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Viszeral- und Transplantationschirurgie, Medizinische Hochschule, Hannover, Germany (H.S., J.K.); and Center of Drug Research and Medical Biotechnology, Fraunhofer Institut für Toxikologie und Experimentelle Medizin, Hannover, Germany (J.B.)
Abstract
Abstract I. Introduction II. The CAAT/Enhancer-Binding Proteins A. The C/EBP Subfamily of the Basic Region Leucine Zipper Family of Transcription Factors 1. C/EBP Homo- and Heterodimers. 2. C/EBPs and Cross-Talk with Other Transcription Factors. 3. Sumoylation May Modulate the Transactivation Activity of C/EBPs. B. C/EBP-{alpha} (Originally Named C/EBP) 1. The Single-Exon C/EBP-{alpha} Gene Is Highly Conserved in Different Species. 2. Tissue-Specific and Species-Specific Autoregulation of the C/EBP-{alpha} Promoter. 3. Thyroid Hormone Positively Regulates C/EBP-{alpha} Expression. 4. Possible Regulatory Function of Alternative C/EBP-{alpha} Translation Products. 5. The Transactivation Domains of C/EBP-{alpha}. 6. RFC140 Acts as a Coactivator for C/EBP-{alpha}. 7. CAAT Displacement Protein as a Competitive Repressor for C/EBP-{alpha}-Mediated Transactivation. 8. Translational Inhibition of C/EBP-{alpha} Expression by Calreticulin. C. C/EBP-{beta} (Formerly Also Named Liver-Activating Protein, Interleukin 6DBP, CRP2, or Nuclear Factor Interleukin 6) 1. C/EBP-{beta} Isoforms Are the Result of Alternative Translation Initiation. 2. Liver-Specific Proteosomal Regulation of C/EBP-{beta} Isoforms by C/EBP-{alpha}. 3. Translational Regulation of C/EBP-{beta} Isoforms by CUGBP1. 4. Translational Inhibition of C/EBP-{beta} Expression by Calreticulin. 5. Regulation of the C/EBP-{beta} Promoter. 6. Tissue-Specific Autoregulation of C/EBP-{beta} Transcription. 7. Negative Regulatory Domains of C/EBP-{beta}. 8. Post-translational Phosphorylation of C/EBP-{beta}. 9. Phosphorylation of C/EBP-{beta} Ser239 leads to C/EBP-{beta} Nuclear Export. 10. Phosphorylation of C/EBP-{beta} Thr217 by p90 Ribosomal S Kinase. D. C/EBP-{gamma} (Also Called Ig/EBP) 1. Structure and Chromosomal Location of C/EBP- {gamma}. E. C/EBP-{delta} 1. Chromosomal Localization. 2. Transcriptional Induction of C/EBP-{delta} by Interleukin 6. 3. Regulation of C/EBP-{delta} by Phosphorylation. 4. Autoregulation of C/EBP-{delta}. 5. Inhibition of C/EBP-{delta} Gene Expression by C/EBP-{beta} and C/EBP-{zeta}. F. C/EBP-{zeta} (Also Called GADD153 or CHOP-10) 1. C/EBP-{zeta} as a Dominant Negative Regulator of C/EBPs. 2. Chromosomal Localization and Characteristics of the Promoter of C/EBP-{zeta}. 3. Regulation of C/EBP-{zeta} in Response to Oxidative Stress and DNA Damage. 4. Phosphorylation of C/EBP-{zeta}. 5. Autoregulation of GADD153 Expression. 6. Differential Regulation of C/EBP-{zeta} 7. C/EBP-{zeta}-C/EBP-{beta} Heterodimer-Mediated Gene Regulation. G. Hormones, Metabolic Hepatic Functions, and the C/EBPs 1. C/EBP-{alpha} and Energy Metabolism. 2. C/EBP-{alpha} in Gluconeogenesis and Detoxification of Ammonia and Bilirubin. 3. C/EBP-{beta} and Energy Metabolism. 4. Insulin and Glucocorticoids Regulate Gluconeogenesis via C/EBP-{beta} Isoforms. 5. Thyroid Hormone and Retinoic Acid Regulate C/EBP-{alpha} and -{beta} in the Liver. 6. The C/EBPs and the Growth Hormone-Regulated Network of Transcription Factors. 7. C/EBPs and the Control of Cytochrome P450 Gene Expression. H. The Role of C/EBPs in the Acute Phase Response 1. Differential Expression Patterns of the C/EBPs during the Acute Phase Response. 2. Differential Expression of C/EBP-{alpha} and C/EBP-{beta} Isoforms. 3. Acute Phase Response-Related mRNA/Protein Interaction leads to Liver-Enriched Transcriptional Inhibitory Protein Translation. 4. Increased Liver-Enriched Transcriptional Inhibitory Protein Translation Leads to Reduced C/EBP-{alpha} RNA Levels. 