Galectin-3 regulates the adhesive interaction between breast carcinoma cells and elastin.

Galectin-3 is a beta-galactoside binding lectin whose precise physiological role is not yet defined. In the present studies, we questioned whether galectin-3 plays a role in the adhesion of breast carcinoma cells to elastin. The impetus for this analysis was the initial observation that the cellular receptor for elastin, the 67 kDa elastin/laminin protein may have galectin-like properties (Mecham et al. [1989] J. Biol. Chem. 264:16652-16657). We therefore analyzed the adhesion of breast carcinoma cells to microtiter wells coated with elastin under conditions which eliminate integrin participation in adhesion. The adhesion assay was done in the absence and presence of purified recombinant galectin-3. We hereby demonstrate that high concentrations of galectin-3 ligate breast carcinoma cells to microtiter wells coated with elastin. Galectin-3 also demonstrated a specific binding interaction with purified elastin in a dose and lactose dependent manner. Furthermore we demonstrated by immunoprecipitation that endogenous galectin-3 in breast carcinoma cells is associated with tropoelastin. Lastly, the breast carcinoma cells which expressed galectin-3 on their surface, demonstrated enhanced cellular proliferation on elastin compared to galectin-3 null expressing cells. These studies suggest that galectin-3 is capable of regulating the interactions between cells and elastin.     

Growth Stimulation of Human Skin Fibroblasts by Elastin-Derived Peptides

Elastin-derived peptides (kappa-elastin: KE, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12). Coated 44.4 μg/cm²insoluble elastin (iE) exhibited the same action; coated iE or KE significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed. Increased ³H thymidine incorporation and proliferative effect of HSF by KE (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density. Optimal proliferative effect was obtained at KE 8.5 10^-10M, a value close to the dissociation constant (k_D= 2.7 10^-10M) of KE to HSF. Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGWGA) also significantly stimulated, optimally at 7.0 10^-10M, HSF proliferation. It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF.     

Growth Stimulation of Human Skin Fibroblasts by Elastin-Derived Peptides

Elastin-derived peptides (kappa-elastin: KE, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12). Coated 44.4 μg/cm²insoluble elastin (iE) exhibited the same action; coated iE or KE significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed. Increased ³H thymidine incorporation and proliferative effect of HSF by KE (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density. Optimal proliferative effect was obtained at KE 8.5 10^?10M, a value close to the dissociation constant (k_D= 2.7 10^?10M) of KE to HSF. Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGWGA) also significantly stimulated, optimally at 7.0 10^?10M, HSF proliferation. It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF.     

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein''s lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.     

The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor

Laminin- and elastin-binding proteins were isolated by ligand affinity chromatography from plasma membranes of fetal bovine auricular chondroblasts and human A2058 melanoma cells. From both cell types, a 67-kDa protein was identified which bound to either elastin or laminin affinity resins. Structural and functional similarities between the elastin and laminin-binding proteins were suggested by 1) cross-reactivity between antibodies directed against the two proteins; 2) elution of the laminin receptor from laminin columns with soluble elastin peptides; and 3) modulation of substrate binding by galactoside sugars. In addition, extraction properties indicate that both receptors are peripheral membrane proteins whose association with the cell surface is mediated by their lectin properties. Mapping of the binding site on laminin suggests that the 67-kDa chondroblast receptor interacts with a hydrophobic elastin-like sequence in domain V of the B1 chain, and chemotaxis studies indicate that cell migration to elastin peptides and laminin involves the same receptor.     

Self-assembly of elastin-based peptides into the ECM: the importance of integrins and the elastin binding protein in elastic fiber assembly

The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.     

Novel mechanism that Trypanosoma cruzi uses to adhere to the extracellular matrix mediated by human galectin-3

Binding of Trypanosoma cruzi trypomastigotes to laminin is enhanced by galectin-3, a beta-galactoside binding lectin. The galectin-3 enhanced binding of trypanosomes to laminin is inhibited by lactose. Co-immunoprecipitations indicate that galectin-3 binds to the 45, 32 and 30 kDa trypanosome surface proteins. Binding of galectin-3 to the 45, 32 and 30 kDa surface proteins is inhibited by lactose. Polyclonal and a monoclonal antibodies to galectin-3 immunoprecipitated a major 64 kDa trypanosome surface protein. T. cruzi monoclonal antibody to mucin recognized the 45 kDa surface protein. The 45, 32 and 30 kDa surface proteins interact with galectin-3 in order to enhance trypanosome adhesion to laminin.     

Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a platelet cell-surface receptor for fibrinogen and fibronectin. A carboxyl-terminal decapeptide of the fibrinogen gamma-chain (Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val LGGAKQAGDV] and a tetrapeptide (Arg-Gly-Asp-Ser (RGDS] from the fibrinogen alpha-chain and the fibronectin cell-binding domain appear to mediate the binding of these ligands to GP IIb-IIIa. The present study was designed to examine the effects of these and related peptides on the structure of purified platelet GP IIb-IIIa. Treatment of GP IIb-IIIa with various synthetic peptides affected the glycoprotein so that GP IIb alpha became a substrate for hydrolysis by thrombin. The order of potency of these peptides was as follows: RGDS greater than LGGAKQAGDV greater than KGDS greater than RGES. This is the same order of potency in which these peptides inhibit fibrinogen binding to platelets. This effect was time-, temperature-, and concentration-dependent; RGDS induced a half-maximal effect at approximately 60 microM. In addition, RGDS, but not RGES, decreased the intensity of the intrinsic protein fluorescence of GP IIb-IIIa. Finally, the decapeptide or RGDS decreased the sedimentation coefficient of GP IIb-IIIa from 8.5 to 7.7 or 7.4 S, respectively, whereas RGES had a minimal effect. This decrease was accompanied by an increase in the Stoke's radius from 74 to 82 A with RGDS or 85 A with the decapeptide, indicating a peptide-induced unfolding of the GP IIb-IIIa complex. This change in conformation may be related to changes in the distribution and function of GP IIb-IIIa on the platelet surface that occur when adhesive proteins or peptides from the GP IIb-IIIa binding domains of these proteins bind to GP IIb-IIIa.     

Identification and signal transduction mechanism of elastin peptide receptor in human leukocytes

The existence of a novel receptor on human polymorphonuclear leukocytes (PMNLs) and monocytes was demonstrated, named soluble elastin peptide receptor. Soluble elastin peptides, like K-elastin, which are liberated from elastin fibres, can be found in the sera, and they possess several biological activities such as chemotaxis. Studying the effects of elastin peptides on leukocytes, it was found that: (i) the elastin peptide stimulates the oxidative burst, the intracellular free Ca2+ elevation through a specific receptor; and (ii) in the signal transduction mechanism of this elastin peptide receptor, the phosphatidylinositol breakdown is involved.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

The elastin receptor complex transduces signals through the catalytic activity of its Neu-1 subunit

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.     

Integrin engagement modulates the phosphorylation of focal adhesion kinase, phagocytosis, and cell spreading in molluscan defence cells

Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, alphaVbeta3 and beta1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120-125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg-Gly-Asp-Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.     

Synergistic activity of αvβ3 integrins and the elastin binding protein enhance cell-matrix interactions on bioactive hydrogel surfaces

Engineering materials suitable for vascular prostheses has been a significant challenge, especially in promoting extracellular matrix (ECM) development within synthetic materials. Herein we have utilized two different elastin mimetic peptide sequences, EM-19 and EM-23, to provide a template for ECM deposition when covalently incorporated into scaffold materials. Both peptides contain the hexapeptide sequence VGVAPG, which interacts with the cell surface receptor known as the elastin binding protein (EBP). Additionally, EM-23 contains an RGDS sequence intended for the peptide's interaction with the α(v)β(3) integrin. We first confirm that the presence of both peptides approximates the synergistic mechanism for elastic fiber assembly in vivo, a process that utilizes both the EBP and α(v)β(3). Peptides were then grafted onto the surface of a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel and their efficacy as templates for promoting cell adhesion, spreading, and elastin deposition was evaluated. Although both peptides were able to encourage smooth muscle cell (SMC) adhesion and elastin deposition over PEG-DA and PEG-RGDS controls, PEG-grafted EM-23 was proven to be the more promising motif for inclusion in synthetic substrates to be used in the engineering of vascular tissues, enhancing cell adhesion 60-fold and elastin content 2-fold compared with PEG-RGDS.     

Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands

In this study, specific interactions between immobilized RGDS (Arg-Gly-Asp-Ser) cell adhesion peptides and cell integrin receptors located on cell membranes are controlled in vitro using stimuli-responsive polymer surface chemistry. Temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(IPAAm-co-CIPAAm)) copolymer grafted onto tissue culture grade polystyrene (TCPS) dishes permits RGDS immobilization. These surfaces facilitate the spreading of human umbilical vein endothelial cells (HUVECs) without serum depending on RGDS surface content at 37 degrees C (above the lower critical solution temperature, LCST, of the copolymer). Moreover, cells spread on RGDS-immobilized surfaces at 37 degrees C detach spontaneously by lowering culture temperature below the LCST as hydrated grafted copolymer chains dissociate immobilized RGDS from cell integrins. These cell lifting behaviors upon hydration are similar to results using soluble RGDS in culture as a competitive substitution for immobilized ligands. Binding of cell integrins to immobilized RGDS on cell culture substrates can be reversed spontaneously using mild environmental stimulation, such as temperature, without enzymatic or chemical treatment. These findings are important for control of specific interactions between proteins and cells, and subsequent "on-off" regulation of their function. Furthermore, the method allows serum-free cell culture and trypsin-free cell harvest, essentially removing mammalian-sourced components from the culture process.     

Binding of elastin to Staphylococcus aureus.

Many pathogenic bacteria specifically bind to components of the extracellular matrix. In this study, we report the specific association of Staphylococcus aureus with elastin, a major structural component of elastic tissue. Competition assays in which the binding of radiolabeled tropoelastin was inhibited by excess unlabeled elastin peptides, but not by other proteins, established the specificity of the interaction. Kinetic studies showed that tropoelastin binding to the bacteria was rapid and saturable. Scatchard analysis of the equilibrium binding data indicated the presence of a single class of high affinity binding sites (KD approximately 4-7 nM) with approximately 1000 sites per organism. Protease susceptibility suggested that the elastin binding moiety on S. aureus was a protein, which was confirmed by the isolation of a 25-kDa elastin-binding protein from S. aureus extracts through affinity chromatography. Using a truncated form of tropoelastin, the bacterial binding domain on elastin was mapped to a 30-kDa fragment at the amino end of the molecule. Although the precise amino acid sequence recognized by the staphylococcal elastin receptor has not been characterized, it is clearly different from the region of tropoelastin that specifies binding to mammalian elastin receptors.     

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide

A tetrapeptide Arg-Gly-Asp-Ser (RGDS) has been shown to be a versatile cell recognition signal of extracellular matrix components for the interaction with cells. We introduced the RGDS tetrapeptide into a truncated form of protein A, a staphylococcal immunoglobulin-binding protein, by inserting an oligonucleotide cassette encoding the tetrapeptide into the coding region of the protein A expression vector pRIT2T. The mutagenized protein was capable of not only binding to immunoglobulin G but also mediating cell attachment and spreading onto an inert substrate. Cell adhesion mediated by the mutagenized protein was inhibitable by a synthetic peptide Gly-Arg-Gly-Asp-Ser but not by a related peptide Gly-Arg-Gly-Glu-Ser, confirming that the inserted RGDS tetrapeptide served as a recognition signal for cell adhesion. Furthermore, the RGDS-containing protein was capable of adhering cells onto an immunoglobulin-coated surface which could not by itself support cell adhesion. Thus, the cell adhesive and immunoglobulin binding activities of the mutagenized protein appear to function coordinately. The protocol described here is essentially applicable to any protein and, therefore, provides a general principle in tailoring novel multifunctional proteins having cell adhesive activity.     

肝癌细胞-胞外基质粘附性与粘附识别序列的相关性

以微管吸吮技术研究了人肝癌细胞在ＩＶ型胶原／层粘连蛋白（ＬＮ）／纤维连结蛋白（ＦＮ）裱衬表面的粘附性。进一步,用四种人工合成肽精－甘－天冬－丝（ＲＧＤＳ）、甘－精－甘－天冬－苏－脯ＧＲＧＤＴＰ）、酪－异亮－甘－丝－精（ＹＩＧＳＲ０和半胱－天冬－脯－甘－酪－异亮－甘－丝－精（ＣＤＰＧＹＩＧＳＲ）研究了肝癌细胞粘附性对两种粘附识别序列ＲＧＤ和ＹＩＧＳＲ的依赖性。为了归纳和整理实验结果,根据竞争性抑制的     

Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.     

The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.