Barrett T J, Vedeckis W V
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, USA.
Recept Signal Transduct. 1996;6(3-4):179-93.
The glucocorticoid receptor (GR) and c-jun promoters both contain activator protein-1 (AP-1) sites (GR AP-1 site and c-jun AP-1 site, respectively) that vary from the consensus AP-1 site. Electrophoretic mobility shift assays (EMSAs) were used to monitor GR AP-1 and c-jun AP-1 oligonucleotide binding by nuclear extracts from AtT-20 and L929 cells that were hormone- and vehicle-treated for 1, 6, or 24 h. Both AtT-20 and L929 cell nuclear extracts bound the c-jun AP-1 site somewhat better than the GRAP-1 site and, in the majority of cases, extracts from hormone-treated cells shifted both GRAP-1 and c-jun AP-1 oligonucleotides more than nontreated nuclear extracts. Supershift assays, using Jun and Fos family member-specific antibodies, showed that protein complexes formed by AtT-20 cell nuclear extracts bound to the c-jun AP-1 site were comprised of Jun family members, JunD, JunB, and cJun. No Fos family members were present. However, protein complexes from AtT-20 nuclear extracts that bound the GR AP-1 site were supershifted by JunD, JunB, cJun, and Fra-2 specific antibodies. In L929 cell nuclear extracts, the c-jun AP-1 site is bound by JunD and cJun. No clear association of Fos family members with the c-jun AP-1 site could be demonstrated. The GR AP-1 site bound protein complexes composed of JunD, JunB, Fra-2, and Fra-1 from L929 nuclear extracts. This demonstrates that the composition of the protein complexes that associate with the c-jun AP-1 site differs from those that bind the GR AP-1 site. These data also indicate that the protein complexes that bind the GR and c-jun AP-1 sites are cell-type-specific. Computer analysis also revealed five putative cyclic AMP response elements (CREs) in the GR promoter. Relative mobility shift and binding studies suggest that CRE binding protein (CREB), CREB modulator (CREM), or CREB/CREM may be associated with the c-jun AP-1 and/or GR AP-1 sites, but the association at these sites occurs at a lower binding affinity than for a consensus CRE. Nuclear extracts from AtT-20 and L929 cells were able to shift the CRE, and supershift analysis revealed that Jun family members are part of the protein complexes that bind the CRE. Pan Jun and pan Fos antibodies were able to supershift protein-CRE complexes formed using NIH 3T3 nuclear extracts. These data raise the possibility that the promiscuous binding of CREB and/or CREM to the AP-1 site, and AP-1 transcription factors to one or more CREs, in the GR promoter may contribute to the regulation of GR gene expression.
糖皮质激素受体（GR）和c-jun启动子均含有与共有激活蛋白-1（AP-1）位点不同的AP-1位点（分别为GR AP-1位点和c-jun AP-1位点）。采用电泳迁移率变动分析（EMSA）来监测AtT-20和L929细胞经激素和赋形剂处理1、6或24小时后的核提取物对GR AP-1和c-jun AP-1寡核苷酸的结合情况。AtT-20和L929细胞核提取物与c-jun AP-1位点的结合略优于与GR AP-1位点的结合，并且在大多数情况下，激素处理细胞的提取物使GR AP-1和c-jun AP-1寡核苷酸的迁移率变动比未处理细胞核提取物的更大。使用Jun和Fos家族成员特异性抗体进行的超迁移分析表明，AtT-20细胞核提取物与c-jun AP-1位点结合形成的蛋白复合物由Jun家族成员JunD、JunB和cJun组成。不存在Fos家族成员。然而，AtT-20细胞核提取物中与GR AP-1位点结合的蛋白复合物被JunD、JunB、cJun和Fra-2特异性抗体超迁移。在L929细胞核提取物中，c-jun AP-1位点由JunD和cJun结合。未发现Fos家族成员与c-jun AP-1位点有明确关联。GR AP-1位点结合了由L929细胞核提取物中的JunD、JunB、Fra-2和Fra-1组成的蛋白复合物。这表明与c-jun AP-1位点结合的蛋白复合物的组成与与GR AP-1位点结合的不同。这些数据还表明，与GR和c-jun AP-1位点结合的蛋白复合物具有细胞类型特异性。计算机分析还揭示了GR启动子中有五个假定的环磷酸腺苷反应元件（CRE）。相对迁移率变动和结合研究表明，CRE结合蛋白（CREB）、CREB调节剂（CREM）或CREB/CREM可能与c-jun AP-1和/或GR AP-1位点相关，但这些位点的结合亲和力低于共有CRE。AtT-20和L929细胞的核提取物能够使CRE迁移，超迁移分析表明Jun家族成员是与CRE结合的蛋白复合物的一部分。泛Jun和泛Fos抗体能够使使用NIH 3T3细胞核提取物形成的蛋白-CRE复合物超迁移。这些数据增加了一种可能性，即CREB和/或CREM与AP-1位点的混杂结合，以及AP-1转录因子与GR启动子中一个或多个CRE的结合，可能有助于GR基因表达的调控。
