Glycosaminoglycans and their synthetic mimetics inhibit RANTES-induced migration and invasion of human hepatoma cells.

The CC-chemokine regulated on activation, normal T-cell expressed, and presumably secreted (RANTES)/CCL5 mediates its biological activities through activation of G protein-coupled receptors, CCR1, CCR3, or CCR5, and binds to glycosaminoglycans. This study was undertaken to investigate whether this chemokine is involved in hepatoma cell migration or invasion and to modulate these effects in vitro by the use of glycosaminoglycan mimetics. We show that the human hepatoma Huh7 and Hep3B cells express RANTES/CCL5 G protein-coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans. Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells and to a lesser extent that of Hep3B cells. RANTES/CCL5 also stimulates the tyrosine phosphorylation of focal adhesion kinase and activates matrix metalloproteinase-9 in Huh7 hepatoma cells, resulting in increased invasion of these cells. The fact that RANTES/CCL5-induced migration and invasion of Huh7 cells are both strongly inhibited by anti-CCR1 antibodies and heparin, as well as by beta-d-xyloside treatment of the cells, suggests that CCR1 and glycosaminoglycans are involved in these events. We then show by surface plasmon resonance that synthetic glycosaminoglycan mimetics, OTR4120 or OTR4131, directly bind to RANTES/CCL5. The preincubation of the chemokine with each of these mimetics strongly inhibited RANTES-induced migration and invasion of Huh7 cells. Therefore, targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for human hepatocellular carcinoma.

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization.

BACKGROUND: Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. RESULTS: Using Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process. CONCLUSION: These findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported.

Stromal cell-derived factor-1/chemokine (C-X-C motif) ligand 12 stimulates human hepatoma cell growth, migration, and invasion.

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The aim of this study was to determine whether the chemokine stromal cell-derived factor 1 (SDF-1) induces the growth, migration, and invasion of human hepatoma cells. We show that SDF-1 G protein-coupled receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), and SDF-1 mRNA are expressed in human hepatoma Huh7 cells, which secrete and bind SDF-1. This binding depends on CXCR4 and glycosaminoglycans. SDF-1 associates with CXCR4, and syndecan-4 (SDC-4), a heparan sulfate proteoglycan at the plasma membrane of Huh7 cells, induces the growth of Huh7 cells by promoting their entry into the cell cycle, and inhibits the tumor necrosis factor-alpha-mediated apoptosis of the cells. SDF-1 also reorganizes Huh7 cytoskeleton and induces tyrosine phosphorylation of focal adhesion kinase. Finally, SDF-1 activates matrix metalloproteinase-9, resulting in increased migration and invasion of Huh7 cells. These biological effects of SDF-1 were strongly inhibited by the CXCR4 antagonist AMD3100, by a glycosaminoglycan, heparin, as well as by beta-D-xyloside treatment of the cells, or by c-jun NH(2)-terminal kinase/stress-activated protein kinase inhibitor. Therefore, the CXCR4, glycosaminoglycans, and the mitogen-activated protein kinase signaling pathways are involved in these events. The fact that reducing SDC-4 expression by RNA interference decreased SDF-1-induced Huh7 hepatoma cell migration and invasion strongly indicates that SDC-4 may be an auxiliary receptor for SDF-1. Finally, the fact that CXCR4 is expressed in hepatocellular carcinoma cells from liver biopsies indicates that the in vitro results reported here could be extended to in vivo conditions.

The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXCL12 and mediated by the matrix metalloproteinase-9.

We recently demonstrated that stromal cell-derived factor-1 (SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4 expressed by human primary lymphocytes and macrophages, and HeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signaling molecule. Here, we demonstrate that SDF-1 strongly accelerates the shedding of syndecan-4 ectodomains and to a lesser extent that of syndecan-1 from HeLa cells. The fact that this acceleration was not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4 mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence. Pre-treating the cells with heparitinases I, III, or with the protein kinase C (PKC) inhibitor, bisindolylmaleimide, significantly inhibited this accelerated shedding, which suggests the involvement of both cell-surface heparan sulfate and PKC transduction pathway. In contrast, Map Kinase or NF-kappaB pathway inhibitors had no effect. Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9) mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9 silencing by RNA interference strongly decreases the syndecan-1 and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1 also accelerates in a CXCR4-independent manner, the shedding of syndecan-1 and -4 from human primary macrophages, which is significantly inhibited by anti-MMP-9 antibodies. This strongly indicates the role of MMP-9 in these events occurring in both a tumoral cell line and in human primary macrophages. Because MMP-9 plays a crucial role in extracellular matrix degradation during cancer cell metastasis and invasion, and shed ectodomains of syndecans may likely be involved in tumor cell proliferation, these data further indicate the multiplicity of the roles played by SDF-1 on tumor cell biology.

Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12.

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.

RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44.

We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase--and protein kinase C-dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1).

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.

Interaction of RANTES with syndecan-1 and syndecan-4 expressed by human primary macrophages.

Interaction of RANTES with its membrane ligands or receptors transduces multiple intracellular signals. Whether RANTES uses proteoglycans (PGs) belonging to the syndecan family to attach to primary cells expressing RANTES G-protein-coupled receptors (GPCRs) was investigated. We demonstrate that RANTES specifically binds to high and low affinity binding sites on human monocyte-derived macrophages (MDM). We show by co-immunoprecipitation experiments that RANTES is associated on these cells with syndecan-1 and syndecan-4, but neither with syndecan-2 nor with betaglycan, in addition to CD44 and its GPCRs, CCR5 and CCR1. Glycosaminidases pre-treatment of the monocyte derived-macrophages strongly decreases the binding of RANTES to syndecan-1 and syndecan-4 and also to CCR5, and abolishes RANTES binding to CD44. This suggests that glycosaminoglycans (GAGs) are involved in RANTES binding to the PGs and that such bindings facilitate the subsequent interaction of RANTES with CCR5, on the MDM, characterized by low membrane expression of CCR5. The role of these interactions in the pathophysiology of RANTES deserves further study.

