Recent characterizations of mucins at the molecular level indicate that at least eight mucin genes are expressed by epithelia of mucosal surfaces. The purpose of this study was to determine whether these cloned mucins, designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7, are expressed by epithelia of the female reproductive tract. Northern blot analysis, in situ hybridization, and immunohistochemistry were performed using RNA and tissue from surgically removed human reproductive tract specimens including endocervix, ectocervix, vagina, endometrium, and fallopian tube. Complementary DNA to the tandem repeat regions of MUCs 1, 2, 3, 5AC, 5B, and 6; oligonucleotides to the tandem repeat regions of MUCs 4, 6, and 7; and antibodies that recognize unique mucin tandem repeats were used. The data demonstrate that the endocervical epithelium expresses five mucin genes: MUCs 1, 4, 5AC, 5B, and 6. The ectocervical and vaginal epithelia express MUCs 1 and 4, although vaginal expression of MUC4 appears patchy. Endometrial epithelium expresses MUC1 and low amounts of MUC6. MUC6 immunoreactivity was detected only is scattered endometrial glands located in the basalis region in specimens from pre- and postmenopausal women. The only mucin detected in the fallopian tube was MUC1.
H185 antibody has been shown to recognize a carbohydrate epitope on a membrane-associated mucin in the apical surfaces of the corneal and conjunctival epithelia. The distribution of this antibody is altered on the surfaces of conjunctival epithelial cells of dry eye patients. The purpose of this work was to determine whether the H185 antibody recognizes the recently cloned membrane-associated mucin MUC16 (formerly CA125 antigen).To determine whether ocular surface epithelia express MUC16, the relative expression of the MUC16 mucin gene was determined by real-time PCR on reverse transcription products from RNA isolated from human corneal and conjunctival tissues, as well as from immortalized human corneal-limbal epithelial cell (HCLE) cultures. To determine the distribution of MUC16 mRNA and protein in the ocular surface epithelia, in situ hybridization and immunohistochemistry were performed on sections of corneal and conjunctival epithelia using, respectively, a MUC16 antisense oligoprobe and the antibodies OC125, VK-8, and R16 raised against the MUC16 mucin. Determination of whether MUC1 and MUC16 mucins carry the H185 carbohydrate epitope was achieved with the respective mucins isolated from HCLE protein extracts, using one- or two-step immunoprecipitation assays and immunodepletion experiments followed by Western blot analysis.MUC16 mucin transcripts were detected in the human ocular surface epithelia and in corneal cell cultures. MUC16 mRNA and protein localized to the apical cell layers of the cornea and to the suprabasal region of the conjunctival epithelium. In HCLE cultures, MUC16 protein was detected in apical cells of islands of stratified cells. Immunofluorescence microscopy demonstrated exact colocalization of the MUC16 mucin and the H185 carbohydrate epitope in sections of human corneal tissue. Immunoprecipitated MUC16 mucin was recognized by the H185 antibody and vice versa, indicating that MUC16 mucin carries the H185 epitope. Immunodepletion with H185 antibody resulted in no OC125 antibody reactivity. No cross-reactivity between immunoprecipitated MUC1 and the H185 antibody was observed.This study demonstrates that the membrane-associated mucin MUC16 is expressed by the human ocular surface epithelia and that MUC16 carries the H185 carbohydrate epitope. Future studies on the expression of MUC16 and the characterization of the molecular structure of the H185 carbohydrate epitope will determine their biological significance on the healthy ocular surface and in dry eye syndrome.
The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands.Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry.Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring.The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs.
Recent development of mice null for either Muc5ac or Muc5b mucin allows study of their specific roles at the mouse ocular surface. A recent report indicated that Muc5ac null mice show an ocular surface phenotype similar to that seen in dry eye syndrome. The purpose of our study was to determine the effect of lack of Muc5ac or Muc5b on the ocular surface, and to determine if environmental desiccating stress exacerbated a phenotype.
Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells.Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber.Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface.These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).
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