Noorjahan Panjwani

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins.

Galectins-3 and -7, but not Galectin-1, Play a Role in Re-epithelialization of Wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study, using cornea as a model, we demonstrate for the first time the importance of carbohydrate-binding proteins galectins-3 and -7 in re-epithelialization of wounds. In two different models of corneal wound healing, re-epithelialization of wounds was significantly slower in galectin-3-deficient (gal3−/−) mice compared with wild-type (gal3+/+) mice. In contrast, there was no difference in corneal epithelial wound closure rates between galectin-1-deficient and wild-type mice. Quantitation of the bromodeoxyuridine-labeled cells in gal3+/+ and gal3−/− corneas revealed that corneal epithelial cell proliferation rate is not perturbed in gal3−/− corneas. Exogenous galectin-3 accelerated re-epithelialization of wounds in gal3+/+ mice but, surprisingly, not in the gal3−/− mice. Gene expression analysis using cDNA microarrays revealed that healing corneas of gal3−/− mice contain markedly reduced levels of galectin-7 compared with those of gal3+/+ mice. More importantly, unlike galectin-3, galectin-7 accelerated re-epithelialization of wounds in both gal3−/− and gal3+/+ mice. In corresponding experiments, recombinant galectin-1 did not stimulate the corneal epithelial wound closure rate. The extent of acceleration of re-epithelialization of wounds with both galectin-3 and galectin-7 was greater than that observed in most of the published studies using growth factors. These findings have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Clinically Atypical Granular Corneal Dystrophy with Pathologic Features of Lattice-like Amyloid Deposits: A Study of Three Families

Four patients from families in Pennsylvania, Massachusetts, and Argentina were diagnosed clinically as having granular dystrophy. Results of pathologic examination of the corneal buttons from each patient after penetrating keratoplasty confirmed granular deposits in the anterior third of the stroma. Amyloid was demonstrated within some of these granular deposits by Congo red staining with birefringence and dichroism and by electron microscopy. In addition to the morphologically granular deposits, numerous fusiform deposits identified as amyloid by histochemistry and electron microscopy and morphologically identical to those seen in lattice corneal dystrophy were detected deep to the granular deposits. It was further shown that the histochemical pattern of staining of the granular material by a series of lectins was similar to that present in corneas with lattice dystrophy. Although a relationship between these patients cannot be definitively proven, each family traces its origins to the Italian province of Avellino.

Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells.

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3−/− and Mgat5−/− mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.

Participation of pigment epithelium in ocular immune privilege. 3. Epithelia cultured from iris, ciliary body, and retina suppress T-cell activation by partially non-overlapping mechanisms

Purpose : The ocular microenvironment is immunosuppressive and anti-inflammatory. Since various ocular pigmented epithelia contribute to this microenvironment, we studied the relative capacities of pigment epithelial (PE) cells cultured from the iris, ciliary body, and retina of mouse eyes to suppress T-cell activation in vitro. Methods : Pigment epithelium was cultured from iris, ciliary body, and retina for 14 days, then assayed for the capacity, directly or across transwell membranes, to suppress mixed lymphocyte reactions and anti-CD3 stimulation of T cells. Potential molecules responsible for suppression were examined by attempting to block suppression with appropriate reagents, and by using mice with pertinent mutant or disrupted genes. Results : We found that PE cells from all three ocular tissue sources profoundly suppressed T-cell activation in vitro. While iris PE suppressed poorly when separated from T cells by a transwell membrane (implying that cell contact is necessary), retina PE suppressed fully even in the presence of such a membrane (implying that soluble factors were responsible). Ciliary body PE used both soluble factors as well as cell contact to achieve suppression. Suppression could not be ascribed to TGFß, IFN?, TNFa, CD48, or ICAM-1, or to interactions between CD40 and CD154, or CD95 and CD95 ligand. Galectin-1, a galactoside-binding protein, was found to be expressed on all cultured PE cells, but only retinal pigment epithelium (RPE) from galectin-1 KO mice showed reduced capacity to inhibit T-cell activation. Conclusions : Cultured pigment epithelia from iris, ciliary body, and retina comparably suppress T-cell activation in vitro, but by partially different mechanisms. Although RPE cells suppress in part through expression of galectin-1, the molecular mediators of suppression by iris and ciliary body PE remain to be identified.

Pathogenesis of Acanthamoeba Keratitis

Acanthamoeba keratitis (AK) is a serious infection of the cornea. At present, diagnosis of the disease is not straightforward and treatment is very demanding. While contact lens wear is the leading risk factor for A K, Acanthamoeba parasites are increasingly recognized as an important cause of keratitis in non-contact lens wearers. The first critical step in the pathogenesis of infection is the adhesion of the microbe to the surface of the host tissues. Acanthamoebae express a major virulence protein, the mannose-binding protein (MBP), which mediates the adhesion of amoebae to the surface of the cornea. The MBP is a transmembrane protein with characteristics of a typical cell surface receptor. Subsequent to the MBP-mediated adhesion to host cells, the amoebae produce a contact-dependent metalloproteinase and several contact-independent serine proteinases. These proteinases work in concert to produce a potent cytopathic effect (CPE ) involving killing of the host cells, degradation of epithelial basement membrane and underlying stromal matrix, and penetration into the deeper layers of the cornea. In the hamster animal model, oral immunization with the recombinant MBP protects against AK, and this protection is associated with an increased level of anti-MBP IgA in tears of protected animals. Normal human tear fluid contains IgA antibodies against Acanthamoeba MBP that is likely to provide protection by inhibiting the adhesion of parasites to host cells. Indeed, in in vitro CPE assays, even a low concentration of tears (10 microL of undiluted tears per milliliter of media) almost completely inhibits Acanthamoeba-induced CPE . In addition to adherence-inhibiting, IgA-mediated protection, human tears also contain IgA-independent factors that provide protection against Acanthamoeba-induced CPE by inhibiting the activity of cytotoxic proteinases. Characterization of the CPE-inhibitory factors of human tears should lead to a better understanding of the mechanism by which the tissues of the host resist the infection and also help decode circumstances that predispose to Acanthamoeba infections.

