Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea.
Department of Physiology, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea.
Cell Death Dis. 2023 May 20;14(5):334. doi: 10.1038/s41419-023-05851-8.
Degenerative changes of the retinal pigment epithelium (RPE) triggered by transforming growth factor-β2 (TGF-β2) and oxidative stress play a critical role in the progression of age-related macular degeneration (AMD). The expression of α-klotho, an antiaging protein, declines with age, increasing the risk factors for age-related diseases. Here, we investigated the protective effects of soluble α-klotho on TGF-β2-induced RPE degeneration. The morphological changes induced by TGF-β2, including epithelial-mesenchymal transition (EMT), were attenuated in the mouse RPE by the intravitreal injection (IVT) of α-klotho. In ARPE19 cells, EMT and morphological alterations by TGF-β2 were attenuated by co-incubation with α-klotho. TGF-β2 decreased miR-200a accompanied by zinc finger e-box binding homeobox1 (ZEB1) upregulation and EMT, all of which were prevented by α-klotho co-treatment. Inhibitor of miR-200a mimicked TGF-β2-induced morphological changes, which were recovered by ZEP1 silencing, but not by α-klotho, implying the upstream regulation of α-klotho on miR-200a-ZEP1-EMT axis. α-Klotho inhibited receptor binding of TGF-β2, Smad2/3 phosphorylation, extracellular signal-regulated protein kinase 1/2 (ERK1/2)-a mechanistic target of rapamycin (mTOR) activation and oxidative stress via NADPH oxidase 4 (NOX4) upregulation. Furthermore, α-klotho recovered the TGF-β2-induced mitochondrial activation and superoxide generation. Interestingly, TGF-β2 upregulated α-klotho expression in the RPE cells, and genetic suppression of endogenous α-klotho aggravated TGF-β2-induced oxidative stress and EMT. Lastly, α-klotho abrogated senescence-associated signaling molecules and phenotypes induced by long-term incubation with TGF-β2. Hence, our findings indicate that the antiaging α-klotho plays a protective role against EMT and degeneration of the RPE, demonstrating the therapeutic potential for age-related retinal diseases, including the dry type of AMD.
转化生长因子-β2（TGF-β2）和氧化应激引起的视网膜色素上皮（RPE）退行性变化在年龄相关性黄斑变性（AMD）的进展中起着关键作用。抗衰老蛋白α-klotho 的表达随着年龄的增长而下降，增加了与年龄相关疾病的风险因素。在这里，我们研究了可溶性α-klotho 对 TGF-β2 诱导的 RPE 变性的保护作用。TGF-β2 诱导的形态变化，包括上皮-间充质转化（EMT），在小鼠 RPE 中通过玻璃体内注射（IVT）α-klotho 得到减弱。在 ARPE19 细胞中，α-klotho 共孵育可减弱 TGF-β2 引起的 EMT 和形态改变。TGF-β2 降低了 miR-200a，同时上调锌指 E 盒结合同源盒 1（ZEB1）并引起 EMT，这些都可以通过 α-klotho 共处理来预防。miR-200a 抑制剂模拟了 TGF-β2 诱导的形态变化，这些变化可以通过 ZEP1 沉默恢复，但不能通过 α-klotho 恢复，表明 α-klotho 对 miR-200a-ZEP1-EMT 轴的上游调节。α-klotho 通过 NADPH 氧化酶 4（NOX4）上调抑制 TGF-β2 的受体结合、Smad2/3 磷酸化、细胞外信号调节蛋白激酶 1/2（ERK1/2）-雷帕霉素的机制靶点（mTOR）激活和氧化应激。此外，α-klotho 恢复了 TGF-β2 诱导的线粒体激活和超氧化物生成。有趣的是，TGF-β2 上调了 RPE 细胞中的α-klotho 表达，而内源性α-klotho 的遗传抑制加重了 TGF-β2 诱导的氧化应激和 EMT。最后，α-klotho 消除了长期与 TGF-β2 孵育诱导的衰老相关信号分子和表型。因此，我们的研究结果表明，抗衰老的α-klotho 对 EMT 和 RPE 变性具有保护作用，为包括干性 AMD 在内的与年龄相关的视网膜疾病的治疗提供了潜力。
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