Abstract
ObjectiveTo 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).Materials and MethodsPrimary 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.ResultsImmunohistochemistry 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.ConclusionsOur 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.
Highlights
Transglutaminases (TGs) are a big class of intra- and extracellular enzymes with at least 8 members, all of which catalyze the formation of epsilon - (c-glutamyl) lysine isopeptide linkages between peptide substrates
Real time PCR showed TG-1, 2, 5 transcript levels to be down regulated 3 fold (p,0.05) in cultured human and mouse scleral fibroblasts (SF) after incubation with atropine and this was reversed by carbachol
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
Summary
Transglutaminases (TGs) are a big class of intra- and extracellular enzymes with at least 8 members, all of which catalyze the formation of epsilon - (c-glutamyl) lysine isopeptide linkages between peptide substrates. These enzymes are tightly regulated, and are involved in processes such as inflammation, reepithelialization, neovascularization, synthesis and stabilization of a fibrous extracellular matrix (ECM) [1,2,3]. Different types of TGs are found in various cellular compartments. TGs-3 and 5 are restricted to the cytosolic compartment. Since TGs are found in different sub-cellular locations, it is not surprising that they sub-serve different functions in these locations
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call