5. Tumor Necrosis Factor {alpha}-Mediated Post-translational Regulation of C/EBPs. 6. Interleukin 6-Mediated Post-translational Phosphorylation of C/EBP-{beta}. 7. Influence of Interleukin 6 on C/EBP-{beta} Transcription. 8. Transcriptional Induction of C/EBP-{delta} after Interleukin 6 Stimulus. 9. A Pivotal Role for C/EBP-{alpha} in the Acute Phase Response. 10. Interactions of C/EBP-{alpha} and C/EBP-{beta} with the Nuclear Matrix. 11. Protein/Protein Interaction between Nuclear Factor {kappa}B p65 and C/EBP-{beta}. 12. Nucleolin Is an Antagonist to C/EBP-{beta} in the Acute Phase Response. 13. Nuclear Factor {kappa}B and Nopp140 Act as Coactivators for C/EBP-{beta}. 14. Heterogeneous Nuclear Ribonucleoprotein K as a Negative Regulator of C/EBP-{beta}-Mediated Gene Activation. 15. The Role of the Hypothalamic-Pituitary-Adrenal Axis in the Acute Phase Response. I. C/EBPs and Cell Cycle Control 1. C/EBP-{alpha} Expression and Growth Arrest. 2. The Glucocorticoid-Induced G1 Cell Cycle Arrest Is Mediated by C/EBP-{alpha}. 3. Protein/Protein Interactions among p21, cdk2, cdk4, and C/EBP-{alpha}. 4. C/EBP-{alpha} and p107 Protein/Protein Interaction Disrupts E2F Complexes. 5. C/EBP-{beta} Arrests the Cell Cycle before the G1/S Boundary. 6. Transforming Growth Factor {alpha}-Mediated Phosphorylation of C/EBP-{beta} Leads to Hepatocyte Proliferation. J. C/EBPS and Cellular Differentiation 1. Transcription Factors during Liver Development and Oval Cell Differentiation. 2. Transdifferentiation of Pancreas to Liver and C/EBP-{beta} Induction. K. C/EBPs and Apoptosis 1. Tumor Necrosis Factor {alpha}-Mediated Apoptosis and C/EBP-{beta}. 2. Fas-Induced Apoptosis and C/EBP-{beta}. L. The Role of C/EBPs in Development 1. Differential Regulation of C/EBPs and Their Isoforms during Development. 2. C/EBP-{alpha} and the Developmental Expression of Essential Metabolic Genes. 3. Differential CYP3A7 and CYP3A4 Expression during Development. 4. Hepatic Expression of the Rat CYP2D5 Gene Is Regulated by C/EBP-{beta}. M. The Role of C/EBPs in Liver Regeneration 1. Liver Regeneration after Concanavalin A-Induced Liver Injury. 2. Hepatocyte Growth Factor Stimulates C/EBP-{beta} and Nuclear Factor {kappa}B Expression. 4. Transcriptional Regulation of C/EBP-{alpha} in Liver Regeneration. 5. C/EBP-{beta} Isoforms in Liver Regeneration. 6. C/EBP-{alpha} and C/EBP-{beta} Expression after Partial Hepatectomy. 7. Influence of Age on Liver Regeneration. 8. Influence of Obstructive Jaundice on Liver Regeneration. 9. Cyclooxygenase 2 Contributes to Liver Regeneration. N. C/EBPs and their Role in Liver Tumor Biology 1. The Ratio of C/EBP-{alpha} and C/EBP-{beta} Expression in Chemical Carcinogenesis. 2. Protein/Protein Interaction between Mutant p53 and C/EBP-{beta} in Liver Cancer. 3. Regulatory Role of Liver-Enriched Transcription Factors in Liver Cancer. 4. Repression of C/EBP-{alpha}-Mediated Transactivation by CAAT Displacement Protein in Human Liver Cancer? O. Human Disease with Causative C/EBP Involvement 1. Essential Hypertension in African-Americans. III The D Site-Binding Protein A. Chromosomal Localization and Interspecies Conservation B. Genomic Structure of D Site-Binding Protein C. The Transactivation Domain of D Site-Binding Protein D. Cotranscriptional and Post-transcriptional Splicing of D Site-Binding Protein E. The Proline and Acidic-Rich Domain and the p300 Coactivator Are Involved in Transactivation by D Site-Binding Protein F. D Site-Binding Protein Forms Homo- and Heterodimers with Proline and Acidic-Rich Protein Family Members G. The Rhythm of D Site-Binding Protein Expression Beats the Drum for Circadian Gene Regulation H. Regulation of the D Site-Binding Protein Promoter by CLOCK I. Changes of Feeding Times Influence Circadian D Site-Binding Protein Expression Patterns J. Influence of Glucocorticoids on Hepatic Circadian Gene Expression K. Rat CYP2C6 Expression in Development Is Positively Regulated by D Site-Binding Protein L. Circadian Expression of Rat CYP7 Is Positively Regulated by D Site-Binding Protein IV. Toxicogenomics as an Emerging Science in Toxicology V. Conclusion
