Hyaluronan binding properties of a CD44 chimera containing the link module of TSG-6

CD44, a cell-surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, can mediate leukocyte rolling on hyaluronan substrates and has been implicated in leukocyte migration to sites of inflammation. CD44-mediated binding to hyaluronan is of low affinity, and effective cell/matrix interaction depends on multiple interactions with the multivalent ligand. We replaced the Link module of CD44 with the homologous region of TSG-6, a hyaluronan-binding protein secreted in response to inflammation whose Link module has a higher affinity for ligand. Monoclonal antibodies raised against the CD44/TSG-6 chimera recognized recombinant human TSG-6 and native mouse TSG-6 and blocked hyaluronan binding to these proteins. Cells expressing the CD44/TSG-6 molecule bound hyaluronan with higher avidity than cells expressing CD44. This resulted in changes in the hyaluronan binding properties characteristic of cells expressing CD44 such as requirements for threshold levels of receptor expression and for hyaluronan of high molecular mass. In parallel plate flow assays used to model leukocyte rolling, cells expressing CD44/TSG-6 failed to roll on hyaluronan. Instead, they stuck and remained "tethered" to the substrate under fluid flow. This result argues that the low affinity of CD44 for its ligand is important for rolling, an early phase of leukocyte extravasation from the blood.     

Mapping the hyaluronan-binding site on the link module from human tumor necrosis factor-stimulated gene-6 by site-directed mutagenesis

Link modules are hyaluronan-binding domains found in extracellular proteins involved in matrix assembly, development, and immune cell migration. Previously we have expressed the Link module from the inflammation-associated protein tumor necrosis factor-stimulated gene-6 (TSG-6) and determined its tertiary structure in solution. Here we generated 21 Link module mutants, and these were analyzed by nuclear magnetic resonance spectroscopy and a hyaluronan-binding assay. The individual mutation of five amino acids, which form a cluster on one face of the Link module, caused large reductions in functional activity but did not affect the Link module fold. This ligand-binding site in TSG-6 is similar to that determined previously for the hyaluronan receptor, CD44, suggesting that the location of the interaction surfaces may also be conserved in other Link module-containing proteins. Analysis of the sequences of TSG-6 and CD44 indicates that the molecular details of their association with hyaluronan are likely to be significantly different. This comparison identifies key sequence positions that may be important in mediating hyaluronan binding, across the Link module superfamily. The use of multiple sequence alignment and molecular modeling allowed the prediction of functional residues in link protein, and this approach can be extended to all members of the superfamily.     

LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.     

Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.     

Characterization of a Functional Hyaluronan-Binding Domain from the Human CD44 Molecule Expressed inEscherichia coli

The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6). In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region. Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module. In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues inEscherichia coli.Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography. We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species. This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy.     

Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction

Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The molecular details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equivalent to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.     

Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6

BACKGROUND: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA-protein interactions remains poorly understood. RESULTS: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA(4)-HA(16)). All oligomers bound (except HA(4)) with K(d) values ranging from 0.2-0.5 microM at 25 degrees C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan-protein interactions. The HA(8) recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the beta4/beta5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. CONCLUSIONS: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.     

TSG-6 modulates the interaction between hyaluronan and cell surface CD44

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.     

Hyaluronan recognition mode of CD44 revealed by cross-saturation and chemical shift perturbation experiments

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first alpha-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first alpha-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.     

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan: USE OF DEFINED OLIGOSACCHARIDES TO PROBE STRUCTURE AND FUNCTION*

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was ¹³C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.     

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.     

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System: INSIGHTS INTO THE DISTINCT BINDING PROPERTIES OF HIGH AND LOW MOLECULAR WEIGHT HYALURONAN*

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.     

Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.     

Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999) J. Cell Biol. 144, 789-801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.     

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.     

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.     

Characterization of the interaction between tumor necrosis factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin in extracellular matrix microenvironments

TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and site-directed mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-alpha-inhibitor (IalphaI). Experiments using the purified components of IalphaI indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6.IalphaI complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.     

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.     

Regulation of the release and function of tumor cell-derived soluble CD44

CD44, a major receptor for glycosaminoglycan hyaluronan (HA), is a broadly distributed cell surface glycoprotein implicated in multiple functions, including tumor growth and dissemination. The affinity of surface CD44 for HA is subject to regulation at several levels. CD44 is found in multiple phases, including as an integral transmembrane protein and as soluble fragment of the extracellular domain found in the circulation and other body fluids. Transmembrane CD44 and its ability to interact with HA have been a focus of numerous studies in the past, but the function of soluble CD44 remains obscure. Interestingly, malignant diseases are often associated with an increase in the plasma level of CD44. The delineation of the HA binding capacity of tumor-derived soluble CD44 is an important step toward understanding the biological function of this molecule. In this study, we demonstrate that tumor cells activated to bind HA by cytokines rapidly release CD44 upon treatment with phorbol ester (PMA). The affinity for HA of the soluble CD44 released in response to PMA varied depending on the cytokine pretreatment. These results suggest that the function of tumor-derived soluble CD44, like the transmembrane form of the receptor, can be regulated.     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.