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Epithelial cells are closely connected to each other and to the extracellular matrix by a set of adhesive contacts that provide tissues with unique barrier properties and play a prominent role in cell morphology, tissue physiology, and cell signaling. This review highlights advances made in understanding the contributions of galectin-3, a carbohydrate-binding protein with affinity toward β-galactosides, as a modulator of epithelial junction assembly and function. The interactions of galectin-3 within adhesive structures are discussed in relation to wound healing and tumor progression.
The presence of specialized cell-cell and cell-extracellular matrix junctions within a polarized epithelium provides tissues with unique barrier functions, and is critical for cell morphology, tissue physiology, and cell signaling.1,2 Adhesive structures connected to intermediate filaments (desmosomes) and to microfilaments (adherens junctions and tight junctions) ensure the appropriate stability and protection of epithelial sheets.3 Desmosomes and adherens junctions provide the mechanical strength to the epithelial cell contacts, whereas tight junctions (zonula occludens) form a selective permeable barrier to ions and small molecules.4 Other types of junctions include the gap junction, which provides direct chemical communication between adjacent cells, and the hemidesmosomes, specialized junctional complexes that contribute to the attachment of epithelial cells to the underlying basement membrane. Loss of cell polarity and reorganization of adhesion junctions are hallmarks of wound repair and cancer,5-7 and are intimately associated with the transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial–mesenchymal transition.8
It has become increasingly clear that members of the galectin class of β-galactoside-binding proteins play a critical role as modulators of wound re-epithelialization and tumor progression.9,10 Based on molecular characterization, galectins (previously known as S-type lectins) are defined by the presence of a canonical carbohydrate recognition domain (CRD) of approximately 130 amino acids containing a conserved sequence motif known to make contacts with β-galactoside ligands.11 They are named and numbered chronologically in order of discovery, and so far, 15 have been found in mammals. These have been classified into 3 groups according to their structure: prototypical, chimeric, and tandem repeat. The prototypical galectins (galectin-1, -2, -5, -7, -10, -11, -13, -14 and -15) contain a single CRD that may associate to form homodimers; chimeric galectins, of which galectin-3 is the only known species found in vertebrates, contain a single CRD and a large amino-terminal domain that contributes to self-aggregation; and tandem-repeat galectins (galectin-4, -6, -8, -9 and -12) contain 2 CRDs within a single polypeptide. Each galectin recognizes different types of glycans in a complex manner that depends on the multivalency and oligomeric state of the galectins, the multivalency of their glycoconjugate ligands, and the mode of presentation of the glycans.11
Galectins can be found in numerous cell types and tissues, within intracellular (nucleus, mitochondria and cytoplasm) and extracellular (cell surface and extracellular matrix) compartments, where they modulate a variety of biological functions. Functions related to their intracellular localization have now been documented, and include modulation of cell growth and apoptosis, and pre-mRNA splicing.12 Galectins also mediate signal transduction events on the cell surface as well as a variety of extracellular processes such as cell adhesion and migration.13,14 It is well known that epithelial cells display dynamic behaviors important to the rearrangement, movement and shape of the cell, during regenerative processes such as wound healing or during pathogenetic processes such as cancer invasion.7 As evidenced by the number of comprehensive reviews of the field, significant progress has been made in the identification of the roles of galectins in epithelia.15-19 In this review, we summarize recent progress made in establishing the contribution of galectin-3 as a regulator of the epithelial junction.
