NEDD8-mediated neddylation is required for human endometrial stromal proliferation and decidualization
NEDD8-mediated neddylation is required for human endometrial stromal proliferation and decidualization
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NEDD8 介导的 neddylation 是人子宫内膜基质增殖和蜕膜化所必需的
DOI：
10.1093/humrep/dev117
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发表时间：
2015-07-01
影响因子：
6.1
通讯作者：
Wang, Haibin
中科院分区：
文献类型：
--
作者：
Liao, Yixin;Jiang, Yaling;Wang, Haibin
研究问题：NEDD 8介导的neddylation是否调节人子宫内膜间质的增殖和蜕膜化？总结回答：选择性NEDD 8激活酶抑制剂MLN 4924对Neddylation的抑制可显著损害人子宫内膜基质细胞（HESC）的增殖和蜕膜化，并通过p21积累促进细胞衰老。Neddylation在胚胎发生和肿瘤发生过程中调节细胞增殖和组织重塑，而人子宫内膜基质经历连续的增殖、分化，研究设计、规模、持续时间：我们首先分析了来自50名受试者的人子宫内膜组织中NEDD 8的表达，然后探讨MLN 4924对HESC增殖、蜕膜化和细胞衰老的neddylation抑制的后果。收集50例月经周期早期增殖期至晚期分泌期的子宫内膜组织，采用实时荧光定量PCR（qRT-PCR）和免疫组织化学染色方法检测NEDD 8在子宫内膜中的表达和细胞定位。还使用类似的方法来探索NEDD 8在永生化人子宫内膜基质细胞系（HESC）中在增殖和蜕膜化期间的mRNA和蛋白表达（N = 6）。进行MTS试验，以评价MLN 4924抑制neddylation对HESC增殖的影响。进行流式细胞术和BrdU掺入试验，以确定增殖期间对MLN 4924暴露的响应的HESC细胞周期进展。我们还通过鬼笔环肽染色和qRT-PCR分析了F-actin分布和蜕膜标记基因表达，以评估neddylation抑制对HESC蜕膜化的影响。免疫印迹分析cullin 1和p21蛋白，SA-β-半乳糖苷酶染色，以揭示HESC增殖、蜕膜化和细胞衰老受损的分子基础。应用siRNA技术敲低p21表达以测试p21积累的清除是否会纠正来自neddylation抑制的HESC缺陷。主要结果和机会的作用：我们证明NEDD 8在月经周期中在人子宫内膜包括腔上皮、腺上皮和基质细胞中普遍表达，以及在培养的HESC增殖和分化期间。采用多种分子、细胞和药理学方法，我们进一步观察到MLN 4924的neddylation抑制作用显著减弱HESC增殖（P值< 0.05），损害蜕膜转化（P值，0.05），并促进细胞衰老。MLN 4924暴露后的这些异常HESC活性伴随cullin 1 neddylation减少和p21异常蓄积。虽然清除p21积累的siRNA敲低可以部分恢复人胚胎干细胞增殖和细胞活力，它未能纠正蜕膜化deficiency.Limitations，理由回避：由于NEDD 8也强烈表达在子宫内膜上皮细胞，这是有趣的，通过分离和培养上皮细胞进一步研究其在基质-上皮相互作用的潜在作用。p21 siRNA敲低实验表明，有差异的分子机制，而不是p21，这是受到neddylation调节HESC增殖与分化相比。这一替代机制值得进一步研究。研究结果的更广泛意义：我们的研究结果首次增加了新的证据，表明NEDD 8介导的neddylation是正常人类子宫内膜功能所必需的，这提高了接近neddylation系统用于诊断和治疗女性不孕症的可能性。这项工作得到了中国国家基础研究计划（2011 CB 944400 to H.W.）的部分支持。和国家自然科学基金（81130009，81330017 to H.W.，81170575至S.Q.和S.Z.的31471106）。提交人声明不存在利益冲突。
STUDY QUESTION: Does NEDD8-mediated neddylation regulate human endometrial stromal proliferation and decidualization?SUMMARY ANSWER: Neddylation inhibition by a selective NEDD8-activating enzyme inhibitor, MLN4924, significantly impairs human endometrial stromal cell (HESC) proliferation and decidualization and facilitates cell senescence, via p21 accumulation.WHAT IS KNOWN ALREADY: Neddylation regulates cell proliferation and tissue remodeling during embryogenesis and tumorigenesis, while human endometrial stroma undergoes sequential proliferation, differentiation, as well as dynamic tissue remodeling during each menstrual cycle.STUDY DESIGN, SIZE, DURATION: We first analyzed the expression of NEDD8 in human endometrial tissues from 50 subjects, and then explored the consequence of neddylation inhibition by MLN4924 on HESCs proliferation, decidualization and cellular senescence.PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected 50 dated human endometrial tissues from early proliferative stage to late secretory phase of the menstrual cycle and analyzed the NEDD8 expression and cellular location in human endometrium by employing quantitative real-time PCR (qRT-PCR) and immunohistochemistry staining. Similar approaches were also used to explore the mRNA and protein expression of NEDD8 in an immortalized human endometrial stromal cell line (HESC) during proliferation and decidualization (N = 6). An MTS assay was performed to evaluate the effects of neddylation inhibition by MLN4924 on HESC proliferation. Flow cytometry and BrdU incorporation assay were conducted to determine the HESC cell cycleprogression in response to MLN4924 exposure during proliferation. We also analyzed F-actin distribution by phalloidin staining and decidual marker gene expression by qRT-PCR to accesses the consequence of neddylation inhibition on HESC decidualization. Immunoblotting analysis of cullin1 and p21, and SA-beta-Galactosidase staining were performed to reveal the potential molecular basis for the impaired HESC proliferation, decidualization and cellular senescence. The siRNA technique was applied to knock down p21 expression to test whether a clearance of p21 accumulation would correct the HESC defects from neddylation inhibition.MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrated that NEDD8 is ubiquitously expressed in human endometrium including luminal epithelium, glandular epithelium and the stromal cells during the menstrual cycle, as well as in the HESCs during proliferation and differentiation in culture. Employing multiple molecular, cellular and pharmacological approaches, we further observed that neddylation inhibition by MLN4924 significantly attenuates HESC proliferation (P-value < 0.05), impairs decidual transformation (P-value, 0.05), and facilitates cellular senescence. These abnormal HESC activities upon MLN 4924 exposure were accompanied with reduced cullin1 neddylation and an aberrant accumulation of p21. While a clearance of p21 accumulation by siRNA knockdown could partially restore HESC proliferation and cellular viability, it failed to correct the decidualization defects.LIMITATIONS, REASONS FOR CAUTION: Since NEDD8 was also intensely expressed in the endometrial epithelium, it is interesting to further study its potential role in stroma-epithelial interactions through isolating and culturing epithelial cells. p21 siRNA knockdown experiments revealed that there are differential molecular machineries, other than p21, that are subject to neddylation regulation during HESC proliferation compared with differentiation. This alternative mechanism warrants further investigation in future.WIDER IMPLICATIONS OF THE FINDINGS: Our findings add novel evidence showing, for what we believe the first time, that NEDD8 mediated neddylation is required for normal human endometrial functions, which raises the possibility of approaching the neddylation system for diagnosis and treatment of infertility in women.STUDY FUNDING/COMPETING INTEREST(S): This work was supported in parts by the National Basic Research Program of China (2011CB944400 to H.W.) and the National Natural Science Foundation (81130009, 81330017 to H.W., 81170575 to S.Q. and 31471106 to S.Z.). The author declares that there is no conflict of interest.