Ilene K. Gipson

Distribution of mucins at the ocular surface

Mucins are vital for maintenance of a healthy, wet ocular surface. Once only thought to be secreted by goblet cells, mucins are now also known to be of the membrane-associated type. Stratified ocular surface epithelia express at their apical-tear fluid surface a repertoire of membrane-associated mucins including MUC1, MUC4, MUC16. These mucins are concentrated on the tips of the microplicae, forming a dense glycocalyx at the epithelial tear film interface. A major mucin of the secretory class is the goblet-cell-derived gel-forming mucin MUC5AC. A small soluble mucin, MUC7, is expressed by the lacrimal gland acini. Our hypothesis of the role/distribution of the secreted and membrane-associated mucins at the ocular surface is that the secreted mucins are soluble in the tear fluid, and are moved about and shunted to the nasolacrimal duct and by the eyelids during blinking. Thus, in the tears, the secreted mucins act as clean-up/debris removing multimeric networks that at the same time, through their hydrophilic nature, hold fluids in place and harbor defense molecules secreted by the lacrimal gland. Membrane-associated mucins, on the other hand, form a dense barrier in the glycocalyx at the epithelial tear film interface. This barrier prevents pathogen penetrance and is a lubricating surface that allows lid epithelia to glide over the corneal epithelia without adherence. The secreted mucins move easily over the glycocalyx mucins because both have anionic character that creates repulsive forces between them. Little is known regarding regulation of expression and glycosylation of mucins by ocular surface epithelia. Since ocular surface drying diseases alter both goblet cell and mucin production, and production and glycosylation of membrane-associated mucins, studies of mucin gene regulation and glycosylation may yield treatment modalities for these diseases.

Role of Mucins in the Function of the Corneal and Conjunctival Epithelia

The surface of the eye is covered by a tear film, which is held in place by a wet-surfaced, stratified, corneal and conjunctival epithelia. Both are vital for light refraction and protection of vision. Maintenance of tear film on the ocular surface, lubrication, and provision of a pathogen barrier on this wet surface is facilitated by a class of large, highly glycosylated, hydrophilic glycoproteins--the mucins. In the past 15 years, a number of mucin genes have been cloned, and based on protein sequence, categorized as either secreted or membrane associated. Both types of mucins are expressed by ocular surface epithelia. Goblet cells intercalated within the stratified epithelium of the conjunctiva secrete the large gel-forming mucin MUC5AC, and lacrimal gland epithelia secrete the small soluble mucin MUC7. Apical cells of the stratified epithelium of both corneal and conjunctival epithelium express at least three membrane-associated mucins (MUCs 1, 4, and 16), which extend from their apical surface to form the thick glycocalyx at the epithelium-tear film interface. The current hypothesis regarding mucin function and tear film structure is that the secreted mucins form a hydrophilic blanket that moves over the glycocalyx of the ocular surface to clear debris and pathogens. Mucins of the glycocalyx prevent cell-cell and cell-pathogen adherence. The expression and glycosylation of mucins are altered in drying, keratinizing ocular surface diseases.

Membrane-tethered mucins have multiple functions on the ocular surface.

Membrane-tethered mucins are large glycoproteins present in the glycocalyx along the apical surface of all wet-surfaced epithelia of the body, including that of the ocular surface. Originally thought to function only in epithelial surface lubrication and hydration, data now indicate that the mucins are multifunctional molecules, each having unique as well as common functions. This review summarizes current knowledge regarding the three major membrane mucins of the ocular surface, MUC1, MUC4, and MUC16. The mucins vary in their ocular surface distribution, size, structural motifs, and functions. The ectodomains of each are released into the tear film and are, thus, a component of the soluble mucins of the tear film. Both animal and in vitro models for their study are herein described, as are alterations of the mucins in ocular surface disease.

Mucin genes expressed by the ocular surface Epithelium

Abstract Mucins are the glycoproteins which form the viscous, gel-like mucus layer of the tear film. Molecular characterization of mucins has been slow due to their heavy glycosylation and high molecular weight, but recent cloning of human gut and trachea, mammary gland, and salivary gland mucins has begun to shed light on the primary structure of these important protective molecules. To date nine human mucin genes, designated MUCI-8, have been cloned. The presence in the protein backbone of tandem repeats of series of amino acids rich in serine and/or threonine is a feature common to all. Numbers and sequences of amino acids in each tandem repeat varies with each mucin. Numbers of amino acids per repeat vary from 169 (MUC6) to 8 (MUC5AC). Until recently little has been known regarding the expression of these genes by the ocular surface epithelium. This review summarizes recent work from our laboratory aimed at determining the molecular character of mucins expressed by conjunctival and corneal epithelium. Using northern blot analysis and in situ hybridization techniques, we have demonstrated that the stratified epithelium of both cornea and conjunctiva express the transmembrane mucin, MUC1. This widely expressed mucin is the best characterized of the cloned mucins. Using similar methodologies we demonstrated that the conjunctival goblet cell expresses MUC5AC, a gel-forming mucin, which has a cysteine-rich domain responsible for the disulfhydryl bonding between mucin molecules. Conjunctival stratified epithelium expresses MUC4, a relatively uncharacterized mucin, whose function is not known. Are there additional and/or unique mucins expressed by the ocular surface epithelium? We summarize our recent work on characterization of a mucin isolated from rat and human corneal epithelium. Antibodies to these mucins localize to the glycocalyx of the corneal epithelial apical cells. Since MUCI appears to be the only one of the cloned mucins expressed by the corneal epithelium, it is a candidate for comparison to our mucin isolate. Current efforts are directed toward this comparison. Identification of mucin genes expressed by the ocular surface epithelium opens many new avenues for investigation into expression, regulation, and glycosylation of mucins in ocular surface pathologies and into the specific character of these molecules which enhance protection from pathogen invasion.