In the first part of our review (see Pharmacol Rev 2002;54:129-158), we discussed the basic principles of gene transcription and the complex interactions within the network of hepatocyte nuclear factors, coactivators, ligands, and corepressors in targeted liver-specific gene expression. Now we summarize the role of basic region/leucine zipper protein family members and particularly the albumin D site-binding protein (DBP) and the CAAT/enhancer-binding proteins (C/EBPs) for their importance in liver-specific gene expression and their role in liver function and development. Specifically, regulatory networks and molecular interactions were examined in detail, and the experimental findings summarized in this review point to pivotal roles of DBP and C/EBPs in cell cycle control, carcinogenesis, circadian gene regulation, liver regeneration, apoptosis, and liver-specific gene regulation. These regulatory proteins are therefore of great importance in liver physiology, liver disease, and liver development. Furthermore, interpretation of the vast data generated by novel genomic platform technologies requires a thorough understanding of regulatory networks and particularly the hierarchies that govern transcription and translation of proteins as well as intracellular protein modifications. Thus, this review aims to stimulate discussions on directions of future research and particularly the identification of molecular targets for pharmacological intervention of liver disease.
New genomic platform technologies enable the study of complex genomes and proteomes for improved target identification and validation. This leads to high-density data sets on the order of millions of data points per day. Turning data into knowledge will be one of the biggest challenge of the 21st century. Here we describe concisely the role of liver-enriched transcription factors in regulatory gene networks and focus on liver development function and disease. This knowledge will prove to be indispensable for interpretation of high-density genomic data sets that are being produced in pharmaco- and toxicogenomics.
Indeed, numerous studies have established the pivotal role of liver-enriched transcription factors in organ development and cellular function, and there is conclusive evidence that transcription factors act in concert in liver-specific gene expression. During organ development and in progenitor cells, the timely expression of certain transcription factors is necessary for cellular differentiation, and there is overwhelming evidence for hierarchical and cooperative principles in a networked environment of transcription factors. The search for molecular switches that control stem cell imprinting and liver-specific functions has lead to the discovery of many interactions among such different molecules as transcription factors, coactivators, corepressors, enzymes, DNA, and RNA. Many of these interactions either repress or activate liver-specific gene expression. In our first review we provided an overview of the basic principles of gene transcription and focused on the role of hepatic nuclear transcription factors in liver gene regulation (Schrem et al., 2002
). Here we summarize the role of basic region leucine zipper (bZIP1) protein family members and focus on the albumin D site-binding protein (DBP) and the CAAT/enhancer-binding proteins (C/EBPs). Furthermore, we summarize our current knowledge on the roles of the transcription factors in important events like cell cycle control, carcinogenesis, liver regeneration, apoptosis, circadian gene regulation, and liver-specific gene regulation. As shown in Fig. 1, C/EBPs play a major role in diverse physiological processes.
