June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Knockdown of MUC16 Alters Tight Junctions of Corneal Epithelial Cells Resulting in Decreased Transepithelial Resistance
Author Affiliations & Notes
  • Ilene Gipson
    Harvard Med Sch/Dept Ophthal, Schepens Eye Research Inst/MEEI, Boston, MA
  • Sandra Spurr-Michaud
    Harvard Med Sch/Dept Ophthal, Schepens Eye Research Inst/MEEI, Boston, MA
  • Ann Tisdale
    Harvard Med Sch/Dept Ophthal, Schepens Eye Research Inst/MEEI, Boston, MA
  • Footnotes
    Commercial Relationships Ilene Gipson, None; Sandra Spurr-Michaud, None; Ann Tisdale, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 554. doi:https://doi.org/
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      Ilene Gipson, Sandra Spurr-Michaud, Ann Tisdale; Knockdown of MUC16 Alters Tight Junctions of Corneal Epithelial Cells Resulting in Decreased Transepithelial Resistance. Invest. Ophthalmol. Vis. Sci. 2013;54(15):554. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: MUC16 is a major membrane associated mucin of the human corneal epithelium. We have shown that MUC16 is a barrier to rose bengal dye penetrance and pathogen adherence. We also demonstrated that MUC16’s cytoplasmic tail associates with actin through the ezrin, radixin, moesin family of actin linking proteins. Because MUC16 may be associated with actin filaments that insert into the apical web of actin filaments that terminate in tight junctions, the purpose of this study was to determine the role of MUC16 in tight junction formation and function as measured by transepithelial resistance (TER).

Methods: An immortalized human corneal limbal epithelial cell line (HCLE) (Gipson et al, 2003) and the HCLE cell line stably transfected with siRNA to MUC16 in which MUC16 levels were reduced by 70% and a vector control line were used. Cells were cultured for optimal mucin expression and stratification. Assays comparing the cell lines included measurement of apical cell size, localization and mRNA levels of Z01, a tight junction protein, and TER using an Evum2 Epithelial Voltohmmeter.

Results: HCLE cells knocked down (KD) for MUC16 showed a significant 2-3 fold (p<0.001) increase in apical cell size as compared to vector and non-transfected controls. ZO1 localization in the MUC16 KD cells showed long discontinuities along apical cell peripheries as well as regions of punctate localization as compared to the control cell lines. Corroboratively, message levels of ZO1 were significantly lower in the MUC16 KO cells (p<0.01 to controls). The alterations in ZO1 localization and expression in the MUC16 KD cells correlated with a significant decrease in TER (39 +3 ohms) compared to controls (127 + 5 ohms, p<0.001).

Conclusions: A decrease in MUC16 in corneal apical cells results in tight junction discontinuities and a decrease in junction function as measured by TER. These data suggest that MUC16 through its association to actin within the apical cytoskeletal web that terminates laterally in tight junctions, influences tight junction formation and function. We have previously shown that as apical cells increase in size with time of residence at the ocular surface, the amount of MUC16 per unit membrane area on the cell decreases. Perhaps MUC16 diminution leads to disruption of tight junctions that in turn leads to cell desquamation.

Keywords: 485 cornea: surface mucins • 482 cornea: epithelium • 480 cornea: basic science  
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