title: Transglutaminases (TGs) in ocular and periocular tissues: effect of muscarinic agents on TGs in scleral fibroblasts.
abstract: OBJECTIVE
  To investigate the expression of transglutaminases (TGs) in the ocular surface, the eyelid margin and associated glands and to determine effect of muscarinic agents on TGs in scleral fibroblasts (SF).
  
  
  METHODS
  Primary SFs cultured from mouse and human sclera were treated with atropine and carbachol for 5 days. Lysed cell RNA was used for real-time PCR, protein was used for Western blot analysis and TG-2 transamidase activity was measured by ELISA. Immunohistochemistry was done to determine the expression of TGases.
  
  
  RESULTS
  Immunohistochemistry and western blot confirmed the expression of TGs-1, 2, 3 and 5 proteins in cultured SFs and eye tissues. Real time PCR showed TG-1, 2, 5 transcript levels to be down regulated 3 fold (p<0.05) in cultured human and mouse SFs after incubation with atropine and this was reversed by carbachol. However, TG-3 expression was increased with atropine and decreased with carbachol at all concentrations. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. TGs-1, 3, 5 were localized in the entire mouse corneal epithelium, stroma and endothelium but TG-2 was present only in the corneal subepithelium and stroma. All TGs were localized in mouse Meibomian glands however TG-2 had a weak expression.
  
  
  CONCLUSIONS
  Our results confirm that TGs-1, 2, 3 and 5 are expressed in human SF and murine ocular tissues, eyelid and associated Meibomian glands. Real-time PCR and Western blot results showed that muscarinic antagonist down-regulates TGs-1, 2 and 5 in both cultured human and mouse SFs and upregulates TG-3. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. These results suggest that manipulation of TGs by way of muscarinic receptor acting drugs may be a plausible method of intervention in wound healing and scleral remodeling.
authors.affiliation: Ocular Disease Model Research Group, Singapore Eye Research Institute, Singapore, Singapore. vabarathi97@mac.com
authors.forename: V A
authors.initials: VA
authors.lastname: Barathi
authors.forename: Sung R
authors.initials: SR
authors.lastname: Weon
authors.forename: Queenie S W
authors.initials: QS
authors.lastname: Tan
authors.forename: Kwan J
authors.initials: KJ
authors.lastname: Lin
authors.forename: Louis
authors.initials: L
authors.lastname: Tong
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D018678:Cholinergic Agents / Q000494:pharmacology*; D005136:Eye Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D051379:Mice; D008875:Middle Aged; D009928:Organ Specificity / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012590:Sclera / Q000166:cytology*; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018678:Cholinergic Agents; D005136:Eye Proteins; D012333:RNA, Messenger; D001285:Atropine; D002217:Carbachol; D011503:Transglutaminases