Generation of mice for evaluating endogenous p16Ink4a protein expression
Generation of mice for evaluating endogenous p16Ink4a protein expression
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生成用于评估内源性 p16Ink4a 蛋白表达的小鼠
DOI：
10.1016/j.bbrc.2022.02.005
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发表时间：
2022
影响因子：
3.1
通讯作者：
Yamada Yasuhiro
中科院分区：
文献类型：
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作者：
Shimada-Takayama Yui;Yasuda Takayuki;Ukai Tomoyo;Taguchi Jumpei;Ozawa Manabu;Sankoda Nao;Ohta Sho;Yamada Yasuhiro
细胞周期蛋白依赖性激酶抑制剂p16 Ink 4a在体外细胞衰老中起着重要作用。尽管以往的研究表明细胞衰老是机体衰老的系统机制之一，但p16 Ink 4a在组织中的定位和动态变化仍知之甚少，这阻碍了p16 Ink 4a在体内背景下的作用的揭示。原因之一是缺乏可靠的试剂;我们在此也证明了常用的抗人p16 INK 4抗体几乎不能识别其鼠直系同源物。在此，我们建立了一个小鼠模型，其中内源性p16 Ink 4a在其N-末端被HA标记，以探索在生物体水平上p16 Ink 4a的蛋白表达。p16 Ink 4 a在蛋白水平沿着小鼠胚胎成纤维细胞的衰老过程中被诱导，与其转录水平一致。然而，值得注意的是，在暴露于促衰老条件（包括遗传毒性应激和致癌信号通路的激活）的小鼠的组织中未检测到p16 Ink 4a，这表明仅存在细微的p16 Ink 4a蛋白诱导。在我们的小鼠模型中的这些结果强调了在体内评估p16 Ink 4a蛋白表达时需要谨慎。
The cyclin-dependent kinase inhibitor p16Ink4aplays a central role in cellular senescencein vitro. Although previous studies suggested cellular senescence is integrated in the systemic mechanisms of organismal aging, the localization and the dynamics of p16Ink4ain tissues remain poorly understood, which hinders uncovering the role of p16Ink4aunder thein vivocontext. One of the reasons is due to the lack of reliable reagents; as we also demonstrate here that commonly used antibodies raised against human p16INK4Abarely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16Ink4ais HA-tagged at its N-terminus, to explore the protein expression of p16Ink4aat the organismal level. p16Ink4awas induced at the protein level along the course of senescence in primary embryonic fibroblasts derived from the mice, consistently to its transcriptional level. Remarkably, however, p16Ink4awas not detected in the tissues of the mice exposed to pro-senescence conditions including genotoxic stress and activation of oncogenic signaling pathways, indicating that there is only subtle p16Ink4aproteins induced. These results in our mouse model highlight the need for caution in evaluating p16Ink4aprotein expressionin vivo.