Resistance to apoptosis and autophagy leads to enhanced survival in Sertoli cells
Resistance to apoptosis and autophagy leads to enhanced survival in Sertoli cells
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DOI：
10.1093/molehr/gax022
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发表时间：
2017-06-01
影响因子：
4
通讯作者：
Bhushan, Sudhanshu
中科院分区：
文献类型：
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作者：
Aslani, Ferial;Sebastian, Tim;Bhushan, Sudhanshu
研究问题：支持细胞（SC）抵抗细胞死亡的潜在机制是什么？结论：高表达促生存b细胞淋巴瘤-2 （BCL2）蛋白和抑制细胞凋亡和自噬可延长应激刺激下SC的存活。已知情况：在人类和睾丸炎的实验模型中，耐受性SC在应激条件下存活，而生殖细胞则经历大量凋亡。一般来说，非分裂的高分化细胞倾向于通过激活促生存机制和抑制细胞死亡途径来抵抗应激条件更长时间。研究设计、规模、持续时间：在本横断面研究中，采用刺激凋亡和自噬的条件诱导原代大鼠SC细胞死亡，以原代大鼠小管周围细胞（PTC）和永生化大鼠93RS2 SC为对照。每个细胞分离计为1个实验（n = 1），每个实验重复3 ~ 6次。参与者/材料、环境、方法：使用来自不育或不育患者的睾丸活检样本和来自实验性自身免疫性睾丸炎大鼠的睾丸样本进行免疫组织学分析。从19日龄雄性Wistar大鼠中分离原代SC。为了保持细胞纯度，细胞在无血清培养基中培养进行凋亡实验，在添加1%血清的培养基中培养进行自噬分析。为了诱导细胞凋亡，分别用staurosporine、borrelidin、顺铂和依托泊苷刺激细胞4或24小时。通过免疫印迹和酶活性测定检测Caspase 3的激活情况。用四甲基罗丹明甲酯测定线粒体膜电位，然后用流式细胞术分析。免疫荧光法检测细胞色素c的释放。采用甲基噻唑四氮唑法测定细胞活力。为了监测自噬通量，用Hank's平衡盐溶液剥夺细胞营养1、2和3小时。通过免疫印迹、免疫荧光标记和超微结构分析分析自噬体的形成。评估参与细胞凋亡和自噬调控基因的相对mRNA水平。采用ELISA法测定细胞外高迁移率组盒蛋白1作为坏死标志物。主要结果和作用：SC在人睾丸和实验性自身免疫性睾丸炎的体内炎症条件下存活。在离体大鼠SC中，诱导凋亡的化疗药物治疗未引起caspase - 3激活，而且，staurosporine治疗未改变线粒体膜电位和细胞色素c的线粒体定位，这表明SC中线粒体前阻断细胞凋亡。在mRNA和蛋白水平上，SC中促生存BCL2家族成员的表达水平均显著高于PTC。此外，营养饥饿后，SC中自噬信号被启动，磷酸化的unc -51样激酶-1 （ULK1）水平下降。然而，轻链3 II （LC3 II）和sequestoome -1 （SQSTM1）的水平保持不变，表明自噬通量被阻断。溶酶体蛋白酶抑制剂在SC中LC3 II的积累表明，溶酶体活性完好无损，表明自噬通量的抑制发生在前一阶段。大规模数据：无。局限性和谨慎的原因：在这项研究中，我们使用了来自青春期前大鼠的原代SC。当将我们的研究结果应用于成年动物时，需要注意的是，与其他睾丸细胞和激素因素的串扰也可能在调节SC的存活中发挥作用。研究结果的更广泛含义：我们的研究结果表明，暴露于外部应激刺激后抑制自噬和凋亡可促进SC存活，这可能是解释SC对应激反应稳健性的一种机制。肿瘤患者化疗后SC细胞的死亡抵抗对精子发生的恢复至关重要。此外，了解SC存活的分子机制揭示了有价值的靶蛋白，如BCL2，可以根据疾病背景在治疗上操纵以控制细胞活力。研究经费和竞争利益(S)：本研究由德国研究基金（DFG）资助BH93/1-1，以及由DFG和莫纳什大学资助的墨尔本Justus Liebig大学和莫纳什大学之间的国际研究培训小组（GRK 1871/1）资助。感谢吉森尤斯图斯-李比希大学医学院的支持。作者声明无利益冲突。
STUDY QUESTION: What is the underlying mechanism of Sertoli cell (SC) resistance to cell death?SUMMARY ANSWER: High expression of prosurvival B-cell lymphoma-2 (BCL2) proteins and inhibition of apoptosis and autophagy prolongs SC survival upon exposure to stress stimuli.WHAT IS KNOWN ALREADY: In human and in experimental models of orchitis, tolerogenic SC survive stress conditions, while germ cells undergo massive apoptosis. In general, non-dividing highly differentiated cells tend to resist stress conditions for a longer time by favoring activation of prosurvival mechanisms and inhibition of cell death pathways.STUDY DESIGN, SIZE, DURATION: In this cross sectional study, conditions stimulating apoptosis and autophagy were used to induce cell death in primary rat SC. Primary rat peritubular cells (PTC) and immortalized rat 93RS2 SC were used as controls. Each cell isolation was counted as one experiment (n = 1), and each experiment was repeated three to six times.PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsy samples from infertile or subfertile patients and testis samples from rats with experimental autoimmune orchitis were used for immunohistological analysis. Primary SC were isolated from 19-day-old male Wistar rats. To maintain cell purity, cells were cultured in serum-free medium for apoptosis experiments and in medium supplemented with 1% serum for autophagy analyses. To induce apoptosis, cells were stimulated with staurosporine, borrelidin, cisplatin and etoposide for 4 or 24 h. Caspase three activation was examined by immunoblotting and enzymatic activity assay. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester followed by flow cytometric analysis. Cytochrome c release was monitored by immunofluorescence. Cell viability was determined using the methylthiazole tetrazolium assay. To monitor autophagy flux, cells were deprived of nutrients using Hank's balanced salt solution for 1, 2 and 3 h. Formation of autophagosomes was analyzed by using immunoblotting, immunofluorescence labeling and ultrastructural analyses. Relative mRNA levels of genes involved in the regulation of apoptosis and autophagy were evaluated. Extracellular high mobility group box protein one was measured as a marker of necrosis using ELISA.MAIN RESULTS AND THE ROLE OF CHANCE: SC survive the inflammatory conditions in vivo in human testis and in experimental autoimmune orchitis. Treatment with apoptosis inducing chemotherapeutics did not cause caspase three activation in isolated rat SC. Moreover, mitochondrial membrane potential and mitochondrial localization of cytochrome c were not changed by treatment with staurosporine, suggesting a premitochondrial blockade of apoptosis in SC. Expression levels of prosurvival BCL2 family members were significantly higher in SC compared to PTC at both mRNA and protein levels. Furthermore, after nutrient starvation, autophagy signaling was initiated in SC as observed by decreased levels of phosphorylated UNC-51-like kinase -1 (ULK1). However, levels of light chain 3 II (LC3 II) and sequestosome-1 (SQSTM1) remained unchanged, indicating blockade of the autophagy flux. Lysosomal activity was intact in SC as shown by accumulation of LC3 II following administration of lysosomal protease inhibitors, indicating that inhibition of autophagy flux occurs at a preceding stage.LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we have used primary SC from prepubertal rats. Caution should be taken when translating our results to adult animals, where crosstalk with other testicular cells and hormonal factors may also play a role in regulating survival of SC.WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that inhibition of autophagy and apoptosis following exposure to extrinsic stress stimuli promotes SC survival, and is a possible mechanism to explain the robustness of SC in response to stress. Cell death resistance in SC is crucial for the recovery of spermatogenesis after chemotherapy treatment in cancer patients. Additionally, understanding the molecular mechanisms of SC survival unravels valuable target proteins, such as BCL2, that may be manipulated therapeutically to control cell viability depending on the context of the disease.STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant BH93/1-1, and by the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) funded by the DFG and Monash University. The support of the Medical Faculty of Justus-Liebig University of Giessen is gratefully acknowledged. The authors declare no conflict of interest.