Functions of MUC16 in corneal epithelial cells.
OBJECTIVE
The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins.


METHODS
The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium.


RESULTS
MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression.


CONCLUSIONS
These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.
Blalock
Timothy D
TD
Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Spurr-Michaud
Sandra J
SJ
Tisdale
Ann S
AS
Heimer
Susan R
SR
Gilmore
Michael S
MS
Ramesh
Vijaya
V
Gipson
Ilene K
IK
D000199:Actins / Q000378:metabolism; D001422:Bacterial Adhesion / Q000502:physiology; D015153:Blotting, Western; D018394:CA-125 Antigen / Q000502:physiology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism / Q000382:microbiology; D008565:Membrane Proteins / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D000199:Actins; D018394:CA-125 Antigen; D004268:DNA-Binding Proteins; C513242:Etv5 protein, mouse; C451759:MUC16 protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors