多環芳香烴受體基因敲除在視網膜色素上皮細胞中促發炎機轉之探討

The Mechanism of Pro-Inflammatory Response in Aryl Hydrocarbon Receptor Gene Knockout Retinal Pigment Epithelial cell

戴佳樺(Chia-Hua Tai)

指導教授 ： 康照洲

國立臺灣大學/醫學院/毒理學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902011

摘要

黃斑病變(MD)是工業化國家中導致中央視野喪失和失明的主要原因之一。 在MD發展過程中，視網膜色素上皮細胞(RPE)和Bruch's membrane之間的Drusen 形成通常伴隨發炎反應的發生及免疫細胞浸潤。目前的研究顯示，在芳香烴受體 (AHR)缺陷小鼠視網膜中出現 MD-like Drusen、免疫細胞聚集以及 RPE 所介導 的異常免疫活化。然而 RPE 中發炎反應和免疫細胞聚集的機制仍不清楚。本研究 使用 CRISPR 敲除 ARPE-19 細胞株中的 AHR 基因，並通過西方墨點法比較野生 型RPE和AHR-KO RPE之間炎症反應相關蛋白(ICAM-1，COX2和iNOS)及 相關之訊息蛋白表現量的差異。此外本研究也透過單核細胞粘附實驗評估 RPE 所 表達之 ICAM-1 功能，並比較野生型與 AHR-KO 小鼠之視網膜中的 AHR 和 ICAM-1蛋白表現量的表現差異。本研究結果顯示，AHR-KO RPE相較於野生型 RPE，其 ICAM-1，COX2 和 iNOS 以及相關訊息蛋白 integrin αvβ3、PKCα、Rac1、 p38、IκBα 及 p65 表現量均顯著提高，且 PKCα、Rac1、MAPK-p38 和 NF-κB 抑 制劑能夠抑制AHR-KO RPE中上升的ICAM-1，COX2和iNOS蛋白表現量。此 外 AHR-KO RPE 的單核球黏附數量顯著高於野生型 RPE 細胞，且 AHR-KO RPE 的單核球貼附數量同樣因 PKCα、Rac1、MAPK-p38 和 NF-κB 抑制劑而下降。小 鼠組織切片中，AHR-/-小鼠視網膜 RPE 層中 ICAM-1 蛋白表現量較野生型小鼠高。 由結果推論，AHR-KO 會上調整合素 αvβ3 蛋白表現量，並誘導 PKCα、RAC1、 MAPK-p38 和 NF-κB 路徑的活化使 RPE 中 ICAM-1，COX2 和 iNOS 的蛋白質表 現增加。AHR 在調節 RPE 的發炎反應中扮演重要角色，而造成 RPE 中 AHR 蛋 白表現量下降的原因如抽菸、老化、光照及暴露其他 AHR ligand 與 MD 之危險因 子有所重疊。然而，AHR 在 RPE 功能維持以及 MD 發展中所扮演的角色在未來 仍需要更進一步的研究。

關鍵字

黃斑病變 ； 多環芳香烴受體 ； 視網膜色素上皮細胞 ； 發炎反應 ； ICAM-1 ； 整合素 αvβ3 ； 整合素 αvβ5

並列摘要

Macular degeneration (MD) is one of the major causes of central sight losing and blindness in developed countries. During MD development, the formation of drusen, a phenomenon of sub-retinal deposits accumulation between retinal pigment epithelium (RPE) and Bruch's membrane is usually accompanied by inflammatory response and immune cell infiltration. Recent studies have showed that MD-liked drusen and immune cell recruitment were upregulated in the retina of Aryl Hydrocarbon Receptor (AHR) deficient mice, as well as the abnormal immune activation mediated by RPE cells. However, the mechanism of inflammatory response and immune cell recruitment to RPE cells remains unclear. In this study, CRISPR is used to knockout the AHR gene in ARPE-19 cells, for examining the inflammatory response relative proteins (ICAM-1, COX2, and iNOS). The possible signaling that involved ICAM-1, COX2, and iNOS protein expression between wild type and AHR knockout RPE cells were conducted by western blot assay. Also, we use the monocyte adhesion assay for examining the function of RPE-expressed ICAM-1. Moreover, both AHR and ICAM-1 were correlated to a comparison between wild type and AHR-/-mice. For further understanding the upstream regulation of AHR-affected inflammatory factors, we found that AHR knockout may increase integrin αvβ3 protein expression and upper activation of PKCα, RAC1, MAPK-p38, and NF-κB pathways in RPE cells. The results of monocyte adhesion assay and paraffin section support the results mentioned above. The number of THP-1 cells adhesion on AHR knockout RPE cells are significant more than wildtype RPE cells, and inhibitor of PKCα, RAC1, MAPK-p38, and NF-κB reversed increasing THP-1 cell adhesion on AHR knockout RPE cells. ICAM-1 protein expression increased in AHR-/- mice RPE layer. AHR seems to play a critical role for regulating inflammatory response in RPE cells, and the deficiency of AHR in RPE cells can be caused by lots of reasons, such as smoking, aging, light exposure, and other AHR ligands, which overlap the risk factors of MD. By revealing the mechanism of inflammatory response mediated by AHR deficiency in RPE cells, it is much clear that AHR may provide the protection to RPE cells to avoid abnormal inflammatory response, and together with other publish reports, could help maintain the normal function of RPE cells. The results in this study suggest AHR could be a key for MD treatment, but further study about the role of AHR in RPE function is needed.

並列關鍵字

Macular degeneration (MD) ； Aryl Hydrocarbon Receptor (AHR) ； retinal pigment epithelium (RPE) ； inflammatory response ； Intracellular adhesion molecule-1 (ICAM-1) ； integrinαvβ3 ； integrin αvβ5

參考文獻

Aberg, E., Torgersen, K. M., Johansen, B., Keyse, S. M., Perander, M. and Seternes, O. M. Docking of PRAK/MK5 to the atypical MAPKs ERK3 and ERK4 defines a novel MAPK interaction motif. J Biol Chem 2009; 284: 19392-19401.

Ambati, J., Ambati, B. K., Yoo, S. H., Ianchulev, S. and Adamis, A. P. Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Survey of Ophthalmology 2003; 48: 257-293.

Arnold, T. R., Stephenson, R. E. and Miller, A. L. Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function. Experimental Cell Research 2017; 358: 20-30.

Bassi, V., Vitale, M., Feliciello, A., De Riu, S., Rossi, G. and Fenzi, G. Retinoic acid induces intercellular adhesion molecule-1 hyperexpression in human thyroid carcinoma cell lines. Journal of Clinical Endocrinology and Metabolism 1995; 80: 1129-1135.

Beischlag, T. V., Luis Morales, J., Hollingshead, B. D. and Perdew, G. H. The aryl hydrocarbon receptor complex and the control of gene expression. Critical Reviews in Eukaryotic Gene Expression 2008; 18: 207-250.