Awad Hanan, Nolette Nora, Hinton Martha, Dakshinamurti Shyamala
Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Canada.
Pediatr Pulmonol. 2014 Sep;49(9):885-97. doi: 10.1002/ppul.22919. Epub 2013 Oct 25.
Hypoxia and reactive oxygen species (ROS) including H(2)O(2) play major roles in triggering and progression of pulmonary vascular remodeling in persistent pulmonary hypertension. Catalase (CAT), the major endogenous enzyme scavenging H(2)O(2), is regulated in a tissue- and context-specific manner.
To investigate mechanisms by which hypoxia and H(2)O(2) regulate catalase expression, and the role of AMPK-FoxO pathway, in neonatal porcine pulmonary artery smooth muscle (PASMC).
DESIGN/METHODS: PASMC were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 72 hr. We measured catalase activity and lipid peroxidation; CAT, FoxO1, and FoxO3a expression by qPCR; protein contents of CAT, FoxOs, p-AMPK, p-AKT, p-JNK, p-ERK1/2 in whole lysates, and FoxOs in nuclear extracts, by immunoblot; and FoxO-1 nuclear localization by immunocytochemistry, quantified by laser scanning cytometry.
Hypoxia upregulated CAT transcription, content and activity, by increasing CAT transcription factors FoxO1 and FoxO3a mRNA, and promoting nuclear translocation of FoxO1. However, lipid peroxidation increased in hypoxic PASMC. Among candidate FoxO regulatory kinases, hypoxia activated AMPK, and decreased p-Akt and ERK1/2. AMPK activation increased FoxO1 (total and nuclear) and CAT, while AMPK inhibition inhibited FoxO1 and CAT, but not FoxO3a. Exogenous H(2)O(2) decreased p-AMPK and increased p-AKT in hypoxic PASMC. This decreased active FoxO1, and reduced mRNA and protein content of CAT. Hypoxic induction of CAT, AKT inhibition (LY294002), or addition of PEG-catalase partly ameliorated the H(2)O(2) -mediated loss of nuclear FoxO1.
Hypoxia induces catalase expression, though this adaptation is insufficient to protect PASMC from hypoxia-induced lipid peroxidation. This occurs via hypoxic activation of AMPK, which promotes nuclear FoxO1 and thus catalase expression. Exogenous ROS may downregulate cellular antioxidant defenses; H(2)O(2) activates survival factor Akt, decreasing nuclear FoxO1 and thus catalase.
缺氧及包括过氧化氢（H₂O₂）在内的活性氧（ROS）在持续性肺动脉高压中引发和促进肺血管重塑过程中起主要作用。过氧化氢酶（CAT）是清除H₂O₂的主要内源性酶，其表达受组织和环境特异性调控。
研究缺氧和H₂O₂调节过氧化氢酶表达的机制，以及AMPK-FoxO信号通路在新生猪肺动脉平滑肌（PASMC）中的作用。
设计/方法：将PASMC置于缺氧（10% O₂）或常氧（21% O₂）环境中培养72小时。我们检测了过氧化氢酶活性和脂质过氧化水平；通过qPCR检测CAT、FoxO1和FoxO3a的表达；通过免疫印迹检测全细胞裂解物中CAT、FoxOs、p-AMPK、p-AKT、p-JNK、p-ERK1/2的蛋白含量，以及核提取物中FoxOs的蛋白含量；通过免疫细胞化学检测FoxO-1的核定位，并通过激光扫描细胞术进行定量分析。
缺氧通过增加CAT转录因子FoxO1和FoxO3a的mRNA水平以及促进FoxO1的核转位，上调了CAT的转录、含量及活性。然而，缺氧的PASMC中脂质过氧化水平增加。在候选的FoxO调节激酶中，缺氧激活了AMPK，并降低了p-Akt和ERK1/2的水平。AMPK的激活增加了FoxO1（总蛋白和核蛋白）及CAT的水平，而AMPK的抑制则抑制了FoxO1和CAT，但对FoxO3a无影响。外源性H₂O₂降低了缺氧PASMC中p-AMPK的水平并增加了p-AKT的水平。这降低了活性FoxO1的水平，并减少了CAT的mRNA和蛋白含量。缺氧诱导的CAT、AKT抑制（LY294002）或添加聚乙二醇化过氧化氢酶可部分改善H₂O₂介导的核内FoxO1的丢失。
缺氧诱导过氧化氢酶表达，尽管这种适应性变化不足以保护PASMC免受缺氧诱导的脂质过氧化损伤。这是通过缺氧激活AMPK来实现的，AMPK促进核内FoxO1的表达从而促进过氧化氢酶的表达。外源性ROS可能下调细胞抗氧化防御；H₂O₂激活生存因子Akt，降低核内FoxO1的水平，进而降低过氧化氢酶的水平。
