Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells

Interaction of CD44, an adhesion molecule, with its ligand, hyaluronan (HA), in monocytic cells plays a critical role in cell migration, inflammation, and immune responses. Most cell types express CD44 but do not bind HA. The biological functions of CD44 have been attributed to the generation of the functionally active, HA-adhesive form of this molecule. Although lipopolysaccharide (LPS) and cytokines induce HA-adhesive CD44, the molecular mechanism underlying this process remains unknown. In this study, we show that LPS-induced CD44-mediated HA (CD44-HA) binding in monocytes is regulated by endogenously produced tumor necrosis factor (TNF)-alpha and IL-10. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation was required for LPS- and TNF-alpha-induced, but not IL-10-induced, CD44-HA-binding in normal monocytes. To dissect the signaling pathways regulating CD44-HA binding independently of cross-regulatory IL-10-mediated effects, IL-10-refractory promonocytic THP-1 cells were employed. LPS-induced CD44-HA binding in THP-1 cells was regulated by endogenously produced TNF-alpha. Our results also suggest that lysosomal sialidase activation may be required for the acquisition of the HA-binding form of CD44 in LPS- and TNF-alpha-stimulated monocytic cells. Studies conducted to understand the role of MAPKs in the induction of sialidase activity revealed that LPS-induced sialidase activity was dependent on p42/44 MAPK-mediated TNF-alpha production. Blocking TNF-alpha production by PD98059, a p42/44 inhibitor, significantly reduced the LPS-induced sialidase activity and CD44-HA binding. Subsequently, TNF-alpha-mediated p38 MAPK activation induced sialidase activity and CD44-HA binding. Taken together, our results suggest that TNF-alpha-induced p38 MAPK activation may regulate the induction of functionally active HA-binding form of CD44 by activating sialidase in LPS-stimulated human monocytic cells.     

E-cadherin negatively regulates CD44-hyaluronan interaction and CD44-mediated tumor invasion and branching morphogenesis

CD44 is a principal cell-surface receptor for hyaluronan (HA). Up-regulation of CD44 is often associated with morphogenesis and tumor invasion. On the contrary, reduction of cell-cell adhesion due to down-regulation of E-cadherin is associated with the invasive and metastatic phenotype of carcinomas. In our current study, we investigated the functional relationship between CD44 and E-cadherin. We established an inverse correlation between CD44 and E-cadherin indicating that the cells expressing higher levels of E-cadherin display weaker binding affinity between CD44 and HA. By using TA3 murine mammary carcinoma (TA3) cells, which display CD44-dependent HA binding, branching morphogenesis, and invasion, we demonstrated an inverse functional relationship between CD44 and E-cadherin by transfecting exogenous E-cadherin into the cells. Our results showed that increased expression of E-cadherin in TA3 cells, but not ICAM-1, weakens the binding between CD44 and HA and blocks spreading of the cells on HA substratum and CD44-mediated branching morphogenesis and tumor cell invasion. The results reported here demonstrated for the first time that E-cadherin negatively regulated CD44-HA interaction and CD44 function and suggested that balanced function of CD44 and E-cadherin may be essential for normal epithelial cell functions, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely contributes to tumor progression.     

CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.     

Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition

We previously found that the CD44 glycoprotein on some lymphocytes can mediate adhesion to hyaluronate (HA) bearing cells. However, many questions remain about the molecular heterogeneity of CD44 and mechanisms which control its recognition of this ligand. In vitro mutagenesis and DNA sequencing have now been used to investigate the importance of the membrane proximal region of murine CD44 for recognition of soluble or cell surface HA. CD44 with an 83 amino acid deletion in this region mediated binding to soluble ligand and the apparent avidity increased markedly in the presence of a particular antibody to CD44, IRAWB14. The shortened CD44 was however inefficient in mediating adhesion of transfected cells to HA immobilized on cell surfaces. Four new murine isoforms of CD44 were isolated from a carcinoma line by use of the polymerase chain reaction. Only two of them correspond to ones recently discovered in rat and human cells. The longest variant nearly doubled the length of the extracellular portion of the molecule and introduced an additional 20 potential sites for glycosylation. When expressed on T lymphoma cells, all four of the new murine CD44 isoforms were capable of mediating adhesion to HA bearing cells. This result contrasts with a report that a related human CD44 isoform lacks this ability when expressed on B lineage lymphoma cells. The new murine isoforms also conferred the ability to recognize soluble HA and were very responsive to the IRAWB14 antibody. A brief survey of normal murine cell lines and tissues revealed that the hemopoietic isoform was the most abundant species. These findings indicate that the NH2-terminal portion of CD44 is sufficient for HA recognition and that this function is not necessarily abrogated by variations which occur in the membrane proximal domain. They add to the known molecular diversity of CD44 and provide another experimental model in which isoform specific functions can be investigated.     

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.     

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.     

TNFalpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44

Our previous studies have identified TNFalpha as a positive regulator and IL-4 as a negative regulator of human monocyte CD44-HA binding. In order to determine the mechanisms of IL-4- and TNFalpha-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFalpha or IL-4, as well as following monocyte treatment with inhibitors of protein synthesis, N- and O-linked glycosylation, and chondroitin sulfation. IL-4 decreased CD44-HA binding on monocytes initially treated with TNFalpha. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFalpha-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and beta-d-xyloside (chondroitin sulfation inhibitor) all inhibited IL-4-mediated downregulation of TNFalpha-induced monocyte HA binding. Western blot analysis of CD44 from TNFalpha-treated monocytes revealed a 5-10 Mr decrease in the standard isoform of CD44. In contrast, IL-4 treatment of monocytes inhibited CD44-HA binding and reversed the 5- to 10-kDa decrease in monocyte CD44 Mr. Finally, studies with F10.44.2, a CD44 mab that enhances CD44-HA binding, indicated that IL-4 treatment of monocytes not only diminished constitutive HA binding, but also diminished CD44 mab-induced HA binding. Taken together, these data suggested that IL-4-mediated inhibition of TNFalpha-induced monocyte HA binding was dependent not only on protein synthesis, but also on N-linked glycosylation and chondroitin-sulfate modification of either CD44 or, alternatively, another monocyte protein(s) that may regulate the ability of CD44 to bind HA.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml-1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml−1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.     

Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell-substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9-specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA-CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.     

Tumor cells enhance their own CD44 cleavage and motility by generating hyaluronan fragments

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that interacts with cell-surface receptors, including CD44. Although HA usually exists as a high molecular mass polymer, HA of a much lower molecular mass that shows a variety of biological activities can be detected under certain pathological conditions, particularly in tumors. We previously reported that low molecular weight HAs (LMW-HAs) of a certain size range induce the proteolytic cleavage of CD44 from the surface of tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we show that MIA PaCa-2, a human pancreatic carcinoma cell line, secreted hyaluronidases abundantly and generated readily detectable levels of LMW-HAs ranging from approximately 10- to 40-mers. This occurred in the absence of any exogenous stimulation. The tumor-derived HA oligosaccharides were able to enhance CD44 cleavage and tumor cell motility. Inhibition of the CD44-HA interaction resulted in the complete abrogation of these cellular events. These results are consistent with the concept that tumor cells generate HA oligosaccha-rides that bind to tumor cell CD44 through the expression of their own constitutive hyaluronidases. This enhances their own CD44 cleavage and cell motility, which would subsequently promote tumor progression. Such an autocrine/paracrine-like process may represent a novel activation mechanism that would facilitate and promote the malignant potential of tumor cells.     

