Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fenyang Road, Shanghai, 200031, China.
Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai, China.
Mol Med. 2022 Oct 22;28(1):125. doi: 10.1186/s10020-022-00554-w.
Oxidative stress-caused damage to the retinal pigment epithelium (RPE) underlies the onset and progression of age-related macular degeneration (AMD). Impaired mitochondrial biogenesis sensitizes RPE cells to mitochondrial dysfunction, energy insufficiency and death. Src-homology 2 domain-containing phosphatase (SHP)-1 is important in regulating immune responses and cell survival. However, its roles in cell survival are not always consistent. Until now, the effects of SHP-1 on RPE dysfunction, especially mitochondrial homeostasis, remain to be elucidated. We sought to clarify the effects of SHP-1 in RPE cells in response to atRAL-induced oxidative stress and determine the regulatory mechanisms involved.
In the all trans retinal (atRAL)-induced oxidative stress model, we used the vector of lentivirus to knockdown the expression of SHP-1 in ARPE-19 cells. CCK-8 assay, Annexin V/PI staining and JC-1 staining were utilized to determine the cell viability, cell apoptosis and mitochondrial membrane potential. We also used immunoprecipitation to examine the ubiquitination modification of stimulator of interferon genes (STING) and its interaction with SHP-1. The expression levels of mitochondrial marker, proteins related to mitochondrial biogenesis, and signaling molecules involved were examined by western blotting analysis.
We found that SHP-1 knockdown predisposed RPE cells to apoptosis, aggravated mitochondrial damage, and repressed mitochondrial biogenesis after treatment with atRAL. Immunofluoresent staining and immunoprecipitation analysis confirmed that SHP-1 interacted with the endoplasmic reticulum-resident STING and suppressed K63-linked ubiquitination and activation of STING. Inhibition of STING with the specific antagonist H151 attenuated the effects of SHP-1 knockdown on mitochondrial biogenesis and oxidative damage. The adenosine monophosphate-activated protein kinase (AMPK) pathway acted as the crucial downstream target of STING and was involved in the regulatory processes.
These findings suggest that SHP-1 knockdown potentiates STING overactivation and represses mitochondrial biogenesis and cell survival, at least in part by blocking the AMPK pathway in RPE cells. Therefore, restoring mitochondrial health by regulating SHP-1 in RPE cells may be a potential therapeutic strategy for degenerative retinal diseases including AMD.
氧化应激导致的视网膜色素上皮（RPE）损伤是年龄相关性黄斑变性（AMD）发病和进展的基础。线粒体生物发生受损使 RPE 细胞易发生线粒体功能障碍、能量不足和死亡。Src 同源 2 结构域含磷酶（SHP）-1 在调节免疫反应和细胞存活中起重要作用。然而，其在细胞存活中的作用并不总是一致的。到目前为止，SHP-1 对 RPE 功能障碍，特别是线粒体动态平衡的影响仍有待阐明。我们旨在阐明 SHP-1 在全反式视黄醛（atRAL）诱导的氧化应激反应中对 RPE 细胞的影响，并确定涉及的调节机制。
在全反式视黄醛（atRAL）诱导的氧化应激模型中，我们使用慢病毒载体敲低 ARPE-19 细胞中的 SHP-1 表达。CCK-8 检测、Annexin V/PI 染色和 JC-1 染色用于检测细胞活力、细胞凋亡和线粒体膜电位。我们还使用免疫沉淀法检测干扰素基因刺激因子（STING）的泛素化修饰及其与 SHP-1 的相互作用。通过 Western blot 分析检测线粒体标记物、与线粒体生物发生相关的蛋白和参与的信号分子的表达水平。
我们发现 SHP-1 敲低使 RPE 细胞在 atRAL 处理后更容易发生凋亡，加重线粒体损伤，并抑制线粒体生物发生。免疫荧光染色和免疫沉淀分析证实 SHP-1 与内质网驻留的 STING 相互作用，并抑制 STING 的 K63 连接泛素化和激活。用特异性拮抗剂 H151 抑制 STING 可减弱 SHP-1 敲低对线粒体生物发生和氧化损伤的影响。腺苷单磷酸激活蛋白激酶（AMPK）通路作为 STING 的关键下游靶点，并参与调节过程。
这些发现表明，SHP-1 敲低增强了 STING 的过度激活，并抑制了 RPE 细胞的线粒体生物发生和细胞存活，至少部分是通过阻断 AMPK 通路。因此，通过调节 RPE 细胞中的 SHP-1 来恢复线粒体健康可能是包括 AMD 在内的退行性视网膜疾病的一种潜在治疗策略。