Galectin-3, also known as Mac-2, L-29 or CBP35, is a 28- to 35-kD protein identified in the early 1980s on the surface of murine macrophages.20 It has a single CRD domain of approximately 130 amino acids in the C-terminus, and a 110–130 amino acid N-terminus with 7–14 repeats of a 9-amino acid sequence rich in proline, glycine, alanine and tyrosine—sometimes referred to as the collagen-like domain.21 The crystal structure of the galectin-3 CRD has been reported when in association with lactose and N-acetyllactosamine. Similarly to galectins-1 and -2, the galectin-3 CRD consists of a β-sandwich structure made of 6-stranded (named S1−S6) and 5-stranded (F1−F5) β-sheets, with the carbohydrate binding site located within a shallow groove over β-strands S4−S6.22 A significant rearrangement of the protein backbone near the binding site occurs in the absence of ligand, with the S4−S5 and the S5−S6 loops of the binding site moving inward to close up the binding cleft.23
Galectin-3, like other members of the galectin family, lacks both a membrane-anchoring domain and a signal sequence. Instead of being transferred into the endoplasmic reticulum and Golgi compartments for classical secretion, galectin-3 is synthesized on clusters of free ribosomes in the cytoplasm of the cell as an non-glycosylated protein before secretion.24 The N-terminal domain of galectin-3 has been implicated in the non-classical secretion of the protein.25 Interestingly, whether galectin-3 is secreted via basolateral or apical membranes in polarized epithelial cells depends on the cell type and the experimental conditions used. For instance, galectin-3 is primarily secreted from the apical domain in monolayer cultures of Madin-Darby canine kidney (MDCK) cells grown in Transwell inserts,26 but is directed to basolateral surfaces in MDCK cells grown as polarized cysts in Matrigel.27 The signals that drive galectin-3 secretion into different polarity compartments are so far unknown.
Studies on the carbohydrate-binding activity and specificity of galectin-3 have shown that N-acetyllactosamine (Galβ1,4(3)GlcNAc) is the preferential ligand, although it can also accommodate other galactose-containing structures.28 Affinity of galectin-3 toward glycosylated receptors is dependent on the metabolic flux within the target cell. Data collected during the past few years support an important function of the hexosamine pathway in modulating the strength of the association of individual receptors within a cell-surface galectin lattice.29,30 Quantitative precipitation studies also indicate that galectin-3 promptly precipitates as a pentamer in the presence of multivalent N-acetyllactosamine ligands in a process mediated through the N-terminal domain.31 In cell culture, oligomerization of galectin-3 occurs on the cell surface at physiological concentrations of the lectin, resulting in galectin-3 lattices that are robust and resistant to lateral movement of cell surface components.32 Increasing concentrations of multivalent glycoprotein ligands promote the formation of these galectin-3 lattices, now known to be involved in the regulation of receptor clustering, endocytosis and signaling.30 In the absence of saccharide ligands, galectin-3 self-associates in a manner that is dependent on the C-terminal domain.33
The major structural modifications of galectin-3 are phosphorylation and proteolysis, which occur outside the CRD. Phosphorylation of galectin-3 was initially observed in confluent 3T3 fibroblasts in both the cytosol and nucleus.34,35 Huflejt et al. later determined the site of phosphorylation of galectin-3 in confluent polarized MDCK cells, taking advantage of the abundant levels of galectin-3 (about 1% of total soluble cell protein) in these cells.36 Their analyses revealed phosphate incorporation only at serine, primarily at serine 6, with lower amounts at serine 12. Although it has been suggested that phosphorylation may modulate the translocation of galectin-3 between different cellular compartments, mutational studies in BT-549 human breast carcinoma cells show no obvious difference in cellular localization between wild-type and serine 6 mutated transfectants.37 In contrast, it has been proposed that phosphorylation can modulate protein-carbohydrate interactions serving as an “on/off” switch for the glycan binding capabilities of galectin-3.38 This hypothesis is supported by data using synthetic peptides derived from the rather flexible human galectin-3 N-terminal domain, which can interact with the CRD in a serine and tyrosine phosphorylation-dependent manner.39
The N-terminal domain of galectin-3 has been referred to as the collagen-like domain, based on the presence of a motif susceptible to collagenase degradation.21 It is now clear that the Ala62-Tyr63 bond within the galectin-3 N-terminus contains a cleavage site for the matrix metalloproteinases MMP2 (gelatinase A) and MMP9 (gelatinase B).40 The biological implications of the proteolytic degradation of galectin-3 by metalloproteinases are multiple. For example, proteolysis can modulate the biological activities of galectin-3 by reducing the expression of the protein on the cell-surface.40,41 Also, the enzymatic cleavage of the galectin-3 by metalloproteinases occurs within the proline- and glycine-rich N-terminal domain and, therefore, may interfere with its oligomerization properties and the clustering of its receptors.42 Finally, cleavage of galectin-3 by metalloproteinases alters the structure of the CRD to keep this domain in a high affinity conformation.43
