Vll. EPITHELIAL CELLS A.

Distribution

Many, but not all, types of epithelia express CD44. For example, it was prominent on the basal cells of stratified squamous epithelia of the skin, tongue, and esophagus, and on the simple epitheUa lining the oviduct and large intestines. However, it was absent from most mesothelial cells, and was only occasionally observed on endothelial cells during the embryonic development of rodents. B. CD44 is Associated with Proliferation

One of the more unusual aspects of CD44 is that its expression appeared to be directly related to the rate of cell division in the epithelium (Alho and Underbill,

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1989). In the case of the epidermis, the actively dividing cells at the basal regions expressed CD44, while the non-dividing cells in the more superficial layers did not. Similarly, in the epithelium of the large intestines, the dividing cells at the base of the crypts of Lieberkuhn expressed CD44, while the non-proliferating cells further up the crypts and lining the surface did not. It should be noted that this unusual situation holds true only for epithelial cells and is not the case for other cell types (e.g., macrophages). C. Possible Functions of CD44

The biological significance of the CD44 on epithelial cells is still an open question. Earlier, we speculated that CD44 helps to bind the epithelial cells to the basement membrane, which was rich in hyaluronan (Alho and Underbill, 1989). However, we no longer favor this possibility, since closer examination revealed that CD44 was generally absent from the basal surface of the epithelial cells, but instead was present along the lateral surfaces of the cells. Thus, it was not in a position to interact with hyaluronan throughout most of the basement membrane. In the case of the epidermis and other stratified epithelia, CD44 may serve to maintain spaces between the cells so that nutrients can get to the more superficial layers. While hyaluronan often coincides with the presence of CD44 between these cells in the epidermis, it is not clear whether the isoforms of CD44 present on the keratinocytes can bind hyaluronan (Stamenkovic et al., 1991). However, this CD44 may serve as a core protein for the attachment of heparin and chondroitin sulfate side-chains (Carter and Wayner, 1988; Jalkanen and Jalkanen, 1992). These glycosaminoglycans may also function to maintain the spaces between the epithelial cells.

Vlll. TUMOR CELLS A. Association with Metastatic Cancers

Recently, several studies have suggested a connection between the expression of CD44 and the metastatic behavior of tumor cells. For example, high levels of CD44 have been found associated with several types of carcinomas, high grade gliomas, and many non-Hodgkin's lymphomas (Stamenkovic et al., 1989; Horst et al, 1990; Kuppner et al., 1992). Indeed, in the case of lymphomas, large amounts of this protein are correlated with the rapid dissemination and negative prognosis of these tumors (Horst et al., 1990). Thus, CD44 may be a useful marker of the diagnosis of some types of cancer (Matsumura and Tarin, 1992). More direct evidence that the expression of CD44 is related to the metastatic behavior of tumor cells comes from the work of Gunthert et al. (1991). They found that several highly metastatic cell lines from rats express a particular isoform of CD44, which was absent from their non-turmorigenic counterparts. This same

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isoform of CD44 is also expressed in human colorectal carcinomas and adenomatous polyps (Heider et al., 1993). More importantly, when non-metastatic cells were transfected with cDNA for this isoform of CD44, they were converted into a more metastatic phenotype suggesting that this isoform of CD44 is directly responsible for the metastatic behavior of these cells (Gunthert et al., 1991). Other isoforms of CD44 also appear to influence the metastatic behavior of cells. Sy et al. (1991) have shown that when human lymphoma cells were transfected with the hemopoietic isoform of CD44 (CD44H), but not the epithelial isoform (CD44E), there was a marked increase in tumor formation and metastatic behavior. In addition, the growth of these cells in nude mice was inhibited by a soluble fusion protein of CD44 and immunoglobulin, which may competitively inhibit the binding of hyaluronan by CD44 on the surfaces of tumor cells (Sy et al., 1992). However, they also noted that lymphoma cells lacking CD44 formed both primary and metastatic tumors, but at a lower rate. Based on these results, these researchers concluded that expression of CD44H promotes, but is not required for tumor growth and metastasis. B. Possible Mechanisms of Action

There are several possible ways in which CD44 could alter the metastatic behavior of cells. First, the expression of CD44 may allow these cells to bind and degrade hyaluronan in the extracellular matrix, similar to macrophages. Since hyaluronan is a major component of the extracellular matrix surrounding blood vessels, its breakdown may allow tumor cells to enter the circulation. A second possibility is that CD44 allows cells to migrate through the extracellular matrix and thereby establish metastases. Recently, Thomas and coworkers (1992) have shown that melanoma cells expressing the CD44H isoform have an enhanced rate of migration on hyaluronan coated substratum. In addition, this enhanced migration was blocked by treating the cells with blocking antibodies to CD44. In a similar fashion, Faassen et al. (1992) have also found that CD44 is important in the migration of melanoma cells on a substrate composed of Type I collagen. However, in this case, the effect appears to be mediated by chondroitin sulfate residues attached to the CD44 since it could be inhibited by treatment with chondroitinase. Thus, the presence of CD44 on the surfaces of tumor cells may enhance their ability to migrate away and form secondary tumors.

IX. CONCLUSION CD44 impacts upon a number of different areas of research including immunology, developmental biology, and tumor biology. From my perspective, one of the most important aspects of CD44 is its ability to interact with hyaluronan, which is responsible for cell adhesion, and for the ability of cells to take up and degrade hyaluronan. Hyaluronan is a major component of many extracellular matrices and

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is responsible for maintaining the space between cells. The removal of this hyaluronan by a CD44-dependent mechanism can lead to shrinkage of the extracellular space that could be a driving force in morphogenesis.

ACKNOWLEDGMENTS The author thanks Tony Shizari for his help with this manuscript. This work was supported by United States Health Service grants HD26758 and CA35592 and by USAMRMC grant N0.DAMD17-94-J-4284.

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