ABSTRACT

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations.

Abbreviations: 3-MA: 3-methyladenine; ACTA2: actin, alpha 2, smooth muscle, aorta; AKT1: AKT serine/threonine kinase 1; p-AKT1: AKT1 Ser473 phosphorylation; t-AKT1: total AKT serine/threonine kinase 1; ATG4A: autophagy related 4A cysteine peptidase; ATG7: autophagy gene 7; C12FDG: 5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; Ctl: control; DAPI: 4ʹ,6-diamidino-2-phenylindole, dilactate; ECM: extracellular matrix; GSH: L-glutathione reduced; H₂O₂: hydrogen peroxide; HLF: adult human lung fibroblasts; Ho: Hoechst 33342 (2′‐[4‐ethoxyphenyl]‐5‐[4‐methyl‐1‐piperazinyl]‐2.5′‐bi‐1H‐benzimidazole); HSC: hepatic stellate cells; LY: LY294002; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTORC1/2: mechanistic target of rapamycin kinase complex 1/2; N: normal growth medium; NAC: N-acetyl-L-cysteine; PBS: phosphate-buffered saline; PDGFA: platelet derived growth factor subunit A; PRKCA/PKCα: protein kinase C alpha; PtdIns3K: class III phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; R: rapamycin; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SGK1: serum/glucocorticoid regulated kinase 1; shRNA: short hairpin RNA; siCtl: control siRNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SS: serum-free (serum starvation) medium; TP53: tumor protein p53; TUBA: tubulin alpha; V: vehicle.

摘要

持续的巨自噬/自噬有利于成纤维细胞分化为肌成纤维细胞。细胞衰老是应对长期细胞压力的另一种方式，也与肌成纤维细胞分化和纤维化有关。在这里，我们评估了在持续自噬的背景下衰老与肌成纤维细胞分化之间的关系。我们分析了体外成纤维细胞中细胞周期停滞/衰老的标志物，其中自噬是由血清饥饿 (SS) 触发的。自噬成纤维细胞表达衰老生物标志物 CDKN1A/p21 和 CDKN2A/p16，并表现出与衰老相关的 GLB1/β-半乳糖苷酶活性增加。用 3-甲基腺嘌呤、LY294002 或ATG7抑制血清饥饿成纤维细胞的自噬（自噬相关 7）沉默阻止了衰老相关标志物的表达。类似地，使用雷帕霉素或通过沉默RICTOR抑制 MTORC2 激活还防止了衰老标志。免疫荧光显微镜显示衰老和肌成纤维细胞分化在不同细胞中被诱导，表明衰老和肌成纤维细胞分化的激活相互排斥。活性氧 (ROS) 是已知的衰老诱导剂，将成纤维细胞暴露于 ROS 清除剂可减少 SS 期间 ROS 的产生，抑制自噬，并显着降低衰老和肌成纤维细胞分化标志物的表达。ROS 清除剂还抑制了 MTORC2 靶标 Ser473 处的 AKT1 磷酸化，从而确定了 ROS 在促进 MTORC2 激活中的重要性。shRNA 对TP53 / p53和 shRNA CDKN2A / p16的衰老抑制增加肌成纤维细胞分化，表明自噬诱导的肌成纤维细胞分化存在衰老的负反馈循环。总的来说，我们的结果将 ROS 鉴定为慢性自噬过程中 MTORC2 激活的中枢诱导剂，这反过来又促进了不同细胞亚群中的衰老激活和肌成纤维细胞分化。

缩写：3-MA：3-甲基腺嘌呤；ACTA2：肌动蛋白、α2、平滑肌、主动脉；AKT1：AKT 丝氨酸/苏氨酸激酶 1；p-AKT1：AKT1 Ser473 磷酸化；t-AKT1：总 AKT 丝氨酸/苏氨酸激酶 1；ATG4A：自噬相关4A半胱氨酸肽酶；ATG7：自噬基因7；C12FDG：5-十二酰氨基荧光素二-β-D-吡喃半乳糖苷；CDKN1A：细胞周期蛋白依赖性激酶抑制剂 1A；CDKN2A：细胞周期蛋白依赖性激酶抑制剂 2A；ctl：控制；DAPI：4ʹ,6-二脒基-2-苯基吲哚，二乳酸；ECM：细胞外基质；GSH：L-谷胱甘肽减少；H ₂ O ₂：过氧化氢；HLF：成人肺成纤维细胞；Ho: Hoechst 33342 (2'-[4-乙氧基苯基]-5-[4-甲基-1-哌嗪基]-2.5'-bi-1 H-苯并咪唑）；HSC：肝星状细胞；LY：LY294002；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3 β；MTORC1/2：雷帕霉素激酶复合物1/2的机制靶点；N：正常生长培养基；NAC：N-乙酰基-L-半胱氨酸；PBS：磷酸盐缓冲盐水；PDGFA：血小板衍生生长因子亚基A；PRKCA/PKCα：蛋白激酶 C α；PtdIns3K：III类磷脂酰肌醇3-激酶；PTEN：磷酸酶和张力蛋白同源物；R：雷帕霉素；RICTOR：MTOR complex 2 的 RPTOR 独立伴侣；ROS：活性氧；RPTOR：MTOR复合体1的调节相关蛋白；SA-GLB1/β-gal：衰老相关的半乳糖苷酶β1；SGK1：血清/糖皮质激素调节激酶1；shRNA：短发夹RNA；siCtl：对照siRNA；siRNA：小干扰RNA；SQSTM1：螯合体 1；SS：无血清（血清饥饿）培养基；TP53：肿瘤蛋白 p53；TUBA：微管蛋白α；五：车辆。