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).

Materials and 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.

Figure 1. Localization of transglutaminases (TGs) in mouse eye.

(A) To determine the presence of TGs in the mouse eye, the whole eye sections (5 microns) were stained using anti mouse TG-1, TG-2, TG-3 and TG-5 rabbit IgG-fluorescein conjugates. The negative control section was incubated with 2% goat serum without the respective primary antibodies. Positive immunostaining of TGs were identified in the mouse cornea, retina pigment epithelium (RPE), choroid and sclera, as compared to the negative control. The localization of TGase-2 in cornea is different from the other 3 TGs. TG-1, TG-3, TG-5 were localized in the entire mouse corneal epithelium, stroma and endothelium but TG-2 was present only in the corneal subepithelium (“CS”) and stroma (“S) (see white arrows). Error bar = 50 µM. DAPI stains nuclei (indicated by the white circles and the white boundaries) and FITC stains cell membrane and cytoplasm. Magnification at 200×. (B) shows the localization of TG-1, TG-2, TG-3 in the mouse palpebral (P), forniceal (F) and bulbar (B) conjunctiva but not TG-5, by immunofluorescent staining. Error bar = 50 µM. DAPI stains nuclei (indicated by white circles) and FITC stains cell membrane and cytoplasm. Magnification at 200×. (C) shows the localization of TGs in mouse meibomian glands. All TGs were expressed in mouse meibomian glands but TG-2 was weakly detected. Error bar = 50 µM. Arrow indicates the meibomian gland. DAPI stains nuclei (indicated by white circles) and FITC stains cell membrane and cytoplasm. Magnification at 200×.

Figure 2. Localization of TGs in human sclera.

The localization of TG-1, TG-2, TG-3 and TG-5 in the human scleral tissue with scleral staining between the collagen fibre bundles was determined by immunofluorescent staining. No immunostaining was found in the negative control. Error bar = 50 µM. Arrow indicates the scleral fibroblasts. DAPI stains nuclei (indicated by the white circles) and FITC stains cell membrane and cytoplasm. Magnification at 200×.

Figure 3. Expression of TGs in mouse and human scleral fibroblasts.

(A) The cultured mouse scleral fibroblasts expressed all 4 TGs at cellular level. TG-1, TG-3 and TG-5 were located in the cytosolic and membrane compartments only whereas TG-2 was present in cell nucleus along with cytosolic and membrane compartment. Error bar = 50 µM. Magnification at 200×. (B) The cultured human scleral fibroblasts expressed all 4 TGs at cellular level. TG-1, TG-3 and TG-5 were located in the cytosolic and membrane compartments only whereas TG-2 was present in cell nucleus along with cytosolic and membrane compartment. Error bar = 50 µM. Magnification at 200×.

Figure 4. Confirmation of TG proteins by Western blot analysis.

Western blot confirmed the expression of TG-1, TG-2, TG-3 and TG-5 proteins in mouse and human scleral fibroblasts. ß-tubulin was used as a loading control. TG-1: 90 kDa, TG-2: 82 kDa, TG-3: 77 kDa, TG-5: 80 kDa and ß-tubulin (loading control): 55 kDa.

Figure 5. Effect of atropine treatment on TGs in mouse and human scleral fibroblasts.

(A) Western blot image detecting TG proteins in mouse scleral fibroblasts after atropine treatment. P2 cultured mouse scleral fibroblasts were treated with atropine at concentrations of 0.01 µM, 0.1 µM, 1 µM, 5 µM and 10 µM for 5 days. Following 5 days of treatment, the total cellular protein was extracted from these cells and TG proteins detected via Western blot analysis. It can be observed that the TG-1, 2 and 5 protein levels were reduced after atropine treatment. However, TG-3 protein level was increased after receiving atropine. ß-tubulin was used as a loading control. (B) Western blot image detecting TG proteins in human scleral fibroblasts after atropine treatment. P2 cultured human scleral fibroblasts were treated with atropine at concentrations of 0.01 µM, 0.1 µM, 1 µM, 5 µM and 10 µM for 5 days. Following 5 days of treatment, the total cellular protein was extracted from these cells and TG proteins detected via Western Blot analysis. It can be observed that the TG-1, 2 and 5 protein levels were reduced after atropine treatment. However, TG-3 protein level was increased after receiving atropine. ß-tubulin was used as a loading control.

Figure 6. Effect of carbachol treatment on TGs in mouse and human scleral fibroblasts.

