TGF-β₂ in Human Retinal Pigment Epithelial Cells: Expression and Secretion Regulated by Cholinergic Signals In Vitro

Abstract

Purpose: To investigate the (i) effect(s) of cholinergics on the expression and secretion of transforming growth factor (TGF)-β₂ in human retinal pigment epithelium (RPE) and (ii) mechanism of action of atropine in the treatment of myopia.

Materials and Methods: The RPE cell line, D407, was (i) treated with carbachol (10 µM), (ii) treated with atropine (10 nM–100 µM), or (iii) pre-treated with atropine (10 nM–100 µM) and then exposed to carbachol (10 µM). A no-treatment group served as control. Expression of TGF-β₂, after stimulation at different time points (2, 4, 8, 16, 24, and 48 hr), was measured by RT-PCR and Western blot analysis. Secretion of TGF-β₂ was determined by ELISA.

Results: Carbachol induced a time-dependent increase in the levels of TGF-β₂ mRNA and protein in the cytoplasm (p < 0.001). ELISA assays showed a time-dependent increase in levels of TGF-β₂ protein in the supernatant with carbachol treatment (p < 0.001). There was no change of TGF-β₂ in the cytoplasm or supernatant with atropine alone (p > 0.05). The increased expression and secretion of TGF-β₂ caused by carbachol were suppressed by atropine (in the range of 10 nM–100 µM) when compared to treatment with carbachol alone (p < 0.001). The stimulating effect of 10 µM carbachol was inhibited completely by 100 µM atropine.

Conclusions: In RPE cells, atropine inhibits the expression and secretion of TGF-β₂ by blocking the muscarinic acetylcholine receptor (mAChR), which may control the development of myopia.

KEYWORDS::

• Atropine
• Carbachol
• Myopia
• Retinal pigment epithelium
• Transforming growth factor-β2

ACKNOWLEDGMENTS

Declaration of interest: This research was supported by Natural Science Foundation of Human Province, China (No. 08JJ3047). The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.