Comparison of the Transmembrane Mucins MUC1 and MUC16 in Epithelial Barrier Function

Membrane-anchored mucins are present in the apical surface glycocalyx of mucosal epithelial cells, each mucosal epithelium having at least two of the mucins. The mucins have been ascribed barrier functions, but direct comparisons of their functions within the same epithelium have not been done. In an epithelial cell line that expresses the membrane-anchored mucins, MUC1 and MUC16, the mucins were independently and stably knocked down using shRNA. Barrier functions tested included dye penetrance, bacterial adherence and invasion, transepithelial resistance, tight junction formation, and apical surface size. Knockdown of MUC16 decreased all barrier functions tested, causing increased dye penetrance and bacterial invasion, decreased transepithelial resistance, surprisingly, disruption of tight junctions, and greater apical surface cell area. Knockdown of MUC1 did not decrease barrier function, in fact, barrier to dye penetrance and bacterial invasion increased significantly. These data suggest that barrier functions of membrane-anchored mucins vary in the context of other membrane mucins, and MUC16 provides a major barrier when present

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-␣-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. (Invest Ophthalmol Vis Sci

Epidemic Keratoconjunctivitis-Causing Adenoviruses Induce MUC16 Ectodomain Release To Infect Ocular Surface Epithelial Cells

ABSTRACT Human adenoviruses (HAdV), species D in particular (HAdV-D), are frequently associated with epidemic keratoconjunctivitis (EKC). Although the infection originates at the ocular surface epithelium, the mechanisms by which HAdV-Ds bypass the membrane-associated mucin (MAM)-rich glycocalyx of the ocular surface epithelium to trigger infection and inflammation remain unknown. Here, we report that an EKC-causing adenovirus (HAdV-D37), but not a non-EKC-causing one (HAdV-D19p), induces ectodomain release of MUC16—a MAM with barrier functions at the ocular surface—from cultured human corneal and conjunctival epithelial cells. HAdV-D37, but not HAdV-D19p, is also found to decrease the glycocalyx barrier function of corneal epithelial cells, as determined by rose bengal dye penetrance assays. Furthermore, results from quantitative PCR (qPCR) amplification of viral genomic DNA using primers specific to a conserved region of the E1B gene show that, in comparison to infection by HAdV-D19p, infection by HAdV-D37 is significantly increased in corneal epithelial cells. Collectively, these results point to a MUC16 ectodomain release-dependent mechanism utilized by the EKC-causing HAdV-D37 to initiate infection at the ocular surface. These findings are important in terms of understanding the pathogenesis of adenoviral keratoconjunctivitis. Similar MAM ectodomain release mechanisms may be prevalent across other mucosal epithelia in the body (e.g., the airway epithelium) that are prone to adenoviral infection. IMPORTANCE: Human adenoviruses (HAdVs) are double-stranded DNA viruses that cause infections across all mucosal tissues in the body. At the ocular surface, HAdVs cause keratoconjunctivitis (E. Ford, K. E. Nelson, and D. Warren, Epidemiol Rev 9:244–261, 1987, and C. M. Robinson, D. Seto, M. S. Jones, D. W. Dyer, and J. Chodosh, Infect Genet Evol 11:1208–1217, 2011, doi:10.1016/j.meegid.2011.04.031)—a highly contagious infection that accounts for nearly 60% of conjunctivitis cases in the United States (R. P. Sambursky, N. Fram, and E. J. Cohen, Optometry 78:236–239, 2007, doi:10.1016/j.optm.2006.11.012, and A. M. Pihos, J Optom 6:69–74, 2013, doi:10.1016/j.optom.2012.08.003). The infection begins with HAdV entry within ocular surface epithelial cells; however, the mechanisms used by HAdVs to transit the otherwise protective mucosal barrier of ocular surface epithelial cells prior to entry remain unknown. Here, we report that the highly virulent keratoconjunctivitis-causing HAdV-D37 induces release of the extracellular domain (ectodomain) of MUC16, a major component of the mucosal barrier of ocular surface epithelial cells, prior to infecting underlying cells. Currently, there is no specific treatment for controlling this infection. Understanding the early steps involved in the pathogenesis of keratoconjunctivitis and using this information to intercept adenoviral entry within cells may guide the development of novel strategies for controlling the infection

Suppression of Toll-like Receptor-Mediated Innate Immune Responses at the Ocular Surface by the Membrane-associated Mucins MUC1 and MUC16

