title: Notch signaling modulates MUC16 biosynthesis in an in vitro model of human corneal and conjunctival epithelial cell differentiation.
abstract: OBJECTIVE
  Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation.
  
  
  METHODS
  HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction.
  
  
  RESULTS
  Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4.
  
  
  CONCLUSIONS
  These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.
authors.affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Linjie
authors.initials: L
authors.lastname: Xiong
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Woodward
authors.forename: Pablo
authors.initials: P
authors.lastname: Argüeso
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D018394:CA-125 Antigen / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D003984:Dibenzazepines / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D018394:CA-125 Antigen; D003984:Dibenzazepines; C451759:MUC16 protein, human; D008565:Membrane Proteins; C499374:NOTCH3 protein, human; D000071656:Receptor, Notch3; D051880:Receptors, Notch; D053829:Amyloid Precursor Protein Secretases; C527817:dibenzazepine