Park E A, Song S, Olive M, Roesler W J
Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center, Memphis 38163, USA.
Biochem J. 1997 Feb 15;322 ( Pt 1)(Pt 1):343-9. doi: 10.1042/bj3220343.
Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by cAMP, the thyroid hormone tri-iodothyronine (T3) and retinoic acid (RA). Regulation of PEPCK transcription by T3 involves two sites in the promoter including a thyroid-hormone-response element (TRE) and a CCAAT-enhancer-binding protein (C/EBP) binding site called P3I. Mutation of either the TRE or P3I eliminates the T3 response. In this study, we examined the role of C/EBPs in the induction of PEPCK transcription by T3 and RA. PEPCK-CAT vectors were transfected into HepG2 cells. Co-transfection of a dominant negative C/EBP eliminated the T3 stimulation indicating that a member of the C/EBP family is required. To determine which C/EBP isoform was required, Gal4 fusion proteins were created that contained the Gal4 DNA-binding domain ligated to the transcriptional activation domain of C/EBP alpha, C/EBP beta or the cAMP-responsive-element-binding protein. A Gal4 DNA-binding site was introduced into the P3(I) site of the PEPCK-CAT vector. Only co-transfection of the Gal4-C/EBP alpha vector was able to restore T3 responsiveness to the PEPCK-CAT vector. The T3 and RA receptors are members of the nuclear receptor superfamily and bind to repeats of the AGGTCA motif. We found that the RA receptor can bind to sequences within the PEPCK-TRE and contribute to RA responsiveness of the PEPCK gene. However, the RA induction of PEPCK transcription was found to be independent of C/EBPs, further demonstrating the specificity of the involvement of C/EBP alpha in the T3 effect.
磷酸烯醇式丙酮酸羧激酶（PEPCK）基因的转录受环磷酸腺苷（cAMP）、甲状腺激素三碘甲状腺原氨酸（T3）和视黄酸（RA）的刺激。T3对PEPCK转录的调控涉及启动子中的两个位点，包括一个甲状腺激素反应元件（TRE）和一个称为P3I的CCAAT增强子结合蛋白（C/EBP）结合位点。TRE或P3I的突变都会消除T3反应。在本研究中，我们研究了C/EBP在T3和RA诱导PEPCK转录中的作用。将PEPCK-CAT载体转染到HepG2细胞中。共转染显性负性C/EBP消除了T3刺激，表明需要C/EBP家族的一个成员。为了确定需要哪种C/EBP异构体，构建了Gal4融合蛋白，其包含与C/EBPα、C/EBPβ或cAMP反应元件结合蛋白的转录激活域连接的Gal4 DNA结合域。将一个Gal4 DNA结合位点引入PEPCK-CAT载体的P3（I）位点。只有共转染Gal4-C/EBPα载体才能恢复PEPCK-CAT载体对T3的反应性。T3和RA受体是核受体超家族的成员，与AGGTCA基序的重复序列结合。我们发现RA受体可以与PEPCK-TRE内的序列结合，并有助于PEPCK基因对RA的反应性。然而，发现RA对PEPCK转录的诱导独立于C/EBP，进一步证明了C/EBPα参与T3效应的特异性。
