CD44 in inflammation and metastasis

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered. Abbreviations: aa, amino acid(s); CS, chondroitin sulfate; CSPG, chondroitin sulfate containing proteoglycan; CD44H, ‘hematopoietic’, also called ‘standard’, isoform of CD44 which contains none of the alternatively spliced variant exons; CD44-Rg, CD44 receptor globulin, a secreted chimaeric protein composed of the external domain of the adhesion receptor CD44 and the hinge, CH2 and CH3 regions of human immunoglobulin-G heavy chain; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; PB, peripheral blood; PBL, peripheral blood lymphocytes This revised version was published online in November 2006 with corrections to the Cover Date.     

A crucial role for CD44 in inflammation

Current therapies for chronic inflammatory diseases typically act through the nonspecific downregulation of immune cell activation. However, it is becoming increasingly evident that parenchymal cells are also active participants in the inflammatory process. Future prospects for the treatment of inflammation should therefore include the targeting of specific inflammatory pathways in both immune cells and parenchymal cells. CD44, a cell-adhesion molecule that is ubiquitously expressed on leukocytes and parenchymal cells, has been implicated, together with its ligand hyaluronan (HA), in several inflammatory diseases. The mechanisms of action of CD44-HA interactions in inflammation might provide potential targets for therapy.     

CD44: functional relevance to inflammation and malignancy

CD44 is a principal cell surface receptor for hyaluronan, a major component of extracellular matrices. Cells are surrounded by and encounter matrix in vivo, which in turn serves a variety of cell functions through the direct adhesion via their receptors. CD44 communicates cell-matrix interactions into the cell via "outside-in signaling" and has an important role in biological activities. The interaction of CD44 with fragmented hyaluronan on rheumatoid synovial cells induces expression of VCAM-1 and Fas on the cells, which leads to Fas-mediated apoptosis of synovial cells by the interaction of T cells bearing FasL. On the other hand, engagement of CD44 on tumor cells derived from lung cancer reduces Fas expression and Fas-mediated apoptosis, resulting in less susceptibility of the cells to CTL-mediated cytotoxicity through Fas-FasL pathway. Thus, although the CD44-mediated signaling differs among cells and circumstances, we here propose the functional role of CD44 in inflammatory processes and tumor susceptibility and the rational design of future therapeutic strategies including the exploitation of CD44-mediated pathway in vivo.     

FTY720 suppresses CD4+CD44highCD62L- effector memory T cell-mediated colitis

FTY720, a sphingosine-derived immunomodulator, causes immunosuppression via enhancement of lymphocyte sequestration into secondary lymphoid organs, thereby preventing their antigen-activated T cell egress to sites of inflammation. FTY720 is highly effective in inhibiting autoimmunity in various animal models. However, there is little known about how FTY720 controls the migration property of memory T cells. Here, we demonstrated that FTY720 prevents the development of colitis induced by the adoptive transfer of lamina propria (LP) colitogenic effector memory CD4+ T cells (TEM cells; CD45RB(low)CD44(high)CD62L-) into severe combined immunodeficiency (SCID) mice and suppresses interferon-gamma, interleukin-2, and tumor necrosis factor-alpha production by LP CD4+ T cells. The numbers of spleen, peripheral blood, mesenteric lymph node, and LP CD4+ T cells in FTY720-treated mice were significantly reduced compared with those in control mice. Notably, LP CD4+ TEM cells as well as splenic CD4+CD45RBhigh T cells expressed several spingosine-1-phosphate receptors that are targets for FTY720. Furthermore, FTY720 also prevented the development of colitis induced by the adoptive transfer of splenic CD4+CD45RBhigh T cells into SCID mice. Collectively, the present data indicate that FTY720 treatment may offer the potential not only to prevent the onset of disease but also to treat memory T cell-mediated autoimmune diseases including inflammatory bowel diseases.     

Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.     

