Alam J, Smith A
Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, Louisiana 70121.
J Biol Chem. 1992 Aug 15;267(23):16379-84.
Hemopexin-mediated heme transport into mouse hepatoma (Hepa) cells and human promyelocytic (HL-60) cells stimulates the expression of heme oxygenase via transcriptional activation (Alam, J., and Smith, A. (1989) J. Biol. Chem. 264, 17637-17640). Incubation of both these cell types in serum-free medium containing heme-hemopexin is shown here also to increase the steady-state level of metallothionein (MT) mRNA in a time- and dose-dependent manner. Heme-hemopexin is a far more effective inducer (12-fold) of the MT isozyme 1 (MT-1) in Hepa cells than nonprotein-bound heme (4-fold). Apohemopexin has no effect on MT-1 expression, and incubation with heme-hemopexin of mouse L fibroblasts that lack hemopexin receptors does not affect MT-1 expression. Thus, an interaction between the heme-hemopexin complex and its receptor is necessary for increased accumulation of MT-1 transcripts. In vitro nuclear "run-on" analysis indicates that the heme-hemopexin-mediated accumulation of MT-1 mRNA is regulated primarily at the level of initiation of transcription. A highly labile protein is required for constitutive MT-1 gene expression and acts to repress transcription. Transcriptional activation by heme or metals may require decreased concentrations or inactivation of the repressor as well as an additional inducer-specific trans-acting factor. Inhibition of protein synthesis augments the heme-hemopexin-mediated accumulation of MT-1 mRNA. Activation of heme oxygenase (HO) gene transcription by heme requires the synthesis of one (or more) heme-inducible proteins that are labile or become labile upon cycloheximide-sensitive processing or activation. Our comparison of MT and HO points to significant differences in the mechanisms of gene regulation by heme. The concomitant regulation of gene expression of MT-1 and HO in response to heme-hemopexin appears to be a concerted adaptive response of the cells, mediated at the level of the plasma membrane hemopexin receptor, and may relate to the proposed role of MT as an intracellular antioxidant or to a need to sequester zinc which otherwise would compete with iron and occupy sites on regulatory proteins such as the iron-responsive elements.
血红素结合蛋白介导的血红素转运进入小鼠肝癌（Hepa）细胞和人早幼粒细胞（HL - 60）细胞，通过转录激活刺激血红素加氧酶的表达（阿拉姆，J.，和史密斯，A.（1989年）《生物化学杂志》264，17637 - 17640）。本文还显示，将这两种细胞类型在含有血红素 - 血红素结合蛋白的无血清培养基中孵育，会以时间和剂量依赖的方式增加金属硫蛋白（MT）mRNA的稳态水平。在Hepa细胞中，血红素 - 血红素结合蛋白是MT同工酶1（MT - 1）的一种比非蛋白结合血红素更有效的诱导剂（12倍对4倍）。脱辅基血红素结合蛋白对MT - 1表达没有影响，并且用缺乏血红素结合蛋白受体的小鼠L成纤维细胞与血红素 - 血红素结合蛋白孵育不会影响MT - 1表达。因此，血红素 - 血红素结合蛋白复合物与其受体之间的相互作用对于MT - 1转录本的积累增加是必要的。体外细胞核“连续转录”分析表明，血红素 - 血红素结合蛋白介导的MT - 1 mRNA积累主要在转录起始水平受到调控。一种高度不稳定的蛋白质对于组成型MT - 1基因表达是必需的，并且起到抑制转录的作用。血红素或金属的转录激活可能需要降低阻遏物的浓度或使其失活，以及一种额外的诱导剂特异性反式作用因子。蛋白质合成的抑制增强了血红素 -血红素结合蛋白介导的MT - 1 mRNA积累。血红素对血红素加氧酶（HO）基因转录的激活需要合成一种（或多种）血红素诱导蛋白，这些蛋白不稳定，或者在环己酰亚胺敏感的加工或激活后变得不稳定。我们对MT和HO的比较指出了血红素在基因调控机制上的显著差异。MT - 1和HO基因表达对血红素 - 血红素结合蛋白的伴随调控似乎是细胞的一种协同适应性反应，在质膜血红素结合蛋白受体水平介导，并且可能与MT作为细胞内抗氧化剂的假定作用有关，或者与螯合锌的需求有关，否则锌会与铁竞争并占据诸如铁反应元件等调节蛋白上的位点。
