May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Post–Translation Regulation of Muc4 in Cultured Corneal Epithelium Cells by Proteolytic Degradation
Author Affiliations & Notes
  • J. Lomako
    Bascom Palmer, Miami, FL
    Cell Biology and Anatomy,
  • W.M. Lomako, V
    Bascom Palmer, Miami, FL
    Cell Biology and Anatomy,
  • C.A. Carothers– Carraway
    Bascom Palmer, Miami, FL
    Biochemistry and Molecular Biology,
  • K.L. Carraway, II
    Bascom Palmer, Miami, FL
    Cell Biology and Anatomy,
  • Footnotes
    Commercial Relationships  J. Lomako, None; W.M. Lomako, None; C.A. Carothers– Carraway, None; K.L. Carraway, None.
  • Footnotes
    Support  NIH : EY 12343
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1608. doi:
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      J. Lomako, W.M. Lomako, V, C.A. Carothers– Carraway, K.L. Carraway, II; Post–Translation Regulation of Muc4 in Cultured Corneal Epithelium Cells by Proteolytic Degradation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1608.

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

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Purpose: : Previous evidence indicates that membrane–bound mucin Muc4 in cultured corneal epithelium accumulates at very high concentration at the most superficial layer as well as in desquamated cells. It was proposed that such over–accumulation of membrane bound Muc4 disrupts cell–cell adhesions that may lead to desquamation. This study was initiated to determine whether the level of Muc4/SMC in corneal epithelial cells depends on proteolytic degradation through proteasomal and lysosomal pathways.

Methods: : The Muc4 level was measured in multilayer epithelial cell sheets and in desquamated cells by immunoblotting. Localization of Muc4 and lysosomes within the cells was determined by confocal immunofluorescence microscopy. Ubiquitination of Muc4 was investigated by sequential immunoprecipitation followed by immunoblotting and by confocal immunofluorescence microscopy. The microscopy study was performed on both nonpermeabilized and permeabilized cells.

Results: : Double staining confocal fluorescence microscopy (CFM) revealed that in nonpermeabilized control cell layers, Muc4 is localized primarily at the membrane of the cells undergoing desquamation. There was high intensity staining in the intracellular compartment of desquamating cells after permeabilization. This staining was diminished in medial layers and practically non–existent in basal layers. Treatment of cultivated corneal epithelium with proteasomal inhibitors, lactacystin or N–CBZ–ILE–GLU(O–t–BUTYL)ALA–LEUCINAL (PSI), increased slightly the amount of Muc4 in the cultures as measured by Western blotting, and CMF revealed increased amounts of intracellular Muc4 in the most superficial cells as well as in the medial and basal layers compared to control. Cells treated with proteasome inhibitors accumulated Muc4 in the form of intracellular mini–aggregates. The transmembrane subunit of Muc4 is ubiquitinated, probably as part of the proteasomal degradation mechanism. The lysosome inhibitor chloroquine increased Muc4 levels and, similar to proteasome inhibitors, induced accumulation of cytoplasmic Muc4 in deeper layers of corneal epithelium.

Conclusions: : These studies suggest that proteosomal and lysosomal degradation play a role in both quality and quantity contol of Muc4 synthesis and also represent, to our knowledge, the first evidence for proteosome and lysosome involvement in mucin distribution among cell layers of squamous epithelia.

Keywords: cornea: epithelium • cornea: surface mucins • cornea: basic science 

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