Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011. © 2010 Wiley‐Liss, Inc.     

The CD44 Receptor of Lymphoma Cells: Structure-Function Relationships and Mechanism of Activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

The CD44 receptor of lymphoma cells: structure-function relationships and mechanism of activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

Regulation of CD44-hyaluronan interactions in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid B cells by PMA and interleukin-4.

In this study, we investigated the regulation of CD44-hyaluronan (HA) interactions in a panel of EBV+ Burkitt's lymphoma (BL) and lymphoblastoid B cell lines (B-LCL) generated by in vitro EBV transformation of normal human B cells. The results show that among B cell mitogens, phorbol 12-myristate 13-acetate (PMA) alone induced strong HA recognition in EBV+ BL-30/B95-8 cells. Among the cytokines that affect B cell growth and differentiation, IL-4 alone induced HA recognition in BL-30/B95-8 cells. Attempts to delineate the molecular mechanism for this increased HA adhesion in BL-30/B95-8 cells revealed an enhanced expression of CD44 H, isoforms containing V3, V6, and V9 exons, alterations in the splicing pattern of the V4 exon, and the increased electrophoretic mobility of the CD44 H protein. In contrast, the ability to recognize HA was not observed in B-LCL cells stimulated with either PMA or IL-4, even though these cells respond to IL-4, as observed by upregulation of CD23 expression. The molecular pathways that regulate CD44 expression and CD44-mediated HA binding may be selectively inactivated in B-LCL cells. These results may have implications with respect to the generation and spread of B cell tumors.     

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.     

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.     

Optical fan for single‐cell screening

The single‐cell screening has attracted great attentions in advanced biomedicine and tissue biology, especially for the early disease diagnosis and treatment monitoring. In this work, by using a specific‐designed fiber probe with a flat facet, we propose an “optical fan” strategy to screen K562 cells at the single‐cell level from a populations of RBCs. After the 980‐nm laser beam injected into the fiber probe, the RBCs were blown away but holding target K562 cells in place. Further, multiple leukemic cells can be screened from hundreds of red blood cells, providing an efficient approach for the cell screening. The experimental results were interpreted by the numerical simulation, and the stiffness of optical fan was also discussed.     

Hyaluronan cell surface binding is induced by type I collagen and regulated by caveolae in glioma cells

Hyaluronan (HA) is a component of the brain extracellular matrix environment that is synthesized and secreted by glioma cells. The primary cell surface receptor for HA is CD44, a membrane glycoprotein that is functionally regulated by a membrane type 1 matrix metalloproteinase (MT1-MMP). Both CD44 and MT1-MMP are partially located in Triton X-100-insoluble domains, but no functional link has yet been established between them. In the present study, we studied the regulation of HA cell surface binding in U-87 glioma cells. We show that an MMP-dependent mechanism regulates the intrinsic cell surface binding of HA as ilomastat, a broad MMP inhibitor, increased HA binding to glioma cells. HA binding was also rapidly and specifically up-regulated by 3-fold by type I collagen in U-87 cells, which also induced a significant morphological reorganization associated with the activation of a latent form of MMP-2 through a MT1-MMP-mediated mechanism. Interestingly, caveolae depletion with a cell surface cholesterol-depleting agent beta-cyclodextrin triggered an additional increase (9-fold) in the binding of HA, in synergy with type I collagen. On the other hand, HA cell surface binding was diminished by the MEK inhibitor PD98059 and by the overexpression of a recombinant, wild type MT1-MMP, whereas its cytoplasmic-deleted form had no effect. Taken together, our results suggest that MT1-MMP regulates, through its cytoplasmic domain, the cell surface functions of CD44 in a collagen-rich pericellular environment. Additionally, we describe a new molecular mechanism regulating the invasive potential of glioma cells involving a MT1-MMP/CD44/caveolin interaction, which could represent a potential target for anti-cancer therapies.     

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFkappaB by hyaluronan oligosaccharides. NFkappaB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-alpha/beta. The link between activation of NFkappaB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFkappaB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFkappaB and p38 MAP kinase.     

Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14(+) peripheral blood monocytes.

Activation of T cells by Ag or stimulation of monocytes with inflammatory cytokines induces CD44 to bind to hyaluronan (HA), an adhesion event implicated in leukocyte-leukocyte, leukocyte-endothelial cell, and leukocyte-stromal cell interactions. We have previously shown that TNF-alpha induces CD44 sulfation in a leukemic cell line, which correlated with the induction of HA binding and CD44-mediated adhesion. In this study, we establish that TNF-alpha and IFN-gamma induce HA binding and the sulfation of CD44 in CD14(+) PBMC, whereas no induced HA binding or CD44 sulfation was observed in CD14(-) PBMC stimulated with TNF-alpha. Treatment of cells with NaClO(3), an inhibitor of sulfation, prevented HA binding in a significant percentage of CD14(+) PBMC induced by TNF-alpha, LPS, IL-1beta, or IFN-gamma. Furthermore, stimulation with TNF-alpha or IFN-gamma in the presence of NaClO(3) reduced the ability of isolated CD44H to bind HA, demonstrating a direct effect of CD44H sulfation on HA binding. In contrast, the transient induction of HA binding in T cells by PHA was not affected by NaClO(3), suggesting that activated T cells do not use sulfation as a mechanism to regulate HA binding. Overall, these results demonstrate that inducible sulfation of CD44H is one mechanism used by CD14(+) peripheral blood monocytes to induce HA binding in response to inflammatory agents such as TNF-alpha and IFN-gamma.     

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.     

Glycosylation Provides Both Stimulatory and Inhibitory Effects on Cell Surface and Soluble CD44 Binding to Hyaluronan

Glycosylation has been implicated in the regulation of CD44-mediated cell binding of hyaluronan (HA). However, neither the relative contribution of N- and O-linked glycans nor the oligosaccharide structures that alter CD44 affinity for HA have been elucidated. To determine the effect of selective alteration of CD44 oligosaccharide composition on the affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant CD44–immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis. Using this cell line, a panel of recombinant glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surface CD44, the effect of any given glycosylation change on the ability of cell surface and soluble CD44 to bind HA could be compared. Four distinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal α2,3-linked sialic acid on N-linked oligosaccharides inhibited binding; (b) the first N-linked N-acetylglucosamine residue enhanced binding; (c) O-linked glycans on N-deglycosylated CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non–N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the intrinsic CD44 affinity for HA (K_d = 5 to >150 μM). The fourth augmented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.     

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

Hyaluronic acid complexed to biodegradable poly L‐arginine for targeted delivery of siRNAs

Background

Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively‐charged HA and cationic poly L ‐arginine (PLR) for siRNA delivery systems.

Methods

Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA.

Results

Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP‐mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS‐7 cells, in parallel with CD44 surface densities of these cell lines. TC₅₀ values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC₅₀ values that were 2‐fold and 23‐fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target‐specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)‐expressing B16F10 cells with RFP‐specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP‐specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues.

Conclusions

HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migration

In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH(2)-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393-738 of Tiam1) and an in vitro binding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K(d)) of 0.2 nM, which is comparable with CD44 binding (K(d) = approximately 0.13 nM) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression.     

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.