BACKGROUND Disturbed flow (d-flow)-induced senescence and activation of endothelial cells (ECs) have been suggested to have critical roles in promoting atherosclerosis. Telomeric repeat-binding factor 2 (TERF2)-interacting protein (TERF2IP), a member of the shelterin complex at the telomere, regulates the senescence-associated secretory phenotype (SASP), in which EC activation and senescence are engendered simultaneously by p90RSK-induced phosphorylation of TERF2IP S205 and subsequent nuclear export of the TERF2IP-TERF2 complex. In this study, we investigated TERF2IP-dependent gene expression and its role in regulating d-flow-induced SASP. METHODS A principal component analysis and hierarchical clustering were used to identify genes whose expression is regulated by TERF2IP in ECs under d-flow conditions. Senescence was determined by reduced telomere length, increased p53 and p21 expression, and increased apoptosis; EC activation was detected by NF-κB activation and the expression of adhesion molecules. The involvement of TERF2IP S205 phosphorylation in d-flow-induced SASP was assessed by depletion of TERF2IP and mutation of the phosphorylation site. RESULTS Our unbiased transcriptome analysis showed that TERF2IP caused alteration in the expression of a distinct set of genes, including rapamycin-insensitive companion of mTOR (RICTOR) and makorin-1 (MKRN1) ubiquitin E3 ligase, under d-flow conditions. In particular, both depletion of TERF2IP and overexpression of the TERF2IP S205A phosphorylation site mutant in ECs increased the d-flow and p90RSK-induced MKRN1 expression and subsequently inhibited apoptosis, telomere shortening, and NF-κB activation in ECs via suppression of p53, p21, and telomerase (TERT) induction. CONCLUSIONS MKRN1 and RICTOR belong to a distinct reciprocal gene set that is both negatively and positively regulated by p90RSK. TERF2IP S205 phosphorylation, a downstream event of p90RSK activation, uniquely inhibits MKRN1 expression and contributes to EC activation and senescence, which are key events for atherogenesis.

中文翻译：

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内皮细胞衰老相关的分泌表型（SASP）由Makorin-1泛素E3连接酶调节。

背景技术扰动的血流（d-flow）诱导的衰老和内皮细胞（EC）的活化已被认为在促进动脉粥样硬化中起关键作用。端粒重复结合因子2（TERF2）相互作用蛋白（TERF2IP）是端粒中庇护蛋白复合物的成员，调节衰老相关的分泌表型（SASP），其中p90RSK诱导的EC激活和衰老同时发生。 TERF2IP S205的磷酸化，以及随后的TERF2IP-TERF2复合物的核输出。在这项研究中，我们调查了TERF2IP依赖性基因表达及其在调节d-flow诱导的SASP中的作用。方法采用主成分分析和层次聚类法鉴定在d-flow条件下EC中其表达受TERF2IP调控的基因。通过减少端粒长度，增加p53和p21表达以及增加细胞凋亡来确定衰老。EC激活通过NF-κB激活和粘附分子的表达进行检测。通过消耗TERF2IP和磷酸化位点的突变来评估TERF2IP S205磷酸化在d流诱导的SASP中的参与。结果我们的无偏转录组分析表明，TERF2IP在d流条件下引起了一组不同基因的表达变化，包括对雷帕霉素不敏感的mTOR（RICTOR）和makorin-1（MKRN1）泛素E3连接酶的表达。特别是，EC中TERF2IP的耗竭和TERF2IP S205A磷酸化位点突变体的过度表达均增加了d-flow和p90RSK诱导的MKRN1表达，并随后抑制了细胞凋亡，端粒缩短，通过抑制p53，p21和端粒酶（TERT）诱导EC中的NF-κB活化。结论MKRN1和RICTOR属于不同的互惠基因集，受p90RSK负向和正向调控。TERF2IP S205磷酸化是p90RSK激活的下游事件，它独特地抑制MKRN1表达并促进EC激活和衰老，这是动脉粥样硬化的关键事件。