Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA
Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA
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DOI：
10.1016/j.bbrc.2011.01.034
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发表时间：
2011-02-18
影响因子：
3.1
通讯作者：
Takigawa, Masaharu
中科院分区：
文献类型：
--
作者：
Kondo, Seiji;Kubota, Satoshi;Takigawa, Masaharu
CCN 2/结缔组织生长因子（CTGF）可被缺氧诱导并促进肿瘤血管生成。我们的前期研究表明，在人软骨肉瘤细胞系HCS-2/8中，低氧诱导的人ccn 2 mRNA的基因表达受到转录后调控，其中位于ccn 2 mRNA 3 '非翻译区（UTR）的一个最小顺式元件（CAESAR）和一个35 kDa的蛋白质对应物通过决定ccn 2 mRNA的稳定性发挥重要作用。在本研究中，我们利用RNA亲和层析结合质谱鉴定了这个相应的蛋白为甘油醛-3-磷酸脱氢酶（GAPDH）。RNA结合试验的结果显示GAPDH与该顺式元件的特异性结合。为了进一步表征GAPDH和ccn 2 mRNA之间的相互作用，我们研究了氧化还原条件和糖酵解辅酶在GAPDH与ccn 2 mRNA结合中的作用。氧化剂，二酰胺，废除GAPDH-RNA相互作用的浓度依赖性的方式，而这种效果可以通过随后的治疗与2-巯基乙醇（2-ME）逆转。另外，GAPDH的辅酶烟酰胺腺嘌呤二核苷酸（NAD）可抑制GAPDH与RNA的结合。综上所述，这些发现表明，糖酵解酶GAPDH通过充当氧化应激和氧化还原信号的传感器来反式调节CCN 2 mRNA的基因表达，导致CCN 2在缺氧条件下过表达并促进血管生成。(C)2011 Elsevier Inc. All rights reserved.
CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3'-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis. (C) 2011 Elsevier Inc. All rights reserved.