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Notably, several C/EBPs were discovered and characterized independently in different laboratories and given distinct names (Landschulz et al., 1988; Akira et al., 1990; Chang et al., 1990; Descombes et al., 1990; Poli et al., 1990, Roman et al., 1990; Ron and Habener, 1992; Takiguchi, 1998; Niu et al., 1999; Ramji and Foka, 2002). We followed the proposal by Cao et al. (1991), who suggested a systematic nomenclature in which members of the C/EBP subfamily are designated as C/EBP followed by a Greek letter indicating the chronological order of their discovery.
II. The CAAT/Enhancer-Binding Proteins
A. The C/EBP Subfamily of the Basic Region Leucine Zipper Family of Transcription Factors
A heat-stable DNA-binding protein found in rat liver nuclei was shown to be capable to bind selectively to the CCAAT motif of several viral promoters, as well as to the "core homology" sequence of several viral enhancers. Hitherto, this protein was termed CCAAT/enhancer-binding protein, or C/EBP (Landschulz et al., 1988). Later, it was recognized that several related proteins form a distinct subfamily of transcription factors called C/EBPs with several members (C/EBP-
, C/EBP-
, C/EBP-
, C/EBP-
, C/EBP-
, and C/EBP-
). The C/EBP subfamily of transcription factors belongs to the larger family of bZIP transcription factors. Furthermore, important members of the bZIP family of transcription factors include c-jun, c-fos (AP-1), and the cAMP responsive element binding protein (CREB). The bZIP family of proteins is one of the largest and most conserved groups of eukaryotic transcription factors (Takiguchi, 1998; Niu et al., 1999).
The DNA-binding motif of transcription factors belonging to the bZIP family is bipartite, consisting of a dimerization interface termed "leucine zipper" and a DNA contact surface termed the "basic region." A common feature of the C/EBP subfamily members is that they consist of three structural components in a modular fashion that include a C-terminal leucine-zipper, a basic DNA-binding region, and an N-terminal transactivating region (Agre et al., 1989; Metallo and Schepartz, 1994; Niu et al., 1999; Ramji and Foka, 2002) (see also Fig. 2). The molecular chaperone bZIP-enhancing factor (BEF) was shown to increase DNA binding of transcription factors that contain a bZIP DNA-binding domain. BEF stimulates DNA binding by recognizing the unfolded leucine zipper and promoting the folding of bZIP monomers to dimers. Anti-sense experiments indicate that BEF is required for efficient transcriptional activation by bZIP proteins in vivo (Virbasius et al., 1999).
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1. C/EBP Homo- and Heterodimers.
Many transcription factors bind DNA to form dimeric (2:1) protein-DNA complexes. Examples include bZIP proteins and basic region helix-loop-helix zipper proteins. These two families of transcription factors follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA (Kohler et al., 1999). Dimerization of these transcription factors stabilizes the protein-DNA complexes and can lead either to homodimers with the same transcription factor or to heterodimers with other members of the same family of transcription factors (Horiuchi et al., 1997).
Noticeably, heterodimers between members of the C/EBP family and the CREB/ATF family of transcription factors (e.g., ATF-2, ATF-3, ATF-4, and C/ATF) were identified by several investigators (Vallejo et al., 1993; Shuman et al., 1997; Wolfgang et al., 1997; Fawcett et al., 1999). Both families belong to the larger group of bZIP proteins. The formation of C/EBP-ATF heterodimers causes altered DNA binding selectivity when compared with C/EBP and ATF homodimers. C/EBPATF heterodimers bind to a so-called C/EBP-ATF composite site in the promoter region of regulated genes. In particular, C/EBP and ATF-2 homodimers bind cAMP response elements (CRE sites), but ATF-2-C/EBP- heterodimers do not. Heterodimers bind an asymmetric sequence composed of one consensus half-site for each monomer and thus have a unique regulatory function (Shuman et al., 1997). C/ATF homodimers do not bind to typical C/EBP DNA sites. Instead, they bind to palindromic CRE sites such as that of the somatostatin gene. C/ATF-C/EBP- heterodimers bind to a subclass of asymmetric cAMP response elements exemplified by those in the phosphoenolpyruvate carboxykinase and proenkephalin genes (Vallejo et al., 1993).
