Mechanisms of Retinoic Acid-AP-1 Signaling Pathway Modulating Secretion of TGF-β2 by Retinal Pigment Epithelial Cells / 中山大学学报(医学科学版)

ABSTRACT

@#【Objective】To investigate the effects of up-regulating RA signal and inhibiting AP-1 transcriptional activity on TGF-β2 secretion by RPEs and its possible pathways.【Methods】① To investigate the effects of ATRA treat⁃ ment，human retinal pigment epithelial cell line ARPE-19 cells were divided into 5 groups：control group and 4 interven⁃ tion groups（6 h，12 h，24 h and 48 h after RA treatment）. Western blot，RT-qPCR and immunofluorescence staining were carried out to analyze RARβ and c-Fos expression. ②To investigate the effects of RARβ inhibitor LE540 treatment on expression of RARβ and c-Fos that were induced by ATRA，ARPE-19 cells were divided into 4 groups：control group，ATRA group，LE540 group and ATRA+LE540 group. RARβ and c-Fos expression was assessed by western blot and RT-qPCR. ③ To investigate the effects of AP-1 inhibitor T-5224 treatment，ARPE-19 cells were divided into 4 groups：control group and treatment groups（12 h，24 h and 48 h after T-5224 treatment）. EMSA was carried out to ana⁃ lyze the AP-1 DNA binding activity. ④To investigate the effects of LE540 and T-5224 administration on ATRA- induced TGF-β2 secretion，ARPE-19 cells were divided into 4 groups：control group，ATRA group，ATRA+LE540 group and ATRA+LE540 group. Western blot and ELISA were carried out to analyze TGF-β2 secretion in ARPE-19 cells.【Results】 RARβ level in ARPE-19 cells was significantly higher in treatment group than in control group after being treated with ATRA for 24 and 48 hours（P＜0.05）. C-Fos level was first up-regulated and then decreased. After treatment with ATRA for 6 and 12 hours，c-Fos expression were significantly upregulated（P＜0.01），but at 48 h after treatment，their expression were significantly decreased to the level which had no statistical difference compared with the control group （P＞0.05）. The AP-1 DNA binding activity was significantly decreased in ARPE-19 cells after being treated with T-5224 for 24 and 48 hours（P＜0.01）. Compared with ATRA group，TGF-β2 secretion was statistically down-regulated after being treated with LE540 and T-5224 for 48 hours（P＜0.05）.【Conclusion】ATRA can induce TGF-β2 secretion in RPE cells through affecting RARβ expression and AP-1 transcriptional activity.