Membrane-associated mucins (MAMs) expressed on the ocular surface epithelium form a dense glycocalyx, which is hypothesized to protect the cornea and conjunctiva from external insult. In this study, the hypothesis that the MAMs MUC1 and MUC16, expressed on the apical surface of the corneal epithelium, suppress Toll-like receptor (TLR)-mediated innate immune responses was tested. Using an in vitro model of corneal epithelial cells that are cultured to express MAMs, we show that reduced expression of either MUC1 or MUC16 correlates with increased message and secreted protein levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α following exposure of cells to the TLR2 and TLR5 agonists, heat killed Listeria monocytogenes and flagellin, respectively. Since mice express MUC1 (but not MUC16) in the corneal epithelium, a Muc1-/- mouse model was used to extend in vitro findings. Indeed, IL-6 and TNF-α message levels were increased in the corneal epithelium of Muc1-/- mice, in comparison to wild type mice, following exposure of enucleated eyes to the TLR2 and TLR5 agonists. Our results suggest that the MAMs MUC1 and MUC16 contribute to the maintenance of immune homeostasis at the ocular surface by limiting TLR-mediated innate immune responses

A Metalloproteinase Secreted by Streptococcus pneumoniae Removes Membrane Mucin MUC16 from the Epithelial Glycocalyx Barrier

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection

MUC 16 mucin is expressed by the human ocular surface epithelia and carries the H185 carbohydrate epitope. Invest Ophthalmol Vis Sci.

PURPOSE. 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). METHODS. 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 corneallimbal 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. RESULTS. 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. CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2003;44:2487-2495) DOI:10.1167/ iovs.02-0862 T he wet-surfaced epithelia, including the corneal and conjunctival epithelia, produce a group of highly glycosylated protective glycoproteins termed mucins. 1,2 On the ocular surface, mucins lubricate the apical surfaces of the epithelium during the eyelid blink, provide a barrier against pathogen invasion, and, due to their hydrophilic nature, prevent the desiccation of the preocular tear film. 3,4 Structurally, mucins are high molecular weight glycoproteins that contain a variable number of tandem-repeat domains rich in proline, serine and threonine. N-acetylgalactosamine is O-linked to the serine and threonine residues and serves to anchor a variety of carbohydrates, which ultimately constitute up to 80% of the mass of the mature mucin molecule, as reviewed by Gendler and Spicer. 1 Based on their protein structure, two types of mucins, secreted and membrane-associated, have been identified. Secreted mucins are synthesized and secreted onto surfaces of epithelia by goblet cells. Five of these mucins have been cloned. Four are large gel-forming mucins, MUC2, -5AC, -5B, and -6, and one is a small monomeric mucin MUC7, as reviewed by Moniaux et al. -11 Several years ago, our laboratory produced a monoclonal antibody, designated H185, that recognized a carbohydrate epitope present on a mucinlike glycoprotein expressed on apical cells of corneal and conjunctival epithelia. 2 Numerous attempts to clone and characterize the molecule have not been successful, in part because of insufficient starting material and its heavy glycosylation. Efforts to characterize the H185 antigen have continued, however, especially because it has an altered distribution on apical cells of conjunctival epithelia of patients with dry eye

Decreased Levels of the Goblet Cell Mucin MUC5AC in Tears of Patients with Sjögren Syndrome

purpose. To determine whether the relative amounts of mucin mRNA in the conjunctival epithelium and mucin protein in the tears are altered in patients with Sjögren syndrome compared with healthy individuals. methods. Tear fluid was collected from the inferior fornix of normal subjects (n = 17) and patients with Sjögren syndrome (n = 11) after instillation of 60 μL sterile water onto the ocular surface. Immediately after tear fluid collection, conjunctival epithelium was obtained by filter paper–stripping from the bulbar temporal region for mRNA isolation. Primers to nontandem repeat sequences of the gel-forming mucin MUC5AC and the membrane-spanning mucins MUC1 and MUC4 were used in real-time RT-PCR to determine relative abundance of MUC mRNA in patients with Sjögren syndrome in relation to that of normal subjects. Enzyme-linked immunosorbent assay was performed on neuraminidase-treated tears, using a polyclonal antibody against a synthetic peptide mimicking the deduced amino acid sequence from the D3 region of MUC5AC. results. The number of RNA transcripts for the goblet cell–specific mucin MUC5AC in the conjunctival epithelium of patients with Sjögren syndrome was significantly lower than in normal individuals. No significant changes were detected when analyzing the mRNA levels of the mucins expressed by the stratified epithelium of the conjunctiva, MUC1 and MUC4. Protein levels of the goblet cell mucin MUC5AC were significantly reduced in the tear fluid of patients with Sjögren syndrome, corroborating mRNA data obtained using real-time RT-PCR. conclusions. The tear fluid of patients with Sjögren syndrome has reduced levels of the goblet cell–specific mucin MUC5AC, which correlates to decreased levels of conjunctival MUC5AC mRNA. The authors propose that deficiency of MUC5AC mucin in tears constitutes one of the mechanisms responsible for tear film instability in Sjögren syndrome

Knockdown of MUC16 results in disruption of the actin cytoskeleton associated with tight junctions and reduces surface microplicae.

