Transcriptional regulation and characterization of the promoter region of the human ABCC6 gene
Transcriptional regulation and characterization of the promoter region of the human ABCC6 gene
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DOI：
10.1038/sj.jid.5700065
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发表时间：
2006-02-01
影响因子：
6.5
通讯作者：
Uitto, Jouni
中科院分区：
文献类型：
--
作者：
Jiang, Qiujie;Matsuzaki, Yasushi;Uitto, Jouni
ABCC 6是腺苷5 '-三磷酸结合盒基因家族的成员，编码多药耐药相关蛋白6，这是一种推定的跨膜转运蛋白，主要在肝脏中表达，在其他组织中表达程度显著较低。ABCC 6突变导致弹性纤维性假黄瘤，一种具有可变表型表达的多系统遗传性结缔组织疾病。为了研究该基因的转录调控和组织特异性表达，我们克隆了2.6 kb的人ABCC 6启动子，并开发了一系列与荧光素酶报告基因连接的50缺失构建体。在多种不同来源的培养细胞系中的瞬时转染鉴定了特异性NF-κ B样序列（-235/-226），其在HepG 2肝癌细胞中赋予高水平表达，推断肝脏特异性。启动子片段的功能性通过尾静脉注射随后荧光素酶报告基因测定在体内确认。选定的细胞因子的测试显示，转化生长因子（TGF）-β上调，而肿瘤坏死因子（TNF）-α和干扰素（IFN）-γ下调HepG 2细胞中的启动子活性。对TGF-β的反应性主要位于-58至-49的Sp1/Sp3同源结合位点内。ABCC 6启动子的表达也被证明是显着增强的Sp1蛋白，证明了通过共转染的ABCC 6启动子荧光素酶结构和Sp1表达载体在果蝇SL 2细胞，这是缺乏内源性Sp1。此外，蛋白质/DNA阵列显示，另外四种转录因子及其存在于DNA中的同源结合序列与2.6 kb启动子片段结合。总的来说，结果表明，人ABCC 6显示组织特异性基因表达，这可以通过促炎细胞因子调节。这些发现可能对涉及ABCC 6基因异常的遗传性和获得性疾病的表型表达有影响。
ABCC6, a member of the adenosine 5'-triphosphate-binding cassette family of genes, encodes multidrug resistance-associated protein 6, a putative transmembrane transporter expressed primarily in the liver and to a significantly lower extent in other tissues. Mutations in ABCC6 result in pseudoxanthoma elasticum, a multisystem heritable connective tissue disorder with variable phenotypic expression. To examine the transcriptional regulation and tissue-specific expression of this gene, we cloned 2.6 kb of human ABCC6 promoter and developed a series of 50-deletion constructs linked to luciferase reporter gene. Transient transfections in a number of cultured cell lines of diverse origin identified a specific NF-kappa B-like sequence (-235/-226), which conferred high level of expression in HepG2 hepatoma cells, inferring liver specificity. The functionality of the promoter fragments was confirmed in vivo by tail vein injection followed by luciferase reporter assay. Testing of selected cytokines revealed that transforming growth factor (TGF)-beta upregulated, while tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma downregulated the promoter activity in HepG2 cells. The responsiveness to TGF-beta was shown to reside primarily within an Sp1/Sp3 cognate-binding site at -58 to -49. The expression of the ABCC6 promoter was also shown to be markedly enhanced by Sp1 protein, as demonstrated by cotransfection of ABCC6 promoter-luciferase constructs and an Sp1 expression vector in Drosophila SL2 cells, which are devoid of endogenous Sp1. Furthermore, four additional transcription factors, with their cognate-binding sequences present in DNA, were shown to bind the 2.6-kb promoter fragment by protein/DNA array. Collectively, the results indicate that human ABCC6 displays tissue-specific gene expression, which can be modulated by proinflammatory cytokines. These findings may have implications for phenotypic expression of heritable and acquired diseases involving abnormality in the ABCC6 gene.