Molecular basis for heme-dependent induction of heme oxygenase in primary cultures of chick embryo hepatocytes. Demonstration of acquired refractoriness to heme.
Molecular basis for heme-dependent induction of heme oxygenase in primary cultures of chick embryo hepatocytes. Demonstration of acquired refractoriness to heme.
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DOI：
10.1111/j.1432-1033.1993.tb17835.x
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发表时间：
1993-05
期刊：
影响因子：
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通讯作者：
Kishore K. Srivastava;E. Cable;S. Donohue;Herbert L. Bonkovsky
中科院分区：
文献类型：
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作者：
Kishore K. Srivastava;E. Cable;S. Donohue;Herbert L. Bonkovsky
在原代培养的鸡胚肝细胞中，研究了血红素对血红素氧合酶-1的mRNA和蛋白质合成的诱导作用。血红素以剂量依赖的方式增加mRNA的量和血红素加氧酶-1-基因转录的速率，在20 μ M血红素时最大增加20倍。最大的增加，在转录速率，核运行的测定，发生在5小时，2小时前的mRNA的量的最大增加，通过密度测定的北方印迹。7-15血红素加氧酶-1mRNA的半衰期为3.5h。相比之下，放线菌酮的加入显着增加了稳定性的消息（半衰期= 18小时），这表明，一个短暂的蛋白质在调节血红素加氧酶-1 mRNA水平起着关键作用。通过[35 S]蛋氨酸标记和免疫沉淀法测定血红素诱导的血红素加氧酶-1蛋白的半衰期为15 h。蛋白质的这种长半衰期可以在很大程度上解释另外的发现，即在加入血红素后，细胞中酶蛋白的量增加了10小时，之后它基本上保持恒定15小时。一个惊人的发现是，在血红素刺激的基因转录的初始爆发后，细胞变得难以进一步血红素介导的诱导。这种获得性抗性不能归因于以下因素：培养时间较长;血红素引起的细胞毒性;培养基或细胞中缺乏血红素;血红素结合蛋白分泌到培养基中，阻止进一步的血红素摄取;诱导细胞血红素催化剂足以耗尽细胞血红素。相反，结果表明，血红素加氧酶-1的血红素依赖性诱导所需的细胞内机制的下调。
The effects of heme on the induction of mRNA and protein synthesis for heme oxygenase-1 have been studied in primary cultures of chick embryo liver cells. Heme increased the amount of mRNA and the rate of heme oxygenase-1-gene transcription in a dose-dependent fashion, with a maximal 20-fold increase occurring at 20 microM heme. The largest increase in the rate of transcription, measured by nuclear run-on assays, occurred at 5 h, 2 h earlier than the maximum increase in the amount of mRNA, measured by densitometry of Northern blots. 7-15 h after heme addition, the half-life of heme-oxygenase-1 mRNA was 3.5 h in the presence or absence of actinomycin D. In contrast, addition of cycloheximide markedly increased the stability of the message (half-life = 18 h), suggesting that a short-lived protein plays a key role in modulating heme oxygenase-1 mRNA levels. The half-life of heme-induced heme-oxygenase-1 protein, measured by [35S]methionine labelling and immunoprecipitation, was 15 h. This long half-life of the protein can largely account for the additional finding that, following addition of heme, the amount of enzyme protein in the cells increased for 10 h, after which it remained essentially constant for 15 h. A striking finding was that, after an initial burst of heme-stimulated gene transcription, the cells became refractory to further heme-mediated induction. This acquired resistance could not be attributed to the following: a longer duration of culture time; cellular toxicity caused by heme; a lack of heme in the medium or the cells; secretion of heme-binding proteins into the medium, preventing further heme uptake; the induction of cellular heme catabolism sufficient to deplete cellular heme. Instead, the results suggest a down-regulation of the intracellular machinery required for heme-dependent induction of heme oxygenase-1.