全反式视黄酸诱导培养的 ARPE-19 细胞中骨形态发生蛋白 2 和基质金属蛋白酶 2 通过视黄酸受体 β 的表达。
The Expression of Bone Morphogenetic Protein 2 and Matrix Metalloproteinase 2 through Retinoic Acid Receptor Beta Induced by All-Trans Retinoic Acid in Cultured ARPE-19 Cells.
机构信息
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, SunYat-sen University, No.54 South Xianlie Road, Guangzhou, 510060, P. R. China.
The First Affiliated Hospital, Sun Yat-sen University, No.58 Zhong Shan Er Road, Guangzhou 510080, P. R. China.
出版信息
PLoS One. 2016 Mar 11;11(3):e0150831. doi: 10.1371/journal.pone.0150831. eCollection 2016.
PURPOSE
All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells.
METHODS
The effects of ATRA (concentrations from 10-9 to 10-5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10-9 to 10-5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ.
RESULTS
RARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10-9 to 10-5 mol/l) with a maximum effect observed at 10-6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10-6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135.
CONCLUSION
ATRA induced upregulation of RARβ in ARPE-19 cells and stimulated these cells to secrete BMP-2 and MMP-2.
目的
全反式视黄酸（ATRA）在眼部发育中起重要作用。先前的研究发现，视黄酸通过促进视网膜色素上皮（RPE）细胞分泌次级信号因子，影响巩膜重塑的代谢。本研究旨在探讨视黄酸是否影响骨形态发生蛋白 2（BMP-2）和基质金属蛋白酶 2（MMP-2）的分泌，并探讨视黄酸在培养的急性视网膜色素上皮 19（ARPE-19）细胞中的信号通路。
方法
采用逆转录-聚合酶链反应（RT-PCR）和 Western blot 检测分别在 mRNA 和蛋白水平上，检测浓度为 10-9 至 10-5mol/L 的 ATRA 对 ARPE-19 细胞中视黄酸受体（RARs）表达的影响。用浓度为 10-9 至 10-5mol/L 的 ATRA 处理 ARPE-19 细胞 24 小时和 48 小时，或用 10-6mol/L ATRA 处理不同时间（6 小时至 72 小时），采用实时定量 PCR（qPCR）和酶联免疫吸附试验（ELISA）检测。用 RARβ 的拮抗剂 LE135 证实 RARβ 诱导的 ARPE-19 细胞的激活作用。
结果
在经 ATRA 处理 24 小时和 48 小时的 ARPE-19 细胞中，RARβmRNA 水平显著增加。这些 RARβmRNA 水平的增加呈剂量依赖性（浓度为 10-9 至 10-5mol/L），在 10-6mol/L 时达到最大效应。RARα和 RARγ的 mRNA 水平没有明显变化。Western blot 分析显示，用 10-6mol/L ATRA 处理 ARPE-19 细胞后，RARβ蛋白水平呈时间依赖性显著增加，在 12 小时至 72 小时时，在 24 小时和 48 小时时显著增加。RARβ 的上调和 ATRA 诱导的 ARPE-19 细胞分泌可被 RARβ 拮抗剂 LE135 抑制。
结论
ATRA 诱导 ARPE-19 细胞中 RARβ 的上调，并刺激这些细胞分泌 BMP-2 和 MMP-2。
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