Ren Y, Smith A
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110, USA.
J Biol Chem. 1995 Oct 13;270(41):23988-95. doi: 10.1074/jbc.270.41.23988.
Heme-hemopexin or cobalt protoporphyrin (CoPP)-hemopexin (a model ligand for hemopexin receptor occupancy) is shown to increase transcription of the metallothionein-1 (MT-1) gene by activation of a signaling pathway. Promoter deletion analysis followed by transient transfection assays show that 110 base pairs (-153 to -43) of 5'-flanking region of the murine MT-1 promoter are sufficient for increasing transcription in response to heme-hemopexin or to CoPP-hemopexin in mouse hepatoma cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), prevented the increase in MT-1 transcription by heme-hemopexin, CoPP-hemopexin, or phorbol 12-myristate 13-acetate, but the protein kinase A inhibitor, HA1004, was without effect. N-Acetylcysteine (NAC) and glutathione, as well as superoxide dismutase and catalase, inhibited both the increase in endogenous MT-1 mRNA and the activation of reporter gene activity by heme-hemopexin, CoPP-hemopexin, and phorbol 12-myristate 13-acetate. In sum, these data suggest that reactive oxygen intermediates are generated by heme-hemopexin via events associated with receptor binding, including protein kinase C activation. Induction of heme oxygenase-1 expression, in contrast to MT-1, is significantly less sensitive to NAC. Deletion and mutation analyses of the MT-1 proximal promoter revealed that the sequence 5'-GTGACTATGC-3' (from -98 to -89 base pairs) is, in part, responsible for the hemopexin-mediated regulation of MT-1 which is inhibited by H7. Regulation via this element is also induced by H2O2 showing that it is an antioxidant response element. Heme itself acts via more distal elements on the MT-1 promoter. In contrast to NAC and glutathione, diethyl dithiocarbamate and pyrrolidine dithiocarbamate, which inactivate reactive oxygen intermediates and chelate Zn(II), synergistically augment the induction of MT-1 mRNA levels and reporter gene activity in response to heme-hemopexin via the antioxidant response element by both metal-responsive element-dependent and -independent mechanisms.
血红素 - 血红素结合蛋白或钴原卟啉（CoPP） - 血红素结合蛋白（一种用于占据血红素结合蛋白受体的模型配体）被证明可通过激活信号通路来增加金属硫蛋白 -1（MT -1）基因的转录。通过瞬时转染实验进行的启动子缺失分析表明，小鼠MT -1启动子5'侧翼区域的110个碱基对（-153至-43）足以在小鼠肝癌细胞中响应血红素 - 血红素结合蛋白或CoPP - 血红素结合蛋白而增加转录。蛋白激酶C抑制剂1 -（5 - 异喹啉磺酰基）-2 - 甲基哌嗪二盐酸盐（H7）可阻止血红素 - 血红素结合蛋白、CoPP - 血红素结合蛋白或佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯引起的MT -1转录增加，但蛋白激酶A抑制剂HA1004则无此作用。N - 乙酰半胱氨酸（NAC）和谷胱甘肽，以及超氧化物歧化酶和过氧化氢酶，均可抑制内源性MT -1 mRNA的增加以及血红素 - 血红素结合蛋白、CoPP - 血红素结合蛋白和佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯对报告基因活性的激活。总之，这些数据表明，血红素 - 血红素结合蛋白通过与受体结合相关的事件产生活性氧中间体，包括蛋白激酶C的激活。与MT -1相反，血红素加氧酶 -1表达的诱导对NAC的敏感性显著较低。MT -1近端启动子的缺失和突变分析表明，序列5'-GTGACTATGC-3'（从-98至-89个碱基对）部分负责血红素结合蛋白介导的MT -1调节，而这种调节被H7抑制。通过该元件的调节也可由H2O2诱导，表明它是一个抗氧化反应元件。血红素本身通过MT -1启动子上更远端的元件起作用。与NAC和谷胱甘肽不同，二乙基二硫代氨基甲酸盐和吡咯烷二硫代氨基甲酸盐可使活性氧中间体失活并螯合Zn（II），它们通过金属反应元件依赖性和非依赖性机制，协同增强通过抗氧化反应元件对血红素 - 血红素结合蛋白的反应中MT -1 mRNA水平的诱导和报告基因活性。
