Groussin L, Massias J F, Bertagna X, Bertherat J
Groupe d'Etude en Physiopathologie Endocrinienne, Centre National de la Recherche Scientifique, UPR1524, Institut Cochin de Génétique Moléculaire, Université René Descartes-Paris V, France.
J Clin Endocrinol Metab. 2000 Jan;85(1):345-54. doi: 10.1210/jcem.85.1.6307.
The pituitary hormone ACTH, acting through the cAMP pathway, plays a key role in proliferation and differentiation of the adrenal cortex. CAMP response element (CRE)-binding protein (CREB) is an ubiquitous transcription factor that binds to the CRE present in the promoter of numerous genes and mediates transcription stimulation by cAMP. Characterization of CRE-binding proteins was performed in the H295R cell line, which is considered a model for human adrenocortical tumor studies. Western blot and RT-PCR studies demonstrated that CREB is not expressed in the human adrenocortical cancer cell line H295R, whereas it is expressed in normal adrenal. During transient transfection experiments, cAMP stimulation of two reporter genes containing canonical CRE was maintained. Cotransfection of the dominant negative inhibitor A-CREB, which prevents transcription factors containing a CREB-like leucine zipper domain to bind DNA, completely inhibited cAMP-induced stimulation of CRE activity. Western blot and RT-PCR studies showed that activating transcription factor-1 (ATF-1), CRE modulator-alpha/gamma (CREMalpha/gamma), and CREMtau2alpha are expressed in H295R cells. High amounts of CREM proteins were present in H295R, demonstrating an overexpression of this transcription factor in the absence of CREB. Furthermore, expression of the activator isoform CREMtau was very high in H295R compared to normal adrenal cortex. Transfection assays demonstrated that CREMtau2alpha is a potent stimulator of CRE activity in H295R. Finally, gel retardation assays showed that CREM and ATF-1 are the nuclear proteins that specifically bind the CRE in H295R cells, whereas CREM binding to CRE is not observed in a CREB-expressing cell line. H295R cells are the first established nontransgenic cell line that does not express the ubiquitous transcription factor CREB. H295R demonstrates that CREMtau up-regulation can compensate for CREB deficiency to maintain CRE regulation by cAMP and is a model of compensation mechanisms between the members of the CREB/ CREM/ATF-1 family of transcription factors. This loss of CREB expression and the overexpression of CREM could be linked to cellular transformation, as the normal adrenal cortex express high levels of CREB and no or low levels of CREMtau.
垂体激素促肾上腺皮质激素（ACTH）通过环磷酸腺苷（cAMP）信号通路发挥作用，在肾上腺皮质的增殖和分化中起关键作用。cAMP反应元件（CRE）结合蛋白（CREB）是一种普遍存在的转录因子，它与众多基因启动子中的CRE结合，并介导cAMP对转录的刺激作用。在H295R细胞系中对CRE结合蛋白进行了表征，该细胞系被认为是人类肾上腺皮质肿瘤研究的模型。蛋白质免疫印迹法（Western blot）和逆转录聚合酶链反应（RT-PCR）研究表明，CREB在人肾上腺皮质癌细胞系H295R中不表达，而在正常肾上腺中表达。在瞬时转染实验中，对含有典型CRE的两个报告基因的cAMP刺激得以维持。共转染显性负性抑制剂A-CREB（可阻止含有CREB样亮氨酸拉链结构域的转录因子与DNA结合）可完全抑制cAMP诱导的CRE活性刺激。蛋白质免疫印迹法和RT-PCR研究表明，激活转录因子-1（ATF-1）、CRE调节因子-α/γ（CREMα/γ）和CREMτ2α在H295R细胞中表达。H295R细胞中存在大量的CREM蛋白，表明在缺乏CREB的情况下该转录因子过表达。此外，与正常肾上腺皮质相比，激活剂亚型CREMτ在H295R中的表达非常高。转染实验表明，CREMτ2α是H295R中CRE活性的有效刺激剂。最后，凝胶阻滞实验表明，CREM和ATF-1是在H295R细胞中特异性结合CRE的核蛋白，而在表达CREB的细胞系中未观察到CREM与CRE的结合。H295R细胞是首个建立的不表达普遍存在的转录因子CREB的非转基因细胞系。H295R表明，CREMτ的上调可以补偿CREB的缺陷，以维持cAMP对CRE的调节，并且是CREB/CREM/ATF-1转录因子家族成员之间补偿机制的模型。CREB表达的缺失和CREM的过表达可能与细胞转化有关，因为正常肾上腺皮质表达高水平的CREB，而CREMτ表达无或低水平。