Similarly to other galectins, the N-terminus of galectin-3 can also be post-translationally modified by cleavage of the N-terminal methionine followed by acetylation at alanine 2—common post-translational modifications found in cytoplasmic proteins.44
Upon secretion, galectins can bind glycosylated receptors on cell surfaces and extracellular matrices to regulate cell-cell and cell-matrix adhesion.19,45 Following binding to receptors (e.g., matrix components such as laminin and integrins on the cell surface), galectins can either inhibit or enhance adhesion through multiple mechanisms that include ligation of glycans and recruitment of signaling molecules.19,46 The role of galectins as agonists or antagonists of adhesion depends on the level of expression and location of the lectin, the glycosylation pattern of the receptors, the binding valency of the interaction, and the repertoire of receptors clustered by the lectin.19 In epithelial tissues, galectins are also thought to have a dual positive or negative regulatory function during the assembly and stabilization of intercellular junctions. As indicated below, contributions of chimeric galectin-3 to the epithelial junction have been described in desmosomes, adherens junctions and tight junctions.
Desmosomes are intercellular junctions that anchor intermediate filaments to the plasma membrane, forming a network of adhesive bonds that provide mechanical strength to tissues. Recent data indicates that galectin-3 enhances epithelial intercellular adhesion through association with the ectodomain of desmoglein-2, a calcium-binding transmembrane glycoprotein component of desmosomes in vertebrate epithelial cells. In experiments using intestinal epithelial cells, Jiang et al. demonstrated that galectin-3 binds to N-linked β-galactosides on the extracellular domain of desmoglein-2.47 Moreover, in the absence of galectin-3, desmoglein-2 is internalized from the plasma membrane and undergoes proteasomal degradation. These events resulted in decreased intercellular adhesion, supporting a role for galectin-3 in controlling desmosome structure and function. In addition to intestinal cells, it is noteworthy to mention that galectin-3-reactive binding sites have also been found to co-localize with desmoglein by immunohistochemical analysis in human corneal and conjunctival epithelia, suggesting that galectin-3 also contributes to desmosome formation in stratified epithelia.48
One of the most important and ubiquitous families of adhesive proteins required for the maintenance of solid tissues are the cadherins, a group of transmembrane calcium-dependent proteins that serve as the major adhesion molecules located within adherens junctions.49 The ectodomain of cadherins mediates intercellular adhesion while the cytosolic region binds to adaptor proteins such as the catenins that connect to the actin cytoskeleton. N-cadherin is found in many different types of intercellular junctions and often replaces E-cadherin at cell-cell junctions of tumor cells that have undergone epithelial-to-mesenchymal transition. Recent evidence by Boscher et al. indicates that galectin-3 lattices are critical regulators of N-cadherin dynamics and cell-cell junction stability in mammary carcinoma cells.50 Using a combination of crosslinking and immunoprecipitation approaches, the authors demonstrated that galectin-3 interacts directly with N-cadherin. Further, they showed that galectin-3 and N-cadherin accumulate at the junctions with the raft marker ganglioside GM1 to promote destabilization of cell-cell junctions. These negative regulatory processes appear to be controlled by N-glycans present on N-cadherin, predominantly by branched N-glycans generated by N-acetylglucosaminyltransferase V (Mgat5) activity. Abrogation of N-cadherin glycosylation by targeting Mgat5 in human fibrosarcoma HT1080 cells resulted in promotion of N-cadherin-mediated cell-cell adhesion and decrease of cell migration and invasion.51,52 Similarly, disruption of the galectin-3 lattice by deletion of Mgat5 in mammary carcinoma cells also resulted in the stabilization of cell-cell junctions, suggesting that interaction of galectin-3 with N-glycans present on N-cadherin enhances the turnover of N-cadherin and negatively regulates intercellular adhesion.50
Tight junctions are intramembrane multiprotein complexes composed of integral plasma membrane linker proteins, such as occludin and claudins, and cytoplasmic adaptor proteins, such as zonula occludens. Occludin, a 65-kDa tetraspan membrane protein, is crucial for maintaining the stability of tight junctions and for regulating actin organization and epithelial migration.53 Studies in our laboratory using the cornea as a model have recently shown that galectin-3 promotes cell–cell detachment and redistribution of occludin through its N-terminal polymerizing domain.54 In these experiments, galectin-3 initiates cell–cell disassembly by inducing matrix metalloproteinase expression in a manner that is dependent on the interaction with and clustering of the matrix metalloproteinase inducer CD147 on the cell surface. The requirement of galectin-3 for matrix metalloproteinase induction suggests that galectin-3 is critical to mitigating the physical constraint to cell movement.