2. C/EBPs and Cross-Talk with Other Transcription Factors.
Transient transfection studies in HepG2 human hepatoma cells and in COS-1 monkey kidney cells indicated that interactions between C/ATF and C/EBP- are the basis for a functional cross-talk between these two families of transcription factors that may be important for the integration of hormonal and developmental stimuli that determine the expression of subsets of genes in specific cellular phenotypes (Vallejo et al., 1993). By forming a complex with C/EBP-, C/ATF directs binding of C/EBP- away from C/EBP sites onto palindromic CREs. The relative rates of production and degradation of C/EBP- and C/ATF might directly influence the regulation of many dependent genes. When the cellular concentrations of C/EBP- are higher than the concentrations of C/ATF, this might favor C/EBP- homodimer formation and, thus as a consequence, for example, angiotensinogen expression, whereas higher cellular C/ATF concentrations could favor C/ATF homodimer formation and, as a consequence, gene expression from genes bearing palindromic CREs, like that encoding somatostatin. Equal amounts of C/ATF and C/EBP- might favor C/ATF-C/EBP- heterodimer formation with the consequence of enhanced somatostatin, enkephalin, or phosphoenolpyruvate carboxykinase (PEPCK) expression (Vallejo et al., 1993). The formation of C/ATF-C/EBP- heterodimers might also be influenced by post-translational modification of C/EBP- by phosphorylation.
NF-
B proteins and C/EBPs belong to distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-B complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-B family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous approximately 300-amino acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. A previously unexpected cross-coupling of members of the NF-B family with three members of the C/EBP family could be demonstrated in 1993. NF-B p65, p50, and Rel functionally synergize with C/EBP-, C/EBP-, and C/EBP-, respectively, via protein/protein interactions. Interestingly, this cross-coupling results in the inhibition of promoters with B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites (Stein and Baldwin, 1993; Stein et al., 1993). Protein/protein interactions of the bZIP region of C/EBP with the Rel homology domain of NF-B could be demonstrated for the regulation of interleukin 8 (IL-8) gene expression (Stein and Baldwin, 1993) as well as for the regulation of the major acute phase reactant serum amyloid A2 (SAA2) gene expression in the liver (Xia et al., 1997) and in a protein complex in avian T cells that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members (Diehl and Hannink, 1994).
3. Sumoylation May Modulate the Transactivation Activity of C/EBPs.
A novel host cell post-translational modification system, termed sumoylation, has recently been characterized. Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO regulates various cellular processes, such as nuclear transport, signal transduction, stress response, and cell cycle progression. But, in contrast to ubiquitylation, sumoylation does not tag proteins for degradation but seems to enhance their stability or modulate their subcellular compartmentalization (for recent reviews, see Muller et al., 2001; Wilson and Rangasamy, 2001). SUMO is highly conserved from yeast to humans. Whereas invertebrates have only a single SUMO gene, three members of the SUMO family have been described in vertebrates. Like ubiquitin, all SUMO forms are initially made as inactive precursors. The processing reaction is catalyzed by a group of cysteine proteases, termed ubiquitin-like protein-processing enzymes or SUMO-specific proteases. In contrast to ubiquitin, SUMO conjugation does not seem to lead to the formation of SUMO-SUMO chains on the substrate. Sumoylation is a dynamic, reversible process. Desumoylation is catalyzed by the ubiquitin-like protein-processing enzyme/SUMO-specific proteases (Muller et al., 2001; Wilson and Rangasamy, 2001).
In C/EBP-, an inhibitory domain termed regulatory domain I was characterized. It could be shown that functionally related domains are present in the liver-enriched transcription factors C/EBP-, C/EBP-, and C/EBP-. These domains contain an evolutionarily conserved five-amino acid motif [the regulatory domain motif (RDM)] that conforms to the consensus sequence [IVL]-K-x-E-P. Mutagenesis studies revealed that the residues at positions 1, 2, and 4 of the RDM are critical for inhibitory domain function. Importantly, the RDM is similar to the recognition sequence for attachment of the ubiquitin-like protein SUMO-1, and it could be shown that the conserved lysine residue of each C/EBP RDM served as an attachment site for SUMO-1. SUMO-1 attachment decreased the inhibitory effect of the C/EBP- regulatory domain, suggesting that sumoylation may play an important role in modulating C/EBP- activity, as well as that of the other C/EBP family members (Kim et al., 2002).
