Regulation of expression of the stress response gene, Osp94:: identification of the tonicity response element and intracellular signalling pathways
Regulation of expression of the stress response gene, Osp94:: identification of the tonicity response element and intracellular signalling pathways
复制标题
DOI：
10.1042/bj20040313
复制
发表时间：
2004-06-15
影响因子：
4.1
通讯作者：
Gullans, SR
中科院分区：
文献类型：
--
作者：
Kojima, R;Randall, JD;Gullans, SR
Osp94 （94 kDa的渗透应激蛋白）在小鼠肾髓内集管（mIMCD3）细胞高渗和热休克应激下上调。为了研究这些胁迫下Osp94基因转录调控的分子机制，我们克隆了该基因的5'侧区并对其进行了表征。对近端4 kb 5′侧区进行序列分析，发现一个含Sp1位点的TATA-less G/C-rich启动子区。我们还发现了与TonE/ORE（强压响应元件/渗透响应元件）和HSE（热冲击元件）相似的上游序列基序。转染含有TonE/ ore样元件（Osp94-TonE; 5′-TGGAAAGGACCAG-3′）和HSE的报告基因构建体的细胞荧光素酶活性在高渗应激和热休克应激下分别增强了报告基因的表达。电泳凝胶迁移分析显示，Osp94-TonE探针上有一个缓慢迁移的条带结合，可能代表TonEBP （TonE结合蛋白）与该增强子元件的结合。此外，用MAPK（丝裂原活化蛋白激酶）抑制剂（SB203580、PD98059、U0126和SP6001125）和蛋白酶体抑制剂（MG132）处理mIMCD3细胞可以抑制高渗NaCl引起的Osp94基因表达的增加。这些结果表明，Osp94基因的5'侧区含有一个高张力敏感的顺式作用元件Osp94- tone，不同于功能性HSE。此外，MAPK和蛋白酶体系统似乎至少部分地通过Osp94- tone参与高渗应激介导的Osp94调控。
Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5'-flanking region of the gene. Sequence analysis of the proximal 4 kb 5'-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus TonE/ORE (tonicity-response element/osmotic response element) as well as the consensus HSE (heat-shock element). Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE; 5'-TGGAAAGGACCAG-3') and HSE enhanced reporter gene expression under hypertonic stress and heat-shock stress respectively. Electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the Osp94-TonE probe, probably representing binding of TonEBP (TonE binding protein) to this enhancer element. Furthermore, treatment of mIMCD3 cells with MAPK (mitogen-activated protein kinase) inhibitors (SB203580, PD98059, U0126 and SP6001125) and a proteasome inhibitor (MG132) suppressed the increase in Osp94 gene expression caused by hypertonic NaCl. These results indicate that the 5'-flanking region of Osp94 gene contains a hypertonicity sensitive cis-acting element, Osp94-TonE, which is distinct from a functional HSE. Furthermore, the MAPK and proteasome systems appear to be, at least in part, involved in hypertonic-stressmediated regulation of Osp94 through Osp94-TonE.