<p>Epithelial cultures of non-transfected controls (A) and those transfected with shMUC16 (B) were double labeled with an antibody to occludin (green) and Phalloidin (red) to localize filamentous actin; note the actin filaments associated with the linear occludin antibody binding in A, and the lack of filamentous actin along the disrupted occludin antibody binding in B. Scanning electron micrographs of control epithelial cultures (C), shMUC16 cultures (D) and native epithelium (E). Note fewer prominent microplicae in the cells knocked down for MUC16 in D and also in the larger darker cell of native epithelium (E), that were shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100393#pone-0100393-g002" target="_blank">Fig. 2F</a>) to bind less antibody to MUC16. The larger darker cells show fewer microplicae than neighboring smaller light cells. Scale bars = 15 µm in A, B, 10 µm in C, D, 5 µm in E.</p

Knockdown of MUC16 results in decreased tight junction function and ZO-1/occludin expression, whereas knockdown of MUC1 has no effect on tight junctions.

<p>(A) Immunofluorescence analysis of occludin localization demonstrated normal linear distribution of occludin in the MUC16 scrambled control (scr16) cells (A) as compared to the disrupted localization seen in the shMUC16 cells (B). (C) A highly significant decrease in transepithelial electrical resistance (TER) was observed in the MUC16 knockdown (shMUC16) cell cultures compared to control cultures and shMUC1 cultures. No difference was seen in TER in the MUC1 knockdown (shMUC1) cells n = 15–30. (D) Analysis of the relative mRNA expression of two tight junction genes (ZO-1, occludin) by qPCR demonstrated a significant reduction in their message in the shMUC16 cells compared to the non-transfected (NT), or scrambled shRNA controls (scr1, scr16) and shMUC1 cells. n = 7, **p<0.01, ns = not significant.</p

Significant knockdown of MUC1 and MUC16 proteins in both cell lysates and on apical cell surfaces following transfection with vectors expressing shMUC1 or shMUC16 sequences.

<p>(A) Western blots demonstrating that <b>MUC1 protein</b> is lower in both cell lysates (upper left) and on apical cell surfaces (lower left) of cell cultures transfected with shMUC1 containing vectors (shMUC1) compared to the non-transfected control (NT), scrambled shRNA (scr1) controls, as well as with shMUC16 containing vector (shMUC16) or its scrambled shRNA control (scr16). Alleles of MUC1 often differ in size and as they are co-dominantly expressed, two distinct protein sizes are evident on western blots. The graphs to the right of each blot, show densitometric analyses of bands demonstrating that MUC1 protein levels are significantly reduced by 71% in the cell lysates and 60% on apical surfaces relative to NT and scr1 controls and that MUC1 protein levels are not significantly reduced by knockdown of MUC16 (shMUC16) or its scrambled shRNA control (scr16). (B) Similarly, on the left are representative Western blots demonstrating that MUC16 protein levels are lower in cell lysates and biotinylated apical cell surface protein isolates of cells transfected with shMUC16 containing vectors compared to non-transfected (NT), or those transfected with scrambled shRNA for either MUC1 or MUC16 (scr1 and scr16) or shMUC1 containing vectors. The graphs on the right show densitometric analyses of blots indicating that MUC16 protein levels are significantly reduced in cell lysates by 70% and on apical surfaces by 51% in cells transfected with shMUC16 containing vectors in comparison to NT and scr16 controls. For both (A) and (B) protein samples from cell lysates were loaded based on equivalent micrograms of protein, and for cell surface proteins on equivalent cm² of cell growth area. Graphic representation of the relative amounts of MUC1 (upper right) and MUC16 (lower right) was derived through densitometric analyses of the blots, cell lysates were normalized to GAPDH, and all data were expressed relative to the non-transfected control (NT). Significant if p<0.01, (**). ns = non-significant, n = 5–10.</p