Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland.
Arthritis Rheumatol. 2014 Jan;66(1):40-8. doi: 10.1002/art.38190.
To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASFs).
RASFs and osteoarthritis synovial fibroblasts (OASFs) were treated with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress, and MG132, a proteasome inhibitor. Then, 3-methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosome inhibitor. Polyubiquitinated proteins, p62, and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy, and immunohistochemistry, respectively. OASFs were transfected with small interfering RNA targeting autophagy-linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase 3 activity assay.
In RASFs, the induction of autophagy by TG and MG132 was increased compared to that in OASFs. Whereas autophagy promoted a caspase 3-independent induction of cell death under ER stress, autophagy had a protective role in apoptosis induced by proteasome inhibition. Treatment of RASFs with 3-methyladenine blocked TG-induced cell death. ER stress induced a strong accumulation of p62-positive polyubiquitinated protein aggregates, accompanied by the formation of large vacuoles in RASFs but not OASFs. Furthermore, TG-induced p62 protein expression was increased, whereas TG-induced ALFY expression was reduced, in RASFs compared to OASFs. ALFY knockdown promoted the accumulation of p62, the formation of polyubiquitinated protein aggregates, and cell death.
Our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. A reduced expression of ALFY and the formation of p62-positive polyubiquitinated protein aggregates promote cell death in RASFs under severe ER stress.
探讨自噬在类风湿关节炎滑膜成纤维细胞（RASFs）细胞死亡调控中的作用。
用内质网应激诱导剂 thapsigargin（TG）和蛋白酶体抑制剂 MG132 处理 RASFs 和骨关节炎滑膜成纤维细胞（OASFs）。然后，用 3-甲基腺嘌呤作为自噬抑制剂，用巴弗洛霉素 A1 作为溶酶体抑制剂。用免疫印迹、免疫荧光显微镜和免疫组织化学分别评估多泛素化蛋白、p62 和自噬诱导。用自噬相关 FYVE 蛋白（ALFY）的小干扰 RNA 转染 OASFs。用流式细胞术和 caspase 3 活性测定评估细胞死亡。
与 OASFs 相比，TG 和 MG132 诱导的 RASFs 自噬增加。虽然自噬在 ER 应激下促进 caspase 3 非依赖性细胞死亡，但自噬在蛋白酶体抑制诱导的细胞凋亡中具有保护作用。用 3-甲基腺嘌呤处理 RASFs 可阻断 TG 诱导的细胞死亡。ER 应激导致 RASFs 中 p62 阳性多泛素化蛋白聚集体大量积聚，同时形成大空泡，但 OASFs 中没有。此外，与 OASFs 相比，TG 诱导的 RASFs 中 p62 蛋白表达增加，而 TG 诱导的 ALFY 表达减少。ALFY 敲低促进了 p62 的积累、多泛素化蛋白聚集体的形成和细胞死亡。
本研究首次提供了自噬在调节 RASFs 死亡途径中的双重作用的证据。在严重的内质网应激下，ALFY 表达降低和 p62 阳性多泛素化蛋白聚集体的形成促进了 RASFs 的细胞死亡。
