视黄酸-AP-1信号通路对视网膜色素上皮细胞分泌TGF-β2的调控机制

Mechanisms of Retinoic Acid-AP-1 Signaling Pathway Modulating Secretion of TGF-β2 by Retinal Pigment Epithelial Cells

【目的】研究上调视黄酸(RA)信号以及抑制活化蛋白-1(AP-1)转录活性对RPE分泌转化生长因子β2(TGF-β2)的影响以及可能的作用途径。【方法】①检测ATRA干预的作用,将ARPE-19细胞分为:正常对照组和6、12、24、48h组,用实时荧光定量PCR(RT-qPCR)、westernblot和免疫荧光检测RARβ和c-Fos的表达。②检测RARβ抑制剂LE540对ATRA诱导后细胞表达RARβ和c-Fos的影响,将ARPE-19细胞分为:正常对照组,ATRA组,LE540组和ATRA+LE540组,用RT-qPCR和westernblot检测RARβ和c-Fos的表达。③检测AP-1抑制剂T-5224干预的作用,将ARPE-19细胞分为:正常对照组和12、24、48h组,用电泳迁移率变动分析法(EM?SA)检测RPE细胞中AP-1活性。④检测LE540和T-5224对ATRA诱导后细胞表达和分泌TGF-β2的影响,将ARPE-19细胞分为正常对照组,ATRA组,ATRA+LE540组和ATRA+T-5224组,用westernblot和酶联免疫吸附试验(ELISA法)检测TGF-β2的表达和分泌量。【结果】ATRA干预24和48h后,RARβ的表达较对照组明显升高(P<0.05),c-Fos的表达先升后降,ATRA干预6和12h后,c-Fos表达明显升高(P<0.01),而干预48h后,c-Fos表达下降,与对照组相比无统计学差异(P>0.05);T-5224干预24和48h后,AP-1的转录活性明显下降(P<0.01)。用LE540和T-5224干预48h后,ATRA+LE540组和ATRA+T-5224组TGF-β2的表达较ATRA组明显下降(P<0.05)。【结论】ATRA通过RARβ影响AP-1的转录活性,进而促进ARPE-19细胞分泌TGF-β2。

【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 treatment,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 analyze 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.