Encouraging progress has been made in the last decade identifying the contribution of galectins to wound repair.10 Galectin-3 has been shown to promote re-epithelialization of wounds in epithelia from the cornea,55 colon,56 and skin.57 The molecular mechanisms, by which galectin-3 modulates wound healing in epithelial cells, involve interaction with Mgat5-modified complex N-glycans on α3β1 integrin and promotion of lamellipodia formation.58 To promote cell migration, galectin-3 activates both focal adhesion kinase, a key regulator of integrin-dependent intracellular signaling, and Rac1 GTPase, a member of the family of Rho GTPases crucial to the reorganization of the actin cytoskeleton and formation of lamellipodial extensions.58
Despite findings supporting a role for galectin-3 in wound repair, the direct contribution of this lectin to the adhesive structures in migrating epithelia remains understudied. Epithelial cells at the leading edge of wounds require significant fluidity to change their shape.59 Abrogation of galectin-3 has been shown to impair matrix metalloproteinase expression in an in vivo model of wound healing, suggesting that galectin-3-mediated matrix metalloproteinase expression is necessary to rearrange the position of the epithelial cell in order to facilitate re-epithelialization.54,55 As mentioned above, this process appears to be mediated by CD147. Both affinity chromatography and confocal microscopy studies in human keratinocytes have revealed that galectin-3 binds to, and promotes cell surface redistribution of CD147, concomitant with cell–cell detachment and occludin redistribution.54
The role of galectin-3 in wound repair does not appear to be associated uniquely to the cell-cell junction, as epithelial cells lacking galectin-3 expression are also known to interact poorly with their extracellular matrices.15 A potential mechanism by which galectin-3 modulates these interactions seem to involve the glycosylation status of integrins and components of the extracellular matrix. This seems to be the case for laminin-332, a basement membrane protein that promotes cell migration and is over-expressed at sites of epidermal wounds.60 Elegant studies from Kariya et al. have shown that galectin-3 significantly enhances the cell motility of keratinocytes through recognition of poly-N-acetyllactosamine-modified laminin-332, but not laminin-332 containing carbohydrates with low galectin-3 affinity.61 The authors further demonstrated that galectin-3 promotes the association of laminin-332 with α6β4 integrin, which is part of the hemidesmosomal adhesion complex,62,63 and epidermal growth factor receptor, to form clusters of cell surface proteins important to the regulation of cellular signaling and cell migration.61 More recently, galectin-3 has been identified as a factor that enhances corneal epithelial wound healing by promoting adhesion to collagen IV, a major structural component of basement membranes.64
Changes in galectin-3 expression and localization are commonly seen in tumors and have been associated with cancer cell growth, transformation, adhesion, invasion and metastasis.65 Overexpression of galectin-3 has been shown to enhance tumor cell adhesion to the extracellular matrix and promote cancer dissemination. Such an effect of galectin-3 is attributed partly through association with a range of the extracellular matrix glycoproteins, such as fibronectin,66,67 collagen IV,67 elastin,68 and laminin.69
Crucial to the understanding of cancer cell invasion and metastasis is the elucidation of the dynamic roles of focal adhesion proteins during cell migration. During the past decade, researchers have provided evidence that supports the involvement of galectin-3 in the stimulation of focal adhesion turnover and tumor cell motility.70 Mgat5-modified N-glycans on integrins were initially proposed as potential candidates in promoting focal adhesion turnover, cell migration and tumor growth.71 It is now established that, at least in mammary tumor cells, galectin-3 interacts with Mgat5-branched N-glycans on integrin α5β1 to promote focal adhesion turnover, phosphatidylinositol 3-kinase signaling and cell spreading.66,72 Interestingly, in vivo studies have also shown that Mgat5−/− cells have stable focal adhesions and express reduced levels of caveolin-1.71,73 Recruitment of tyrosine-phosphorylated caveolin-1 following galectin-3–mediated activation of integrins is now considered a central mechanism to promote focal adhesion disassembly and tumor cell migration.74
