Zhiyi Cao

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.

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.

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...

Pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3

Lymphangiogenesis plays a pivotal role in diverse pathological conditions. Here, we demonstrate that a carbohydrate-binding protein, galectin-8, promotes pathological lymphangiogenesis. Galectin-8 is markedly upregulated in inflamed human and mouse corneas, and galectin-8 inhibitors reduce inflammatory lymphangiogenesis. In the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection. Further, in the murine model of herpes simplex virus keratitis, corneal pathology and lymphangiogenesis are ameliorated in Lgals8−/− mice. Mechanistically, VEGF-C-induced lymphangiogenesis is significantly reduced in the Lgals8−/− and Pdpn−/− mice; likewise, galectin-8-induced lymphangiogenesis is reduced in Pdpn−/− mice. Interestingly, knockdown of VEGFR-3 does not affect galectin-8-mediated lymphatic endothelial cell (LEC) sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtails both galectin-8- and VEGF-C-mediated LEC sprouting. Together, this study uncovers a unique molecular mechanism of lymphangiogenesis in which galectin-8-dependent crosstalk among VEGF-C, podoplanin and integrin pathways plays a key role.

Galectin-1–Mediated Suppression of Pseudomonas aeruginosa–Induced Corneal Immunopathology

Corneal infection with Pseudomonas aeruginosa leads to a severe immunoinflammatory lesion, often causing vision impairment and blindness. Although past studies have indicated a critical role for CD4+ T cells, particularly Th1 cells, in corneal immunopathology, the relative contribution of recently discovered Th17 and regulatory T cells is undefined. In this study, we demonstrate that after corneal P. aeruginosa infection, both Th1 and Th17 cells infiltrate the cornea with increased representation of Th17 cells. In addition to Th1 and Th17 cells, regulatory T cells also migrate into the cornea during early as well as late stages of corneal pathology. Moreover, using galectin-1 (Gal-1), an immunomodulatory carbohydrate-binding molecule, we investigated whether shifting the balance among various CD4+ T cell subsets can modulate P. aeruginosa–induced corneal immunopathology. We demonstrate in this study that local recombinant Gal-1 (rGal-1) treatment by subconjunctival injections significantly diminishes P. aeruginosa–mediated corneal inflammation through multiple mechanisms. Specifically, in our study, rGal-1 treatment significantly diminished corneal infiltration of total CD45+ T cells, neutrophils, and CD4+ T cells. Furthermore, rGal-1 treatment significantly reduced proinflammatory Th17 cell response in the cornea as well as local draining lymph nodes. Also, rGal-1 therapy promoted anti-inflammatory Th2 and IL-10 response in secondary lymphoid organs. Collectively, our results indicate that corneal P. aeruginosa infection induces a strong Th17-mediated corneal pathology, and treatment with endogenously derived protein such as Gal-1 may be of therapeutic value for the management of bacterial keratitis, a prevalent cause of vision loss and blindness in humans worldwide.

Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

Purpose The trabecular meshwork (TM) 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. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling. Methods Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-β1 integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined. Principal Findings We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-β1 integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632. Conclusions/Significance The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow.

Analysis of Differential Expression of Glycosyltransferases in Healing Corneas by Glycogene Microarrays

It is generally accepted that the glycans on the cell surface and extracellular matrix proteins play a pivotal role in the events that mediate re-epithelialization of wounds. Yet, the global alteration in the structure and composition of glycans, specifically occurring during corneal wound closure remains unknown. In this study, GLYCOv2 glycogene microarray technology was used for the first time to identify the differentially expressed glycosylation-related genes in healing mouse corneas. Of approximately 2000 glycogenes on the array, the expression of 11 glycosytransferase and glycosidase enzymes was upregulated and that of 19 was downregulated more than 1.5-fold in healing corneas compared with the normal, uninjured corneas. Among them, notably, glycosyltransferases, beta3GalT5, T-synthase, and GnTIVb, were all found to be induced in the corneas in response to injury, whereas, GnTIII and many sialyltransferases were downregulated. Interestingly, it appears that the glycan structures on glycoproteins and glycolipids, expressed in healing corneas as a result of differential regulation of these glycosyltransferases, may serve as specific counter-receptors for galectin-3, a carbohydrate-binding protein, known to play a key role in re-epithelialization of corneal wounds. Additionally, many glycogenes including a proteoglycan, glypican-3, cell adhesion proteins dectin-1 and -2, and mincle, and mucin 1 were identified for the first time to be differentially regulated during corneal wound healing. Results of glycogene microarray data were confirmed by qRT-PCR and lectin blot analyses. The differentially expressed glycogenes identified in the present study have not previously been investigated in the context of wound healing and represent novel factors for investigating the role of carbohydrate-mediated recognition in corneal wound healing.