Characterization of domains in C/EBPalpha that mediate its constitutive and cAMP-inducible activities.

Structure/function analysis of CCAAT/enhancer binding proteins (C/EBP) alpha and beta have shown that they possess both constitutive and cAMP inducible activities. Three regions conserved between C/EBPalpha and beta were identified which lie within the cAMP inducible domains of each protein. Deletion analysis of these conserved regions within C/EBPalpha show that conserved region 2 plays a particularly critical role in mediating the PKA inducible activity of the protein, however, the constitutive activity of conserved region 2 depends on promoter context. This data supports previous findings that constitutive and cAMP responsiveness are mediated by domains of the protein that do not directly overlap, suggesting that they occur through distinct mechanisms.

Characterization of CCAAT/enhancer-binding protein alpha as a cyclic AMP-responsive nuclear regulator.

The alpha isoform of CCAAT/enhancer-binding protein (C/EBPalpha) is a transcription factor that regulates expression of genes linked to adipose differentiation and hepatic nutrient metabolism. Recently, our laboratory has characterized a role for C/EBPalpha in mediating hormonal responsiveness. For example, the cAMP responsiveness of the phosphoenolpyruvate carboxykinase gene promoter in liver requires synergism among the cAMP response element-binding protein (CREB), C/EBPalpha, and activator protein-1. In the present study, we show that C/EBPalpha can functionally substitute for CREB in this cAMP response unit, i.e. cAMP responsiveness can occur in the absence of CREB. This observation is physiologically relevant since both CREB and C/EBPalpha have been shown to bind with high affinity to the cAMP response element in this particular promoter. Structure/function analysis of C/EBPalpha identified specific mutations that differentially affected its constitutive and protein kinase A-inducible activities. This finding suggests that the mechanism whereby C/EBPalpha mediates constitutive transactivation is distinct from that whereby it mediates cAMP responsiveness. These data support the hypothesis that C/EBPalpha plays a critical role in metabolism, in part by participating in the hormonal regulation of expression of metabolically important genes.

The alpha-isoform of the CCAAT/enhancer-binding protein is required for mediating cAMP responsiveness of the phosphoenolpyruvate carboxykinase promoter in hepatoma cells.

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.

The cAMP response element binding protein synergizes with other transcription factors to mediate cAMP responsiveness.

The cAMP responsiveness of the promoter for phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is mediated by a synergistic interaction between a complex regulatory region, which binds liver-enriched transcription factors, and a typical cAMP response element (CRE). Although a role for the CRE-binding protein (CREB) in the cAMP-responsiveness of this promoter has been generally assumed, some uncertainty remains due to the observations that several C/EBP-related proteins bind with near equal affinity, relative to CREB, to this particular CRE. Thus, a detailed analysis of the involvement of CREB in this synergism was undertaken in HepG2 cells. Gel mobility shift assays demonstrate that a CRE probe is bound by CREB present in HepG2 cells. Furthermore, we show that a dominant repressor of CREB is able to significantly reduce the cAMP responsiveness of the PEPCK promoter in HepG2 cells. Finally, we demonstrate using a GAL4-CREB fusion protein that CREB is able to synergize with the liver-enriched factors bound upstream on the PEPCK promoter to mediate a liver-specific response to cAMP. Examination of several mutant forms of CREB allow us to conclude that the "synergy" domain of CREB resides within amino acid residues 83-203, and that residues 83-145 can mediate a partial synergistic response. This study establishes that CREB is able to synergize with liver-enriched transcription factors to mediate a tissue-specific response to cAMP.

Characterization of the liver-specific component of the cAMP response unit in the phosphoenolpyruvate carboxykinase (GTP) gene promoter.