Bikunin down-regulates heterodimerization between CD44 and growth factor receptors and subsequently suppresses agonist-mediated signaling

We provided evidence previously that bikunin, a Kunitz-type protease inhibitor, can disrupt dimerization of CD44 proteins, which may result in suppression of receptor-mediated MAP kinase signaling. However, to what extent dimerization may alter ligand-induced signaling has not been documented. Given the recent recognition that some growth factor receptors can form heterodimers with CD44, the present study was undertaken to determine whether the CD44 and growth factor receptors (e.g., EGFR, FGFR, HGFR, VEGFR, TGF-betaRI, or TGF-betaRII) can form heterodimers in cancer cells and, if so, to investigate the potential functional consequences of such heterodimerization. We also examined whether bikunin can abrogate these heterodimerizations and inhibit CD44/growth factor-dependent signaling. Here, we show direct evidence for heterodimerization of CD44-FGFR and CD44-TGF-betaRI in human chondrosarcoma HCS-2/8 cells, CD44-EGFR complex in human glioma U87MG cells, and CD44-TGF-betaRI heterodimer in human ovarian cancer HRA cells. Coupling of CD44 and growth factor receptor may be selective, depending on a cell type. Bikunin does not alter the ligand binding, whereas functionally reduces heterodimerization between CD44 and growth factor receptors. The disruption of heterodimerization substantially reduces receptor-induced tyrosine phosphorylation and ERK1/2 activation. Taken together, our data suggest that bikunin-mediated suppression of heterodimerization between CD44 and growth factors may inhibit the agonist-promoted activation of the signaling pathway.     

Klotho suppresses RIG-I-mediated senescence-associated inflammation

It is well known that aged or senescent cells develop a complex senescence-associated secretory phenotype (SASP), which is observed both in culture and in vivo. However, the mechanisms underlying the induction of the SASP are largely unknown. We demonstrate that retinoic-acid-inducible gene-I (RIG-I) is induced through the ataxia telangiectasia mutated-interferon regulatory factor 1 (ATM-IRF1) axis in senescent cells and that RIG-I signalling mediates the expression of two important mediators of inflammation, interleukin-6 (IL-6) and IL-8. Klotho has been associated with ageing. We show here that the intracellular, but not the secreted, form of klotho interacts with RIG-I and that this interaction inhibits RIG-I-induced expression of IL-6 and IL-8 both in vitro and in vivo. Our study uncovers a mechanism in which klotho functions as an anti-ageing factor through the suppression of RIG-I-mediated inflammation.     

MicroRNA-34a suppresses invasion and metastatic in esophageal squamous cell carcinoma by regulating CD44

Cisplatin is a widely used antineoplastic agent in the treatment of head and neck cancer. However, it is highly nephrotoxic. Oxidative stress is the main mechanism responsible for cisplatin-induced nephrotoxicity. The aim of this study was to characterize cisplatin-induced nephrotoxicity, oxidative stress in peripheral blood mononuclear cells, and the relationship between them. Twenty-four patients were included in the study. Patients had their blood collected prior to cisplatin administration, and 5 and 20 days after initiating therapy, to assess renal function and to determine oxidative stress with MitoSOX?Red, H₂DCF-DA, and Amplex^® Red tests. Renal function was assessed by measuring serum creatinine, creatinine clearance, and blood urea nitrogen (BUN). Serum creatinine and creatinine clearance were used to grade nephrotoxicity using Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Compared to baseline values, the mean BUN and serum creatinine increased 135 and 100%, respectively, 5 days after cisplatin infusion. Mean creatinine clearance showed a 43% decrease compared to baseline value. Non-statistically significant changes in superoxide anion (O ₂ ^?? ), hydrogen peroxide (H₂O₂), and general reactive oxygen species production occurred. A higher production of H₂O₂ was correlated with variation in serum creatinine, and was associated with higher grades for serum creatinine increases and creatinine clearance reductions. Linear regression analyses showed an association between H₂O₂ production and serum creatinine, creatinine clearance, and BUN levels. These results were observed for 5 days following cisplatin administration. In conclusion, H₂O₂ production was significantly related to changes in all renal parameters that were evaluated, following the cisplatin infusion.     

