The Stoichiometric Transition from Zn6Cu1-Metallothionein to Zn7-Metallothionein Underlies the Up-regulation of Metallothionein (MT) Expression QUANTITATIVE ANALYSIS OF MT-METAL LOAD IN EYE CELLS
The Stoichiometric Transition from Zn6Cu1-Metallothionein to Zn7-Metallothionein Underlies the Up-regulation of Metallothionein (MT) Expression QUANTITATIVE ANALYSIS OF MT-METAL LOAD IN EYE CELLS
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DOI：
10.1074/jbc.m112.365015
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发表时间：
2012-08-17
影响因子：
4.8
通讯作者：
Coca-Prados, Miguel
中科院分区：
文献类型：
--
作者：
Alvarez, Lydia;Gonzalez-Iglesias, Hector;Coca-Prados, Miguel
我们通过基于微阵列的分析研究了尸体眼人体组织中金属硫蛋白（MT）的基因表达谱。所有的MT 1亚型，除了MT 1B，在透镜和角膜组织中大量表达。与MT 1B一起，在任何组织中均未检测到MT4沿着。MT 1/2的抗体标记角膜上皮细胞和内皮细胞，而MT3标记视网膜神经节细胞。我们研究了锌和细胞因子对角膜上皮细胞系（HCEsv）中MT亚型基因表达的影响。锌引起MT亚型表达的上调，并且在IL 1 α或TNF α存在下，这种作用进一步增强。锌也引起了强烈的下调炎症细胞因子的表达，这种效果被阻断在TNF α或IL 1 α的存在。通过质谱法测定培养的HCEsv中MT、结合锌和MT的金属化学计量的浓度。MT的总浓度为0.24 +/- 0.03 μ M，在锌暴露24小时后，增加至0.96 +/- 0.01 μ M。锌和IL 1 α的组合进一步将MT水平提高至1.13 +/- 0.03 μ M。MT的平均金属化学计量比为Zn 6 Cu 1-MT，在暴露于不同处理后，其变为Zn 7-MT。放线菌素D阻断转录，放线菌酮衰减合成的MTs在存在或不存在的锌，这表明转录调控。总的来说，这些数据提供了锌，MTs和促炎细胞因子在HCEsv细胞中相互作用的分子和分析证据，对基于细胞的炎性眼病具有潜在的影响。
We examined the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based analysis. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1 alpha or TNF alpha. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNF alpha or IL1 alpha. The concentration of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were determined by mass spectrometry. The total concentration of MTs was 0.24 +/- 0.03 mu M and, after 24 h of zinc exposure, increased to 0.96 +/- 0.01 mu M. The combination of zinc and IL1 alpha further enhanced the level of MTs to 1.13 +/- 0.03 mu M. The average metal stoichiometry of MTs was Zn6Cu1-MT, and after exposure to the different treatments, it changed to Zn-7-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide molecular and analytical evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.