In addition to focal adhesions, loss of intercellular cell-cell contacts also contributes to tumor invasion and metastasis, as it allows malignant cells to dissociate from the primary tumor mass. Localization of galectin-3 within intercellular junctional complexes has been reported in tumor cells. As mentioned before, galectin-3 destabilizes cell-cell junctions by enhancing junctional mobility of N-cadherin and other glycoconjugates critical to cell migration in mammary carcinoma cells.50 Further, galectin-3-reactive sites have been found to co-localize with the desmosomal proteins desmoplakin-1 and desmoglein in primary squamous carcinomas, pointing to a potential role of galectin-3 in mediating intercellular contacts in these tumors.75 Similarly, galectin-3 has been shown to co-immunoprecipitate in a carbohydrate-dependent manner with liver-intestine cadherin in ductal adenocarcinoma of the pancreas,76 although the contribution of this association to the junctional complex in tumor cells remains unclear.
Evidence from different model systems has accumulated in recent years implicating galectin-3 in the dynamic regulation of cell-cell and cell-extracellular matrix junctions (Fig. 1). Functions assigned to galectin-3 in adhesion complexes are diverse and include control of cell surface expression, mobility, and integrity, of adhesion proteins. Research on the role of galectin-3 in the epithelial junction is still in its infancy but, based on the multifunctional roles ascribed to galectin-3, it is entirely possible that there are yet more important galectin-3 partners and regulatory functions within the epithelial junction to be discovered. A thorough characterization of these associations and processes is warranted. In addition, further research of the role of galectin-3 on the epithelial junction might provide novel opportunities to a better understanding of the pathophysiology of epithelial diseases. Galectin-3 has been ascribed a plethora of functions during wound healing and cancer, and there is sufficient evidence to suggest that targeting galectins may have a significant effect on pathological processes.77 Through a greater understanding of the role of galectin-3 at the epithelial junction, there is no doubt that novel therapeutic targets will come to light.
An important gap in our knowledge also relates to the role of phosphorylation and proteolysis as modulators of the biological activities of galectin-3 at the epithelial junction. It is tempting to speculate that galectin-3 proteolysis influences re-epithelialization in wound healing and cell migration in cancer progression. This hypothesis is supported by data showing increased MMP7 expression in epithelial cells bordering intestinal ulcerations78 and elegant studies from Puthenedam et al. demonstrating that cleavage of the N-terminal region of galectin-3 by MMP7 impairs galectin-3-mediated wound healing.56 Moreover, galectin-3 cleavage is known to be an active process during tumor progression.79 Considering that phosphorylation and proteolysis can affect the glycan binding capabilities of galectin-3 and the clustering of galectin-3-binding partners on cell surfaces and extracellular matrices, analyses of the post-translational processing of this lectin will become crucial to understanding the dynamics of epithelial adhesion, motility, and invasion.
No potential conflicts of interest were disclosed.
This work was supported by a grant from the National Eye Institute of the National Institutes of Health (R01EY024031 to P. Argüeso).