The α-Isoform of the CCAAT/Enhancer-binding Protein Is Required for Mediating cAMP Responsiveness of the Phosphoenolpyruvate Carboxykinase Promoter in Hepatoma Cells

Abstract

The gene coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) is expressed in all gluconeogenic tissues, but stimulation of its rate of transcription by cAMP is robust only in liver. Evidence has accumulated which suggests that a liver-enriched transcription factor, likely a member of the CCAAT/enhancer binding protein (C/EBP) family, is required along with other ubiquitously expressed transcription factors to mediate this liver-specific response to cAMP. In this study, we examined the ability of C/EBP to participate in the cAMP-mediated activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in hepatoma cells. Expression of a dominant repressor of C/EBP in hepatoma cells significantly inhibited the protein kinase A-stimulated transcription of the PEPCK promoter, suggesting that a C/EBP family member was required for maximal transcriptional activation by protein kinase A. To provide additional support for this hypothesis, we prepared GAL4 fusion proteins containing C/EBP domains. Both C/EBPalpha and C/EBPbeta GAL4 fusion proteins were capable of stimulating transcription from promoters containing binding sites for the DNA-binding domain of GAL4. However, only the GAL4-C/EBPalpha fusion protein demonstrated the ability to synergize with the other transcription factors bound to the PEPCK promoter which are required to mediate cAMP responsiveness. The DNA-binding domain of C/EBPalpha was not required for this activity in hepatoma cells, although in non-hepatoma cells the basic region leucine zipper domain appeared to inhibit the ability of C/EBPalpha to participate in mediating cAMP responsiveness. These results suggest that the liver-specific nature of the cAMP responsiveness of the PEPCK promoter involves the recruitment of C/EBPalpha to the cAMP response unit.

Role of CCAAT Enhancer-binding Protein β in the Thyroid Hormone and cAMP Induction of Phosphoenolpyruvate Carboxykinase Gene Transcription

“…These restriction site overhangs were filled in with Klenow enzyme and religated. Construction of the Gal4-C/EBP␣ vectors is given elsewhere (11,20). The sequence of all Gal4-C/EBP␤ vectors was confirmed by sequence analysis.…”

“…1A) (9,10). Three families of transcription factors that contribute to the cAMP stimulation include the cAMP-responsive element-binding protein (CREB), AP-1 proteins, and C/EBP (11). The P3(I) site is central to both the T 3 and cAMP induction of PEPCK transcription.…”

“…C/EBP␣ is expressed primarily in liver and adipose tissue and regulates the expression of hepatic genes involved in energy metabolism (16,17). In addition to participating in hormonal responsiveness, C/EBP proteins play an important role in PEPCK gene transcription by contributing to the induction of the PEPCK gene at birth and directing liver-specific expression (11,17,18). Our early studies showed that C/EBP␣ and ␤ could bind to the CRE, P3(I), and P4(I) elements in the PEPCK promoter (15,19).…”

“…Our early studies showed that C/EBP␣ and ␤ could bind to the CRE, P3(I), and P4(I) elements in the PEPCK promoter (15,19). C/EBP␣ can participate in the T 3 and cAMP induction of PEPCK transcription, and recent studies have shown that C/EBP␣ can mediate a cAMP induction without CREB being present (8,11,20).…”

Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene

“…Therefore, C\EBP proteins may contribute to the induction of L-CPT I expression in the liver at birth. C\EBPα has been shown to participate in the cAMP induction of the PEPCK gene [45]. However, L-CPT I is widely expressed and C\EBPα has been postulated to induce the expression of genes primarily in liver and adipose tissue.…”