Galectin-3 promotes lamellipodia formation in epithelial cells by interacting with complex N-glycans on α3β1 integrin

Recent studies have shown that galectin-3 (Gal-3; also known as LGALS3), a β-galactoside-binding lectin, promotes cell migration during re-epithelialization of corneal wounds. The goal of this study was to characterize the molecular mechanism by which Gal-3 stimulates cell migration. We demonstrate here that exogenous Gal-3, but not Gal-1 or Gal-8, promotes cell scattering and formation of lamellipodia in human corneal epithelial cells in a β-lactose-inhibitable manner. α3β1 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Preincubation of cells with anti-α3 integrin function-blocking antibody significantly inhibited the induction of lamellipodia by Gal-3. Furthermore, exogenous Gal-3 activated both focal adhesion kinase, a key regulator of integrin-dependent intracellular signaling, and Rac1 GTPase, a member of the family of Rho GTPases, well known for its role in the reorganization of the actin cytoskeleton and formation of lamellipodial extensions. Experiments involving knockdown of β-1,6-N-acetylglucosaminytransferase V, an enzyme that synthesizes high-affinity glycan ligands for Gal-3, revealed that carbohydrate-mediated interaction between Gal-3 and complex N-glycans on α3β1 integrin plays a key role in Gal-3-induced lamellipodia formation. We propose that Gal-3 promotes epithelial cell migration by cross-linking MGAT5-modified complex N-glycans on α3β1 integrin and subsequently activating α3β1-integrin–Rac1 signaling to promote lamellipodia formation.

Molecular basis for MMP9 induction and disruption of epithelial cell–cell contacts by galectin-3

ABSTRACT Dynamic modulation of the physical contacts between neighboring cells is integral to epithelial processes such as tissue repair and cancer dissemination. Induction of matrix metalloproteinase (MMP) activity contributes to the disassembly of intercellular junctions and the degradation of the extracellular matrix, thus mitigating the physical constraint to cell movement. Using the cornea as a model, we show here that a carbohydrate-binding protein, galectin-3, promotes cell–cell detachment and redistribution of the tight junction protein occludin through its N-terminal polymerizing domain. Notably, we demonstrate that 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 (also known as EMMPRIN and basigin) on the cell surface. Using galectin-3-knockout mice in an in vivo model of wound healing, we further show that increased synthesis of MMP9 at the leading edge of migrating epithelium is regulated by galectin-3. These findings establish a new galectin-3-mediated regulatory mechanism for induction of metalloproteinase expression and disruption of cell–cell contacts required for cell motility in migrating epithelia.

The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading

Primary open angle glaucoma (POAG) is a major blindness-causing disease, characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork (TM) lining the aqueous outflow pathway modulates the aqueous outflow facility. TM cell adhesion, cell-matrix interactions, and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. In a recent study, we demonstrated that galectin-8 (Gal8) modulates the adhesion and cytoskeletal arrangement of TM cells and that it does so through binding to beta(1) integrins and inducing Rho signaling. The current study is aimed at the characterization of the mechanism by which Gal8 mediates TM cell adhesion and spreading. We demonstrate here that TM cells adhere to and spread on Gal8-coated wells but not on galectin-1 (Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TM cells to Gal8-coated wells was abolished by a competing sugar, beta-lactose, but not by a noncompeting sugar, sucrose. Also, a trisaccharide, NeuAcalpha2-3Galbeta1-4GlcNAc, which binds specifically to the N-CRD of Gal8, inhibited the spreading of TM cells to Gal8-coated wells. In contrast, NeuAcalpha2-6Galbeta1-4GlcNAc which lacks affinity for Gal8 had no effect. Affinity chromatography of cell extracts on a Gal8-affinity column and binding experiments with plant lectins, Maakia Amurensis and Sambucus Nigra, revealed that alpha(3)beta(1), alpha(5)beta(1), and alpha(v)beta(1) integrins are major counterreceptors of Gal8 in TM cells and that TM cell beta(1) integrins carry predominantly alpha2-3-sialylated glycans, which are high-affinity ligands for Gal8 but not for Gal1 or Gal3. These data lead us to propose that Gal8 modulates TM cell adhesion and spreading, at least in part, by interacting with alpha2-3-sialylated glycans on beta(1) integrins.

BINDING OF ACANTHAMOEBA TO 23 MANNOSE-GLYCOPROTEINS OF CORNEAL EPITHELIUM : EFFECT OF INJURY

Purpose. Acanthamoeba keratitis is a sight-threatening corneal infection. It is known that: (i) more amoebae bind to the surface of injured corneas than to the normal corneal surface and (ii) mannose-containing glycoproteins (GPs) possess binding sites for Acanthamoeba. The present study was undertaken to determine whether subtle corneal surface injury exposes mannose-GPs and whether more amoebae bind to the mannose-GPs of injured corneas than to those of normal corneas.Methods. Corneal cup assays were developed to determine whether corneal surface injury exposes binding sites for a mannose/glucose-specific lectin, succinylated-concanavalin A (s-ConA). To determine whether injury exposes mannose-GPs, corneal surface proteins were biotinylated, biotin-labeled mannose-GPs were allowed to bind to s-ConA-agarose beads and were analyzed by SDS-polyaerylamide gel electrophoresis (PAGE). Amoeba binding to mannose-GPs of corneal epithelia was analyzed by PAGE-blot overlay assays.Results. S-ConA binding site densi...