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44.     

CD44 is a negative regulator of acute pulmonary inflammation and lipopolysaccharide-TLR signaling in mouse macrophages

CD44 is a transmembrane adhesion molecule and hemopoietic CD44 has an essential role in hyaluronan clearance and resolution of noninfectious lung injury. In this study, we examined the role of CD44 in acute pulmonary inflammation and in the regulation of LPS-TLR signaling. Following intratracheally LPS treatment, CD44(-/-) mice demonstrated an exaggerated inflammatory response characterized by increased inflammatory cell recruitment, elevated chemokine expression in bronchoalveolar lavage fluid, and a marked increase in NF-kappaB DNA-binding activity in lung tissue in vivo and in macrophages in vitro. Furthermore, CD44(-/-) mice were more susceptible to LPS-induced shock. Reconstitution of hemopoietic CD44 reversed the inflammatory phenotype. We further found that the induction of the negative regulators of TLR signaling IL-1R-associated kinase-M, Toll-interacting protein, and A20 by intratracheal LPS in vivo and in macrophages in vitro was significantly reduced in CD44(-/-) mice. Collectively, these data suggest CD44 plays a previously unrecognized role in preventing exaggerated inflammatory responses to LPS by promoting the expression of negative regulators of TLR-4 signaling.     

Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes.mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed.CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA.These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.     

CD44s and CD44v6 expression in head and neck epithelia

Background

CD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.

Methods

Expression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.

Results

In normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.

Conclusion

CD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.     

The liberation of CD44

CD44 was once thought to simply be a transmembrane adhesion molecule that also played a role in the metabolism of its principal ligand hyaluronan. Investigations of CD44 over the past approximately 20 yr have established additional functions for CD44, including its capacity to mediate inflammatory cell function and tumor growth and metastasis. It has also become evident that intricate posttranslational modifications of CD44 regulate the affinity of the receptor for its ligands. In this review, we focus on emerging evidence that functional fragments of the cytoplasmic and ectodomain of CD44 can be liberated by enzymatic modification of cell surfaces as well as of cell-associated matrix. Based on the evidence discussed, we propose that CD44 exists in three phases, as a transmembrane receptor, as an integral component of the matrix, and as a soluble protein found in body fluids, each with biologically significant functions of which some are shared and some distinct. Thus, CD44 represents a model for understanding posttranslational processing and its emerging role as a general mechanism for regulating cell behavior.     

CD44 in rheumatoid arthritis

CD44 is a multistructural cell-surface glycoprotein that can theoretically generate close to 800 isoforms by differential alternative splicing. At present, several dozen isoforms are known. The polymorphic nature of CD44 might explain its multifunctionality and its ability to interact with many cell-surface and extracellular ligands, the principal one being hyaluronic acid (HA). Of the many CD44 functions, our review focuses on its involvement in cell–cell and cell–matrix interactions, as well as on its implication in the support of cell migration and the presentation of growth factors to their cognate receptors. Cells involved in pathological activities such as cancer cells and destructive inflammatory cells, and also normal cells engaged in physiological functions, use cell-surface CD44 for their localization and expansion at extravascular sites. This article reviews the evidence that the joint synovium of patients with rheumatoid arthritis (RA) contains considerable amounts of various CD44 isoforms as well as the HA ligand. The review also shows that anti-CD44 monoclonal antibody (mAb) directed against constant epitopes, shared by all CD44 isoforms, can markedly reduce the inflammatory activity of arthritis induced by collagen or proteoglycans in mice. Anti-CD44 mAb also interferes with the migration of RA synovial-like fibroblasts in vitro and is able to disturb the destructive interaction between RA synovial-like fibroblasts and the cartilaginous matrix. However, the transition from the experimental model to the patient's bedside is dependent on the ability to target the CD44 of cells engaged in RA pathology, while skipping the CD44 of normal cells.     

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44- hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44- mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N- linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.