The cAMP response unit of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) gene promoter consists of two independently weak components; the typical cAMP response element and a region of the promoter that contains three binding sites for CCAAT/enhancer-binding proteins. Previous work from our laboratory indicated that all three binding sites were required for the full response to cAMP. However, in the present study, we demonstrate that the activity of this latter component cannot be mimicked by multiple copies of other well characterized CCAAT/enhancer-binding protein binding sites. Re-examination of the premoter region containing the three C/EBP binding sites revealed the presence of an additional cis-element, which is required to mediate the activation by cAMP. This DNA sequence binds a protein in HepG2 nuclear extracts that is distinct from CCAAT/enhancer-binding protein and cAMP response element-binding protein, and evidence is presented which suggests that its identity is activator protein-1. Thus, the robust response of the phosphoenolpyruvate carboxykinase gene promoter to cAMP in liver requires the involvement of three different transcription factors. Utilization of multiple transcription factors to mediate the activation by cAMP likely allows for a tissue-specific response to this signal, a mechanism whereby to fine-tune the extent of the response, and a mechanism whereby to integrate signals from separate signaling pathways into a coordinated response.

Evidence for the involvement of at least two distinct transcription factors, one of which is liver-enriched, for the activation of the phosphoenolpyruvate carboxykinase gene promoter by cAMP.

A detailed analysis of the promoter sequence requirements for the cAMP induction of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was undertaken. It was determined that the cAMP-responsive unit of this promoter consisted of two independently weak or inactive components; the typical cAMP response element (CRE) sequence located at position -85 and a region of the promoter extending from -300 to -230 which contained multiple binding sites for liver-enriched nuclear proteins. A previous study had indicated that multiple binding sites are critical for the activity of this latter region (Liu, J., Park, E. A., Gurney, A. L., Roesler, W. J., and Hanson, R. W. (1991) J. Biol. Chem. 266, 19095-19102). In the present study, we extend this observation by demonstrating that the activity of this region can be effectively substituted for by three copies of just one subsite present in that region. This would suggest that the binding of three molecules of a single liver-enriched factor is what forms this component of the cAMP response unit. The other component of the cAMP response unit, the CRE, has been shown previously to be bound by these same liver-enriched factors as well as by cAMP response element binding protein, leading to some debate as to the identity of the protein mediating the cAMP response through this element. By two different experimental approaches, we show that neither CCAAT/enhancer binding protein nor D-site binding protein is the likely mediator of the cAMP response through the CRE, while cAMP response element binding protein is a possible candidate.

The liver-enriched transcription factor D-site-binding protein activates the promoter of the phosphoenolpyruvate carboxykinase gene in hepatoma cells.

It has been previously demonstrated that the CCAAT/enhancer-binding protein (C/EBP) trans-activates the gene coding for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) and binds to several sites along the promoter. The additional observations that C/EBP is expressed in liver and follows the same developmental profile as PEPCK suggests that C/EBP plays an important role in the regulation of PEPCK gene expression. However, since C/EBP is expressed at high levels in lung, a tissue in which PEPCK is not expressed, it appears that other mechanisms are involved to provide PEPCK with high level expression in liver. We now show that the albumin promoter D-site-binding protein (DBP), a transcription factor whose expression is limited to the liver, is also able to trans-activate the PEPCK promoter through sequence-specific binding. Both recombinant DBP and C/EBP bind with highest affinity to regions located at positions -85 and -245 in the promoter, but display differences in their binding properties at other sites. Using eukaryotic expression vectors for both C/EBP and DBP, we found that with 5'-deletion mutants of the PEPCK promoter, both C/EBP and DBP exerted their effects through similar regions of the promoter. However, the use of internal deletion mutants of the promoter identified distinct differences in the mechanism of activation by C/EBP and DBP. In particular, a region of the promoter between positions -86 and -117 significantly attenuated the level of trans-activation by DBP, but not by C/EBP. Evidence presented also supports a model whereby the relative ratios of C/EBP and DBP in the cell fine-tune the expression of the PEPCK gene. These results demonstrate that DBP and C/EBP, while having similar DNA binding specificities, have distinct functional differences in the context of the PEPCK promoter. These differences, along with the developmental profiles of C/EBP and DBP, may provide a mechanistic explanation for the liver-specific as well as the developmental profile of PEPCK gene expression.