Salazar G, Huang J, Feresin R G, Zhao Y, Griendling K K
Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA; Center for Advancing Exercise and Nutrition Research on Aging (CAENRA), Florida State University, Tallahassee, FL 32306, USA.
Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA.
Free Radic Biol Med. 2017 Jul;108:225-235. doi: 10.1016/j.freeradbiomed.2017.03.032. Epub 2017 Mar 29.
AIMS: The role of oxidative stress and inflammation in the development and progression of cardiovascular diseases (CVD) is well established. Increases in oxidative stress can further exacerbate the inflammatory response and lead to cellular senescence. We previously reported that angiotensin II (Ang II) and zinc increase reactive oxygen species (ROS) and cause senescence of vascular smooth muscle cells (VSMCs) and that senescence induced by Ang II is a zinc-dependent process. Zinc stimulated NADPH oxidase (Nox) activity; however, the role of Nox isoforms in zinc effects was not determined. RESULTS: Here, we show that downregulation of Nox1, but not Nox4, by siRNA prevented both Ang II- and zinc-induced senescence in VSMCs. On the other hand, overexpression of Nox1 induced senescence, which was associated with reduced proliferation, reduced expression of telomerase and increased DNA damage. Zinc increased Nox1 protein expression, which was inhibited by chelation of zinc with TPEN and by overexpression of the zinc exporters ZnT3 and ZnT10. These transporters work to reduce cytosolic zinc, suggesting that increased cytosolic zinc mediates Nox1 upregulation. Other metals including copper, iron, cobalt and manganese failed to upregulate Nox1, suggesting that this pathway is zinc specific. Nox1 upregulation was inhibited by actinomycin D (ACD), an inhibitor of transcription, by inhibition of NF-κB, a known Nox1 transcriptional regulator and by N-acetyl cysteine (NAC) and MitoTEMPO, suggesting that NF-κB and mitochondrial ROS mediate zinc effects. Supporting this idea, we found that zinc increased NF-κB activation in the cytosol, stimulated the translocation of the p65 subunit to the nucleus, and that zinc accumulated in mitochondria increasing mitochondrial ROS, measured using MitoSox. Further, zinc-induced senescence was reduced by inhibition of NF-κB or reduction of mitochondrial ROS with MitoTEMPO. NF-κB activity was also reduced by MitoTEMPO, suggesting that mitochondrial ROS is upstream of NF-κB. INNOVATION AND CONCLUSION: Our data demonstrate that altered zinc distribution leading to accumulation of zinc in the mitochondria increases mitochondrial ROS production causing NF-κB activation which in turn upregulates Nox1 expression inducing senescence of VSMCs.
目的：氧化应激和炎症在心血管疾病（CVD）发生发展中的作用已得到充分证实。氧化应激增加会进一步加剧炎症反应并导致细胞衰老。我们之前报道过，血管紧张素II（Ang II）和锌会增加活性氧（ROS）并导致血管平滑肌细胞（VSMC）衰老，且Ang II诱导的衰老过程是一个锌依赖的过程。锌可刺激NADPH氧化酶（Nox）活性；然而，Nox亚型在锌作用中的角色尚未明确。 结果：在此，我们发现，通过小干扰RNA（siRNA）下调Nox1而非Nox4，可预防Ang II和锌诱导的VSMC衰老。另一方面，Nox1过表达会诱导衰老，这与增殖减少、端粒酶表达降低及DNA损伤增加有关。锌可增加Nox1蛋白表达，用三乙烯四胺（TPEN）螯合锌以及过表达锌转运体ZnT3和ZnT10可抑制这一作用。这些转运体可减少胞质锌，表明胞质锌增加介导了Nox1上调。包括铜、铁、钴和锰在内的其他金属未能上调Nox1，提示该途径具有锌特异性。放线菌素D（ACD，一种转录抑制剂）、抑制已知的Nox1转录调节因子NF-κB以及N-乙酰半胱氨酸（NAC）和线粒体靶向抗氧化剂MitoTEMPO均可抑制Nox1上调，提示NF-κB和线粒体ROS介导了锌的作用。支持这一观点的是，我们发现锌增加了胞质中的NF-κB激活，刺激p65亚基转位至细胞核，且锌在线粒体中蓄积增加了线粒体ROS（使用MitoSox测定）。此外，抑制NF-κB或用MitoTEMPO减少线粒体ROS可减轻锌诱导的衰老。MitoTEMPO也可降低NF-κB活性，提示线粒体ROS位于NF-κB上游。 创新与结论：我们的数据表明，锌分布改变导致锌在线粒体中蓄积，增加了线粒体ROS生成，引起NF-κB激活，进而上调Nox1表达，诱导VSMC衰老。
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