Characterization of the SOCS3 promoter response to prostaglandin E2 in T47D cells
Characterization of the SOCS3 promoter response to prostaglandin E2 in T47D cells
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DOI：
10.1210/me.2007-0030
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发表时间：
2007-10-01
影响因子：
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通讯作者：
Curlewis, Jon D.
中科院分区：
文献类型：
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作者：
Barclay, Johanna L.;Anderson, Stephen T.;Curlewis, Jon D.
细胞因子信号传导抑制因子3（SOCS 3）是细胞因子信号传导的负调节因子，其在乳腺癌细胞中表达，在乳腺癌细胞中其可以通过Janus激酶/信号转导子和转录激活子（JAK/ STAT）途径改变对细胞因子信号传导的敏感性和响应性。尽管广泛接受SOCS 3表达本身受STAT调节，但我们和其他人已经表明，野牡丹素也可以上调SOCS 3表达。在这里，我们使用前列腺素E（2）（PGE（2））处理的T47 D乳腺癌细胞来检查这一途径。T47 D细胞对PGE（2）刺激的反应是SOCS 3 mRNA的显著增加，这与从头蛋白质合成无关。PGE（2）刺激导致STAT 3丝氨酸和酪氨酸磷酸化，尽管SOCS 3启动子上两个先前表征的STAT反应元件中的任何一个突变都不影响PGE（2）对SOCS 3启动子的激活。此外，STAT 3野生型、组成型活性或显性阴性构建体的过表达不影响PGE（2）诱导的SOCS 3启动子激活，表明STAT不太可能是这些细胞中该途径的介导物。PGE（2）是已知的cAMP/蛋白激酶A（PKA）途径的激活剂，并且在T47 D细胞中，PGE（2）对SOCS 3 mRNA的上调通过用PKA抑制剂H89预处理而被消除，并且通过cAMP和毛喉素处理而被增加。与此一致，PGE（2）处理增加cAMP反应元件（CRE）结合蛋白丝氨酸磷酸化。然而，启动子上激活蛋白1/ CRE的突变不影响基础或PGE（2）刺激的激活，表明cAMP/ PKA的作用不依赖于CRE结合蛋白的结合。SOCS 3启动子富含GC的区域（一个假定的Sp1/ Sp3结合位点）的突变消除了基础和PGE（2）刺激的激活。凝胶迁移试验表明，处理后复合物形成增加，这是通过添加Sp1抗体或PKA抑制剂预处理抑制。染色质免疫沉淀试验证实了Sp1与启动子的结合是对PGE的响应（2）。Sp1过表达增加了SOCS 3启动子的激活，并且基础和PGE（2）诱导的SOCS 3 mRNA表达都被Sp1 DNA结合抑制剂光辉霉素所阻止。最后，证明了PGE（2）的生理作用，其中PGE（2）预处理减少脂多糖诱导的STAT 3活化。总的来说，这项研究详细说明了乳腺癌细胞中PGE（2）上调SOCS 3的新机制，该机制似乎是STAT非依赖性的，并涉及Sp1与启动子的结合。这一过程可能与细胞因子反应性和肿瘤进展有关。
Suppressor of cytokine signaling 3 ( SOCS3), a negative regulator of cytokine signaling, is expressed in breast cancer cells where it can modify sensitivity and responsiveness to cytokine signaling through the Janus kinase/ signal transducer and activator of transcription ( JAK/ STAT) pathways. Although it is widely accepted that SOCS3 expression is in itself regulated by STATs, we and others have shown that prostaglandins can also up- regulate SOCS3 expression. Here we used T47D breast cancer cells treated with prostaglandin E(2) ( PGE(2)) to examine this pathway. T47D cells responded to PGE(2) stimulation with a significant increase in SOCS3 mRNA that was independent of de novo protein synthesis. PGE(2) stimulation resulted in STAT3 serine and tyrosine phosphorylation, although mutation of either of the two previously characterized STAT response elements on the SOCS3 promoter did not affect SOCS3 promoter activation by PGE(2). In addition, overexpression of STAT3 wild- type, constitutively active or dominant-negative constructs did not affect PGE(2) induced SOCS3 promoter activation, indicating that STATs are unlikely mediators of this pathway in these cells. PGE(2) is a known activator of the cAMP/ protein kinase A ( PKA) pathway, and in T47D cells, up- regulation of SOCS3 mRNA by PGE(2) was abolished by pretreatment with H89, a PKA inhibitor and increased by cAMP and forskolin treatment. Consistent with this, PGE(2) treatment increased cAMP response element ( CRE)- binding protein serine phosphorylation. However, mutation of the activator protein 1/ CRE on the promoter did not affect basal or PGE(2)- stimulated activation, suggesting a role for cAMP/ PKA that is independent of CRE- binding protein binding. Mutation of the GC- rich region of the SOCS3 promoter, a putative Sp1/ Sp3 binding site, abolished both basal and PGE(2)- stimulated activation. Gel- shift assays showed increased complex formation after treatment, and this was inhibited by the addition of an Sp1 antibody or pretreatment with PKA inhibitor. Chromatin immunoprecipitation assay verified Sp1 binding to the promoter in response to PGE(2). Sp1 overexpression increased SOCS3 promoter activation, and both basal and PGE(2)- induced SOCS3 mRNA expression was prevented by mithramycin, an inhibitor of Sp1 DNA binding. Finally, a physiological role for PGE(2) was demonstrated with PGE(2) pretreatment reducing lipopolysaccharide- induced STAT3 activation. Collectively, this study details a novel mechanism of SOCS3 up- regulation by PGE(2) in breast cancer cells that appears to be STAT independent and involve Sp1 binding to the promoter. This process has possible implications for cytokine responsiveness and tumor progression.