TGF-beta2 in human retinal pigment epithelial cells: expression and secretion regulated by cholinergic signals in vitro.
OBJECTIVE
To investigate the (i) effect(s) of cholinergics on the expression and secretion of transforming growth factor (TGF)-beta(2) in human retinal pigment epithelium (RPE) and (ii) mechanism of action of atropine in the treatment of myopia.


METHODS
The RPE cell line, D407, was (i) treated with carbachol (10 microM), (ii) treated with atropine (10 nM-100 microM), or (iii) pre-treated with atropine (10 nM-100 microM) and then exposed to carbachol (10 microM). A no-treatment group served as control. Expression of TGF-beta(2), 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-beta(2) was determined by ELISA.


RESULTS
Carbachol induced a time-dependent increase in the levels of TGF-beta(2) mRNA and protein in the cytoplasm (p < 0.001). ELISA assays showed a time-dependent increase in levels of TGF-beta(2) protein in the supernatant with carbachol treatment (p < 0.001). There was no change of TGF-beta(2) in the cytoplasm or supernatant with atropine alone (p > 0.05). The increased expression and secretion of TGF-beta(2) caused by carbachol were suppressed by atropine (in the range of 10 nM-100 microM) when compared to treatment with carbachol alone (p < 0.001). The stimulating effect of 10 microM carbachol was inhibited completely by 100 microM atropine.


CONCLUSIONS
In RPE cells, atropine inhibits the expression and secretion of TGF-beta(2) by blocking the muscarinic acetylcholine receptor (mAChR), which may control the development of myopia.
Tan
Jia
J
Department of Ophthalmology, Xiangya Medical College, Central South University, Changsha, China.
Deng
Zhi-hong
ZH
Liu
Shuang-zhen
SZ
Wang
Jian-tao
JT
Huang
Chen
C
D001285:Atropine / Q000494:pharmacology; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D018727:Muscarinic Antagonists / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053781:Transforming Growth Factor beta2 / Q000235:genetics* / Q000378:metabolism*; D015854:Up-Regulation
D018679:Cholinergic Agonists; D018727:Muscarinic Antagonists; D012333:RNA, Messenger; D053781:Transforming Growth Factor beta2; D001285:Atropine; D002217:Carbachol