Molecular mechanism of the transcription regulation by the retinoic acid receptor in human salivary gland cell line HSG.

HSG细胞的生长受维甲酸(RA)的调节。最近发现，在RA信号转导过程中，维甲酸受体(RAR)与CREB结合蛋白(CBP)和p160家族成员蛋白等共激活因子相互作用，促进核心转录因子对DNA的访问。免疫沉淀和Western blotting检测到CBP和类固醇受体共激活因子1(SRC1)的表达，具有组蛋白乙酰转移酶的活性。CBP在HSG细胞中的过表达使依赖RA的转录激活增加了近10倍。CBP反义寡核苷酸的导入抑制了反式激活的增加。这些结果表明，在HSG细胞中表达的CBP与RARs协同介导了生长调节转录激活。我们先前已经克隆了Coup-转录因子I的DNA片段。在本研究中，RT-PCR和Western blotting证实了全长COUP-TFI的表达。为了确定COUP-TFI在RA信号转导中的作用，对报告基因进行了分析。COUP-TFI的过表达抑制了RA诱导的报告基因的转录激活。染色质整合的稳定转染的报告基因系统也显示了类似的结果。COUP-TFI的反义寡核苷酸抑制了RA依赖的生长抑制，这是通过[~3H]胸腺嘧啶核苷掺入来衡量的。根据这些结果，COUP-TFI很可能通过抑制RA诱导的反式激活来调节RA敏感的过程，如细胞的增殖或分化。

Growth of HSG cells is regulated by retinoic acid (RA). Recently, it has been revealed that, in the process of RA signaling, retinoic acid receptors (RAR) interact with coactivators such as CREB-binding protein (CBP) and p160 family member proteins, which facilitates the access of core transcription factors to the DNA.To investigate the relationship of coactivators to the RA signaling in HSG cells, we examined the expression of coactivators. Immunoprecipitation and western blotting revealed the expression of CBP and steroid receptor coactivator 1 (SRC 1), which exhibited the activity of histone acetyltransferase. The overexpression of CBP in HSG cell strongly increased the RA-dependent transcription activation approximately 10-fold. This increase of the transactivation was inhibited by the transfection of the antisense oligonucleotide for CBP.These findings suggest that CBP expressed in HSG cells mediates the growth-regulating transcription activation in concert with RARs.We have previously cloned the DNA fragment of COUP-transcription factor I (COUP-TFI). In this study, the expression of full length COUP-TFI was confirmed by use of RT-PCR and western blotting. To determine the role of COUP-TFI in the RA signaling, the reporter gene analysis was examined. The overexpression of COUP-TFI suppressed the RA-induced transcription activation of the reporter gene. Similar results were shown using a chromatin-integrated stably-transfected reporter gene system. The antisense oligonucleotide for COUP-TFI squelched the RA-dependent growth inhibition which was measured by the [^3H]thymidine incorporation. From these results, COUP-TFI very likely regulates RA-sensitive processes such as proliferation or differentiation of the cells by repressing the RA-induced transactivation.