(A) Western blot image detecting TG proteins in mouse scleral fibroblasts after carbachol treatment. P2 cultured SFs were treated with carbachol at different concentrations for 5 days. Following 5 days of treatment, the total cellular protein was extracted from these cells and TGs proteins detected via Western blot analysis. After carbachol treatment in both SFs, the TGs-1, 2 and 5 protein levels were increased. However, TG-3 protein level was decreased after receiving carbachol. ß-tubulin was used as a loading control. (B) Western Blot image detecting TG proteins in human scleral fibroblasts after carbachol treatment. P2 cultured human scleral fibroblasts were treated with carbachol at concentrations of 0.01 µM, 0.1 µM, 1 µM, 5 µM and 10 µM for 5 days. Following 5 days of treatment, the total cellular protein was extracted from these cells and TG proteins detected via Western blot analysis. It can be observed that the TG-1, TG-2 and TG-5 protein levels were increased after carbachol treatment. However, TG-3 protein level was decreased after receiving carbachol. ß-tubulin was used as a loading control.

Figure 7. Transamidase activity of TG-2 with stimulation of atropine and carbachol in scleral fibroblasts.

Line graph illustrating the results of the transamidase activity of TG-2 in 100 µg of protein lysate from mouse and human cultured scleral fibroblasts. Values were normalized against control values. The mouse and human scleral fibroblasts treated with carbachol and atropine at 0.01, 0.1, 1, 5 and 10 µM for 5 days. The SF treated with carbachol increased the transamidase activity of endogenous cellular TG-2 but this activity was reduced by atropine treatment in a concentration-dependent manner. The values represented the means of three independent samples and error bars represent standard deviation, n = 3, *p<0.05, **p<0.01. Straight lines represent the mouse SF and dashed lines represent the human SF. Open markers are atropine treated and closed markers are carbachol treated SF cells.

Figure 8. Effect of carbachol on TGM mRNA expression in mouse scleral fibrobalsts.

Bar chart illustrating the results of TGM mRNA expression levels of mouse scleral fibroblasts following with stimulation of carbachol. P2 cultured mouse scleral fibroblasts were treated with atropine or carbachol at different concentrations of 0.01 µM, 0.1 µM, 1 µM, 5 µM and 10 µM for 5 days. Following 5 days of treatment, the total RNA was extracted from these cells and TGM transcript level was quantified via qPCR analysis. Height of bars show the means of three independent samples and error bars represent standard deviation. Values are normalized against GAPDH house keeping genes. The TGM-1, 2 and 5 transcript levels were down regulated after receiving atropine (A, B and D respectively) at all concentrations. However, TGM-3 transcript was upregulated in the atropine treated cells (C). The opposite findings were observed with carbachol treatment in both TGM-2 (E) and TGM-3 (F) at all concentrations. However, TGM-1 and 5 were increased only by relatively higher concentrations of carbachol (G and H respectively).

Figure 9. Effect of atropine on TGM mRNA expression in human scleral fibroblasts.

P2 cultured human SFs were treated with atropine at different concentrations for 5 days. Following 5 days of treatment, the total RNA was extracted from these cells and TGM transcript level was quantified via qPCR analysis. Height of bars show the means of three independent samples and error bars represent standard deviation. Values are normalized against GAPDH house keeping genes. The TGM-1, 2 and 5 transcript levels were down regulated after receiving atropine (A, B and D respectively) at all concentrations. However, TGM-3 transcript was upregulated in the atropine treated cells (C). The opposite findings were observed with carbachol treatment in both TGM-2 (E) and TGM-3 (F) at all concentrations. However, TGM-1 and 5 were increased only by relatively higher concentrations of carbachol (G and H respectively).

Figure 10. Effect of atropine and carbachol on TGM mRNA expression.

Bar chart illustrating the results of TGM mRNA expression levels of mouse and human scleral fibroblasts (A–D and E–H respectively) with atropine and carbachol. P2 cultured mouse (A–D) and human SFs (E–H) was pre-treated with atropine (0.01, 0.1, 1, 5 and 10 µM) 4 hours before being incubated with equimolar carbachol. Following 5 days of treatment, the total RNA was extracted from these cells and TGM transcript level was quantified via qPCR analysis. Height of bars show the means of three independent samples and error bars represent standard deviation. Values are normalized against GAPDH house keeping genes. Atropine abrogated the carbachol-induced activation of TGM-1,2,3,5 in a dose-dependent manner.