A critical role for increased labile zinc in reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis
A critical role for increased labile zinc in reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis
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DOI：
10.1152/ajplung.00385.2011
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发表时间：
2012-06-01
影响因子：
4.9
通讯作者：
Pitt, Bruce R.
中科院分区：
文献类型：
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作者：
Thambiayya, Kalidasan;Wasserloos, Karla;Pitt, Bruce R.
我们先前注意到在脂多糖诱导的绵羊肺动脉内皮细胞(SPAEC)的凋亡过程中，细胞内不稳定锌([Zn](I))的减少起着重要的信号作用(ThomZL，Wasserlos KJ，Liu X，Stitt MS，Reynolds IJ，Pitt BR，St Croix CM)。Mol Cell Biochem 234-235：211-217,2002；Thambiayya K，Wasserlos KJ，Huang Z，Kagan VE，St Croix CM，Pitt BR.Am J Physiol肺细胞分子Physiol 300：L624-632,2011)。在本研究中，我们使用小干扰RNA(SiRNA)对锌稳态的重要贡献者[锌进口蛋白SLC39A14或Zrt/irt样蛋白14(ZIP14)；锌结合蛋白金属硫蛋白(MT)]确定了细胞外锌或一氧化氮(NO)增加不稳定[锌](I)[例如，可被N，N，N‘，N’，N‘-四(2-吡啶甲基)乙二胺检测和/或络合的锌敏感荧光团(FluoZin-3)]的分子途径。在无血清培养的SPAEC培养液中加入10 mU/L锌，可增加细胞内[锌](I)浓度，并可阻断脂多糖诱导的细胞凋亡(如膜联蛋白V结合增加)。细胞外锌对ZIP14表达的降低(SiRNA)很敏感，但不受共同敲除绵羊MT主要亚型(SMT-Ia、-Ib、-Ic和-II)的影响。用250mM的NO供体S-亚硝基-N-乙酰青霉胺(SNAP)处理野生型血管内皮细胞，以N，N，N‘，N’-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive的方式增加细胞外锌的活性，降低细胞对脂多糖诱导的细胞凋亡(如caspase-3/7激活)的敏感性。SNAP的抗凋亡作用对ZIP14的siRNA敲除不敏感，但当绵羊MT用siRNA处理SPAEC时，SNAP的抗凋亡作用被消除(伴随着SNAP引起的[Zn](I)升高)。在体外实验中，锌能直接抑制重组caspase-3的活性。总而言之，这些数据表明，不稳定[锌](I)的增加是ZIP14或NO介导的对内毒素诱导的细胞凋亡抵抗的重要组成部分。ZIP14的细胞保护作用次要于细胞外锌的跨细胞移动，而NO介导的保护作用次要于S亚硝化MT和[Zn](I)的重新分布。
We previously noted an important signaling role for decreased labile intracellular zinc ([Zn](i)) in LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) (Tang ZL, Wasserloos KJ, Liu X, Stitt MS, Reynolds IJ, Pitt BR, St Croix CM. Mol Cell Biochem 234-235: 211-217, 2002; Thambiayya K, Wasserloos KJ, Huang Z, Kagan VE, St Croix CM, Pitt BR. Am J Physiol Lung Cell Mol Physiol 300: L624-632, 2011). In the present study, we used small interfering RNA (siRNA) to important contributors of zinc homeostasis [SLC39A14 or Zrt/Irt-like protein 14 (ZIP14), a zinc importer; metallothionein (MT), a zinc binding protein] to define molecular pathways by which extracellular zinc or nitric oxide (NO) increase labile [Zn](i) [e.g., zinc-sensitive fluorophore (FluoZin-3) detectable and/or chelatable by N,N,N',N'- tetrakis(2-pyridylmethyl)ethylenediamine] and reduce the sensitivity of SPAEC to LPS. Addition of 10 mu M zinc to serum-free medium of SPAEC increased [Zn](i) and abolished LPS-induced apoptosis (e. g., increased annexin V binding). The increase in [Zn](i) and the protective effect of extracellular zinc were sensitive to reduction in ZIP14 expression (by siRNA), but not affected by collectively knocking down major isoforms of sheep MT (sMT-Ia, -Ib, -Ic, and -II). Pretreatment of wild-type SPAEC with 250 mu M of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased labile zinc in a relatively similar fashion to addition of extracellular zinc and reduced sensitivity of SPAEC to LPS-induced apoptosis (e.g., caspase-3/7 activation) in a N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive fashion. The antiapoptotic effects of SNAP were insensitive to siRNA knockdown of ZIP14, but were abolished (along with SNAP-induced increase in [Zn](i)) when SPAEC were pretreated with siRNA to sheep MT. Zinc was able to directly inhibit recombinant caspase-3 activity in an in vitro assay. Collectively, these data show that increases in labile [Zn](i) are an important component of ZIP14- or NO-mediated resistance to LPS-induced apoptosis. Cytoprotection via ZIP14 appeared to be secondary to transcellular movement of extracellular zinc, whereas NO-mediated protection was secondary to S-nitrosation of MT and redistribution of [Zn](i).