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T.D. Blalock, S.J. Spurr–Michaud, A. Tisdale, P. Argüeso, I.K. Gipson; Shedding of Membrane–Associated Mucins on the Ocular Surface . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5432.
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© ARVO (1962-2015); The Authors (2016-present)
Three membrane–associated mucins, MUC1, 4 and 16, are known to be expressed by the epithelium on the ocular surface. Since soluble forms of MUC1, 4, and 16 are detected in the tear film, we are identifying the mechanism of shedding of the extracellular domains.
Shedding of the membrane–associated mucins MUC1, 4, and 16 was assessed in an immortalized human corneal–limbal epithelial cell line (HCLE). HCLE cells were exposed to potential inducers of shedding, and protein was separated from conditioned medium samples and cell lysates using agarose gel electrophoresis, followed by immunoblotting using antibodies to MUC1 (HMFG–2), MUC4 (8G7), and MUC16 (OC125). To further study the mechanism of mucin shedding on the ocular surface, a truncated MUC16 construct was produced by fusing a segment of the distal N–terminal portion of the MUC16 molecule with the C–terminal portion (containing the transmembrane domain). The gene construct was inserted into pCMV–Tag1 for expression of an N–terminal FLAG and C–terminal c–myc–labeled protein and transfected into CHO–K1 cells.
Tumor necrosis factor alpha (TNF–α) induced shedding of all three membrane–associated mucins from the surface of HCLE cells. In addition, matrix metalloproteinase–7 (MMP–7), neutrophil elastase, and TNF–α–converting enzyme (TACE) induced shedding of MUC16. Phorbol–12–myristate–13–acetate (PMA), an activator of TACE, also induced shedding of MUC4 and MUC16. The effect of neutrophil elastase and TNF–α on MUC16 shedding in HCLE cells was confirmed by immunofluorescence microscopy using the OC125 antibody. Expression of the MUC16 construct in CHO cells was verified using RT–PCR and detection of the FLAG and c–myc tags were shed into the culture medium.
Several agents induce shedding of specific membrane–associated mucins, suggesting selective mechanisms of mucin release at the ocular surface. The mechanism of MUC16 shedding from the cell surface can be further assessed using the MUC16 construct expressed in cell culture.
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