Interactions of heparin with human skin cells: binding, location, and transdermal penetration.

The development of new materials for tissue engineering of skin substitutes requires an increasing knowledge of their interactions with human skin cells. Since carbohydrate recognition is involved in numerous biologic processes, including skin regeneration, the aim of this study was to identify sugar receptors expressed at the surface of human dermic and epidermic cells. Binding of fluorescent sugar-polyhydroxyethylacrylamide derivatives was analyzed by flow cytofluorimetry on cultured human skin fibroblasts, keratinocytes, and melanocytes. We observed that these three cell types express a membrane receptor specific for GlcNAc6S. Since the polysaccharide heparin contains this sugar moiety, we further investigated the interactions of heparin with skin cells. We analyzed the in vitro cell binding and ex vivo diffusion with the Franz cell of heparin and of two other polysaccharides of similar molecular weight, dextran and chondroitin sulfate. We found evidence of the preferential binding of heparin on keratinocytes and its high transcutaneous penetration of skin. Altogether, our results describe the affinity of heparin for human skin cells and suggest it may be an excellent candidate for use in the skin delivery of drugs or cosmetics and also as an active component in engineered skin.

Binding of the CC-chemokine RANTES to syndecan-1 and syndecan-4 expressed on HeLa cells.

It is believed that proteoglycans influence biological properties of chemokines. We show that the CC chemokine RANTES binds not only to high-affinity binding sites on CCR5-positive HeLa cells but also to low-affinity binding sites on HeLa cells expressing or lacking RANTES G protein-coupled receptors. Coimmunoprecipitation studies demonstrate that RANTES forms complexes with glycanated syndecan (SD)-1 and -4, in addition to CCR5 on the CCR5-positive HeLa cells. Moreover, confocal microscopy analysis shows the colocalization of RANTES with SD-1 and -4. Glycosaminoglycans removal from the cells by glycosaminidases treatment prevented RANTES binding to SD-1 and -4 and decreased RANTES binding to CCR5 on the CCR5-positive HeLa cells. Removal of glycosaminoglycans by glycosaminidases treatment of the complexes, RANTES/SD-1/SD-4/+/-CCR5, immobilized on beads, reversed SD-1 and -4 bindings. Therefore, RANTES bindings to SD-1 and -4 depend on glycosaminoglycans and facilitate RANTES interaction with CCR5. Extracting plasma membrane cholesterol abolished the coimmunoprecipitation of SD-1 with RANTES, suggesting that rafts are involved in RANTES association to SD-1. Confocal microscopy analysis as well as coimmunoprecipitation experiments show a RANTES-independent heteromeric complex on the CCR5-positive HeLa cells, SD-1, SD-4, and CCR5. This complex is likely a functional unit in which proteoglycans may modulate RANTES binding to CCR5.

Human alpha-fetoprotein binds to primary macrophages.

We have previously reported that alpha-fetoprotein (AFP) inhibits infection of human monocyte-derived macrophages (MDM) by R5-HIV-1 strains and that a peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds to CCR5. We demonstrate here that AFP binds high- and low-affinity binding sites of MDM, characterized, respectively, by 5.15 and 100nM K(d) values. Heat denaturation or neuraminidase treatment of AFP inhibits this binding, suggesting the involvement of protein-protein and lectin-carbohydrate interactions. Moreover, AFP displaces V3Cs binding to MDM. In addition, MIP-1beta, the most specific CCR5 ligand, displaces AFP binding to MDM (IC(50)=4.3nM). Finally, we demonstrate that AFP binds to a ligand of HIV-gp120 V3Cs domain, CCR5, expressed by MDM and by HeLa cells expressing CCR5. Such binding is not observed in the presence of HeLa cells lacking CCR5. The present results provide strong evidence that AFP directly binds to CCR5 expressed by human primary macrophages and by transfected CCR5+ HeLa cells.

Role of N-glycans and SDF-1alpha on the coassociation of CD4 with CXCR4 at the plasma membrane of monocytic cells and blood lymphocytes.

CXCR4 is a coreceptor, along with CD4, for human immunodeficiency virus type 1 (HIV-1). Trimolecular complexes between HIV-1 glycoprotein (gp)120, CD4 and CXCR4 constitute a prerequisite for HIV entry. We studied whether CD4 is associated with CXCR4 on CD4+ CXCR4+ cells. Using the conformation-dependent anti-CXCR4 mAb 12G5, CD4 was coimmunoprecipitated with CXCR4 from the membrane of U937 cells which support HIV-1(LAI) efficient infection, and from that of peripheral blood lymphocytes (PBL). CD4 association with CXCR4 increased upon PBL coculture for 5 days with autologous monocytes, decreased upon treatment of the cells or the CD4-CXCR4 complex with either N-glycanase or stromal cell derived factor-1alpha (SDF-1alpha) and was abolished by incubation of the cells with both, N-glycanase and SDF-1alpha. This indicates that glycans are partly involved in CD4 association with CXCR4 and may partly explain the inhibitory effect of SDF-1alpha on HIV infection.