Release of membrane-associated mucins from ocular surface epithelia.
OBJECTIVE
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-alpha-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.
Blalock
Timothy D
TD
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Spurr-Michaud
Sandra J
SJ
Tisdale
Ann S
AS
Gipson
Ilene K
IK
D051722:ADAM Proteins / Q000502:physiology; D000072198:ADAM17 Protein; D000818:Animals; D019921:Biotinylation; D018394:CA-125 Antigen / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism*; D005260:Female; D006801:Humans; D019272:Leukocyte Elastase / Q000502:physiology; D016850:Limbus Corneae / Q000166:cytology; D008297:Male; D020783:Matrix Metalloproteinase 7 / Q000502:physiology; D020780:Matrix Metalloproteinase 9 / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D018396:Mucin-1 / Q000378:metabolism*; D055264:Mucin-4; D009077:Mucins / Q000378:metabolism*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012395:Rose Bengal / Q000493:pharmacokinetics; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
D018394:CA-125 Antigen; D003470:Culture Media; C060192:MUC1 protein, human; C451759:MUC16 protein, human; C524658:MUC4 protein, human; D008565:Membrane Proteins; D018396:Mucin-1; D055264:Mucin-4; D009077:Mucins; D014409:Tumor Necrosis Factor-alpha; D012395:Rose Bengal; D019272:Leukocyte Elastase; D051722:ADAM Proteins; D020783:Matrix Metalloproteinase 7; D020780:Matrix Metalloproteinase 9; D000072198:ADAM17 Protein