Hemin and iron increase synthesis and trigger export of xanthine oxidoreductase from hepatocytes to the circulation.
Hemin and iron increase synthesis and trigger export of xanthine oxidoreductase from hepatocytes to the circulation.
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DOI：
10.1016/j.redox.2023.102866
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发表时间：
2023-11
期刊：
影响因子：
11.4
通讯作者：
Kelley, Eric E.
中科院分区：
文献类型：
--
作者：
Devallance, Evan R.;Schmidt, Heidi M.;Seman, Madison;Lewis, Sara E.;Wood, Katherine C.;Vickers, Schuyler D.;Hahn, Scott A.;Velayutham, Murugesan;Hileman, Emily A.;Vitturi, Dario A.;Leonardi, Roberta;Straub, Adam C.;Kelley, Eric E.
我们最近报道了黄嘌呤氧化还原酶（XOR）在血管内血红素超载中的有益作用，因此肝细胞向循环输出XOR被确定为提供保护的开创性步骤。然而，支持这一过程的细胞信号传导和输出机制尚未确定。在这里，我们提供的新数据显示，肝细胞在暴露于血凝素结合的血红素、血红素或游离铁后，会上调XOR表达/蛋白丰度，并主动将其释放到细胞外腔室。例如，用hemin （10 μM）处理的小鼠（AML-12细胞）肝细胞在没有细胞死亡或膜完整性丧失的情况下输出XOR（2.0±1.0 vs 16±9 μU/mL p < 0.0001）。结果表明，溶酶体运输抑制剂Vaculin-1（84±17 μU/mg vs 24±8 μU/mg vs 5±3 μU/mg对照）与Brefeldin a（84±17 μU/mg vs 24±8 μU/mg）预处理肝细胞抑制XOR释放和促进细胞内XOR积累的途径是不规范的。有趣的是，与血红蛋白相比，游离铁（Fe2+和Fe3+）诱导了类似的XOR上调和释放。相反，与血红素和经典过渡金属螯合剂DTPA （20 μM）或尿酸同时治疗完全阻断XOR释放（p < 0.01）。我们之前发表的时间过程显示，肝细胞的XOR释放可能需要转录上调。因此，我们确定Sp1和NF-kB都被hemin治疗急性激活（两者为2倍>对照，p < 0.05），并且用siRNA沉默TLR4或TLR4可以阻止hemin诱导的XOR上调（p < 0.01）。最后，为了确认这些转录因子对Xdh基因的直接作用，进行了染色质免疫沉淀，表明血红蛋白在转录起始位点附近显著富集（约5倍）Sp1和NF-kB。总之，我们的研究确定了一个以前未知的途径，通过SP1/NF-kB上调XOR，随后输出到细胞外环境。据我们所知，这是第一个从机制上证明异或可以作为血红素/铁过载补偿性反应的开创性步骤专门针对出口的研究。血红素、血红素-血红素复合物和游离铁上调肝细胞XOR并引发胞吐。XOR的血红素介导的胞外分泌是非典型的，依赖于溶酶体的转运。hemin介导的XOR上调依赖于TLR4，并通过p65和SP1介导下游。肝细胞释放XOR可能是对血红素/铁超载的适应性/保护性反应。
We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 μM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 μU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control μU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 μM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload. Hemin, hemin-hemopexin complexes and free iron upregulate hepatocellular XOR and trigger exocytosis. The hemin-mediated exocytosis of XOR is noncanonical and dependent on lysosomal trafficking. Hemin-mediated upregulation of XOR is TLR4 dependent and mediated downstream via p65 and SP1. Hepatocellular release of XOR may be an adaptive/protective response to hemin/Fe overload.
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影响因子：
11.4
作者：
Cantu-Medellin N;Kelley EE
通讯作者：
Kelley EE
影响因子：
11.4
作者：
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影响因子：
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Harmon, Daniel B.;Mandler, W. Kyle;Kelley, Eric E.
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影响因子：
4.4
作者：
Boekhoudt, GH;Guo, Z;Boss, JM
通讯作者：
Boss, JM
影响因子：
4.8
作者：
Godber, BLJ;Doel, JJ;Harrison, R
通讯作者：
Harrison, R