Hemidesmosomes and anchoring fibril collagen appear synchronously during development and wound healing

Bullous pemphigoid antisera and monoclonal antibodies to type VII collagen were used to localize hemidesmosomes and anchoring fibrils, respectively, in tissues of developing eyes and healing corneal wounds of New Zealand white rabbits. In the 17-day fetal rabbit eye, both antibodies colocalize to the epithelial-stromal junction of the lid and conjunctival region, but neither binds to the cornea, and electron microscopy demonstrates hemidesmosomes only where the antibodies bind. By 20 days of fetal development, the antibodies colocalize in cornea, and, by electron microscopy, hemidesmosomes are shown to be present as well. In healing 7-mm corneal wounds, both antibodies colocalize at the wound periphery within 66 h. By electron microscopy, hemidesmosomes along small segments of basal lamina are also shown to be present at the wound periphery at this time. These demonstrations of the synchronous assembly of hemidesmosomes and anchoring fibrils support the hypothesis of linkage of hemidesmosomes through the basement membrane to anchoring fibrils.

Extracellular matrix and growth factors in corneal wound healing.

&NA; Healing of corneal wounds is a complex process involving epithelial, keratocyte, and endothelial interactions that are affected by their associations with wound bed matrix and by cytokine availability and activation. The spectrum of possible cellular‐matrix‐growth factor interactions is indeed great and growing. Several of the significant contributions made during the past year include development of an organotypic organ culture model of the cornea that allows in vitro assembly of the epithelial extracellular matrix‐anchoring complex, demonstration of epithelial synthesis of Bowmans layer collagens, demonstration of transforming growth factor‐&bgr;2s inhibition of stromal cell collagenase synthesis, and demonstration of the paracrine pathway of keratinocyte growth factor action in the cornea.

MUC16 is lost from the uterodome (pinopode) surface of the receptive human endometrium: in vitro evidence that MUC16 is a barrier to trophoblast adherence.

Abstract In order for the preimplantation embryo to implant into the uterus, the trophoblast cells must initially adhere to the uterine epithelial surface. In preparation, the luminal secretory cells of the epithelium lose their nonadhesive character and their surface microvilli and bulge into the lumen, forming uterodomes (pinopodes; uterodome is used instead of pinopode, since in humans the surface membrane exocytoses rather than endocytoses (Murphy, Hum Reprod 2000; 15:2451–2454). Previous research has led to the hypothesis that loss of the nonadhesive membrane-spanning mucin MUC1 from the uterodome surface allows trophoblast adherence. Immunofluorescence microscopic assay of luminal epithelia on human uterine biopsies taken from LH+0 to LH+13 show that another membrane-spanning mucin, MUC16, was lost from uterodome surfaces in all samples taken during the receptive phase, LH+6 to LH+8 (n = 12), and that MUC1 was present on uterodomes in 4 of 12 samples and on all ciliated cells of the epithelium in the receptive phase. Short interfering RNA (siRNA) knockdown of MUC16 in a uterine epithelial cell line ECC-1 that, like uterine epithelium, expresses MUC16 and MUC1 allowed increased adherence of cells of a trophoblast cell line. In parallel experiments, siRNA knockdown of MUC1 did not affect trophoblast cell adherence. These data indicate that MUC16 is a membrane component of the nonreceptive luminal uterine surface, which prevents cell adhesion, and that its removal during uterodome formation facilitates adhesion of the trophoblast.

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.

Cellular origin of mucins of the ocular surface tear film.

In summary, we have demonstrated that the ocular surface epithelia express at least three mucin genes. We suggest that the gel-forming mucin MUC5AC is a major mucin forming the mucus gel of the tear film. We further suggest that MUC1 facilitates the spread of the MUC5-containing mucus on the ocular surface and, along with the mucus gel, prevents cell and debris adhesion to the ocular surface. The function of MUC4 at the ocular surface remains to be elucidated.

Release of Membrane-associated Mucins from Ocular Surface Epithelia

PURPOSE Three membrane-associated mucins (MAMs)--MUC1, MUC4, and MUC16--are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. METHODS An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to the MUC16 cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in the release or shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor [TNF]), TNF-alpha-converting enzyme, and matrix metalloproteinase-7 and -9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure the efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. RESULTS MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced the release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced the release of MUC16 but not of MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16, 78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. CONCLUSIONS Results suggest that the extracellular domains of MUC1, MUC4, and MUC16 can be released from the ocular surface by agents in tears. Neutrophil elastase and TNF, present in higher amounts in the tears of patients with dry eye, may cause MAM release, allowing rose bengal staining.