The fate of low affinity tumor-specific CD8+ T cells in tumor-bearing mice

A major challenge in tumor immunology is how best to activate the relatively low avidity self-specific and tumor-specific T cells that are available in the self-tolerant repertoire. To address this issue, we produced a TCR transgenic mouse expressing a class I-restricted hemagglutinin (HA)-specific TCR (clone 1 TCR) derived from a mouse that expressed HA as a self-Ag in the insulin-producing beta cells of the pancreatic islets (InsHA) mice. Upon transfer of clone 1 TCR CD8(+) T cells into InsHA mice, very few cells were activated by cross-presented HA, indicating that the cells were retained in InsHA mice because they ignored the presence of Ag, and not because they were functionally inactivated by anergy or tuning. Upon transfer into recipient mice in which HA is expressed at high concentrations as a tumor-associated Ag in spontaneously arising insulinomas (RIP-Tag2-HA mice), a high proportion of clone 1 cells were activated when they encountered cross-presented tumor Ag in the pancreatic lymph nodes. However, the activated cells exhibited very weak effector function and were soon tolerized. The few activated cells that did migrate to the tumor were unable to delay tumor progression. However, when HA-specific CD4 helper cells were cotransferred with clone 1 cells into RIP-Tag2-HA recipients and the mice were vaccinated with influenza, clone 1 cells were found to exert a significant level of effector function and could delay tumor growth. This tumor model should prove of great value in identifying protocols that can optimize the function of low avidity tumor-specific T cells.     

NMR fragment-based screening for development of the CD44-binding small molecules

The cell-surface protein CD44, a primary receptor for hyaluronic acid (HA), is one of the most promising targets for cancer therapies. It is prominently involved in the process of tumor growth and metastasis. The possibility of modulating the CD44-HA interaction with a pharmacological inhibitor is therefore of great importance, yet until now there are only few small molecules reported to bind to CD44. Here, we describe the results of the NMR fragment-based screening conducted against CD44 by which we found eight new hit compounds that bind to the receptor with the affinity in milimolar range. The NMR-based characterization revealed that there are two possible binding modes for these compounds, and for some of them the binding is no longer possible in the presence of hyaluronic acid. This could provide an interesting starting point for the development of new high-affinity ligands targeting the CD44-HA axis.     

Differential antigen sensitivity and costimulatory requirements in human Th1 and Th2 antigen-specific CD4+ cells with similar TCR avidity

The differentiation of naive CD4(+) Th cells into Th1 and Th2 phenotypes is influenced by cytokines, concentration of Ag, accessory molecules, and the affinity of the MHC-TCR interaction. To study these factors in human memory T cells, T cell lines with Th1 or Th2 phenotypes specific for the peptide hemagglutinin (HA)(307-319) in the context of DRB1*0401 were established from the peripheral blood of an individual previously vaccinated for influenza virus. Flow cytometric analysis with fluorescent-labeled MHC class II tetramers was used to analyze TCR avidity: the Th2 line bound the HLA-DR*0401-HA(307-319) tetramers with higher mean avidity, although the range of binding avidity largely overlapped with the Th1 line. High-affinity Th1 and Th2 lines were established for further study by FACS sorting. When activated with plate-bound HLA-DR*0401-HA(307-319) monomers, the Th1 line proliferated and produced IFN-gamma without additional costimulation whereas the Th2 line required the addition of soluble anti-CD28 Ab to induce proliferation and IL-5 production, but this requirement could be overcome with high concentrations of plate-bound monomer alone. IL-2 production was dependent on costimulation in both cell lines. These findings demonstrate that upon antigenic rechallenge, Th1 and Th2 cells differ in their response to Ag-specific stimulation. Th2 cells were sensitive to the strength of signal to a greater degree than Th1 cells and required costimulation through CD28 for maximal proliferation. These distinctions between Th1 and Th2 activation are not consistent with a simple avidity model of Ag recognition and indicate both qualitative and quantitative differences in determining cell lineage commitment.     

Role of the extracellular and cytoplasmic domains of CD44 in the rolling interaction of lymphoid cells with hyaluronan under physiologic flow

CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly(305) or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys(295) adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the "non-conserved" membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogeneous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.     

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.