Transglutaminases (TGs) in ocular and periocular tissues: effect of muscarinic agents on TGs in scleral fibroblasts

• Published in: PloS one Vol. 6; no. 4; p. e18326
• Main Authors: Barathi, V A, Weon, Sung R, Tan, Queenie S W, Lin, Kwan J, Tong, Louis, Beuerman, Roger W
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.04.2011; Public Library of Science (PLoS)
• Subjects: Acetylcholine receptors (muscarinic); Activation; Aged; Aged, 80 and over; Analysis; Animals; Atropine; Atropine - pharmacology; Biology; Blotting, Western; Carbachol; Carbachol - pharmacology; Cell adhesion & migration; Cholinergic Agents - pharmacology; Cornea; Disease; Drugs; Endothelium; Enzyme-linked immunosorbent assay; Enzymes; Epithelium; Extracellular matrix; Eye; Eye Proteins - metabolism; Eyelid; Fibroblasts; Fibroblasts - drug effects; Fibroblasts - enzymology; Gene Expression Regulation, Enzymologic - drug effects; Glands; Glaucoma; Growth factors; Humans; Immunohistochemistry; Medicine; Membranes; Mice; Middle Aged; Neurosciences; Organ Specificity - drug effects; Protein Transport - drug effects; Proteins; Real time; Reproducibility of Results; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; Rodents; Sclera - cytology; Stroma; Tissues; Transcription; Transglutaminases - genetics; Transglutaminases - metabolism; Wound healing; Singapore
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0018326

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). 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. 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. 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.