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 P3(I). Mutation of either the TRE or P3(I) 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/EBPα, C/EBPβ 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/EBPα 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/EBPα in the T3 effect.

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