B. C/EBP- (Originally Named C/EBP)
1. The Single-Exon C/EBP- Gene Is Highly Conserved in Different Species.
The C/EBP- gene was found to be syntenic on human chromosome 19, on rat chromosome 1, and on mouse chromosome 7. These results provide further evidence for conservation of synteny (= genome collinearity) on these three chromosomes (Birkenmeier et al., 1989; Szpirer et al., 1992). There is a strong conservation of C/EBP- and its role in gene expression as demonstrated, for instance, by the regulation of the alcohol dehydrogenase gene in the adult body of Drosophila melanogaster and in the human and rat liver (Falb and Maniatis, 1992; Potter et al., 1994). Further, the human C/EBP- gene is 2783 bp long and encodes a 356-amino acid protein, which is of the same length as the gene for rat C/EBP-. Compared with rat C/EBP-, there are two insertions of two amino acids and one deletion of four. The amino acid similarity between the two proteins is over 92% (Antonson and Xanthopoulos, 1995). The C/EBP- mRNAs of chicken, rat, and Xenopus all contain a small 5' open reading frame (5'ORF), whose size (18 nucleotides) and distance (seven nucleotides) to the C/EBP- cistron have been conserved in vertebrate evolution (Calkhoven et al., 1994).
2. Tissue-Specific and Species-Specific Autoregulation of the C/EBP- Promoter.
The human C/EBP- gene was found to be expressed at highest levels in placenta. High expression was also found in liver, lung, skeletal muscle, pancreas, small intestine, colon, and in peripheral blood leukocytes, but the expression was undetectable or very low in brain, kidney, thymus, testis, and ovary (Antonson and Xanthopoulos, 1995). In vitro transcription experiments showed that the basis for the restricted cellular distribution of the mouse C/EBP- mRNA is to be found in the transcriptional regulation of the gene (Xanthopoulos et al., 1989).
The sequence of the C/EBP- promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc+Max heterodimers, and mutation of this site drastically reduces transcription of C/EBP- promoter luciferase constructs both in liver and nonliver cell lines (Legraverend et al., 1993). In addition, three additional protein-binding sites were identified, two of which display similarity to NF-1 and NF-B binding sites. The region located between nucleotides -197 and -178 forms several heat-stable complexes with liver nuclear proteins in vitro, which are recognized mainly by antibodies specific for C/EBP-. Furthermore, transient expression of C/EBP- and, to a lesser extent, C/EBP- expression vectors result in transactivation of a cotransfected C/EBP- promoter-luciferase reporter construct. These experiments support the notion that the gene coding for C/EBP- is regulated by C/EBP-, but other C/EBP-related proteins may also be involved in its regulation (Legraverend et al., 1993).
The human C/EBP- gene promoter shares significant sequence homology with that of the mouse but interestingly has a different mechanism of autoregulation. Activation of the murine promoter by direct binding of C/EBP- to a site within 200 bp of the transcriptional start was shown to elevate activity by approximately 3-fold (Legraverend et al., 1993; Timchenko et al., 1995). Unlike its murine counterpart, the human C/EBP- gene promoter does not contain a cis element that binds the C/EBP- protein. Neither C/EBP- nor C/EBP- binds the human C/EBP- promoter within 437 bp. However, cotransfection studies in human hepatoma-derived Hep3B2 cells show that C/EBP- stimulates transcription of a reporter gene driven by 437 bp of the C/EBP- promoter. The human C/EBP- protein stimulates upstream stimulating factor (USF) to bind to a USF consensus element within the C/EBP- promoter and activates it by 2- to 3-fold (Timchenko et al., 1995). Therefore, it was proposed that the human C/EBP- gene uses the ubiquitously expressed DNA-binding protein factor USF to carry out autoregulation. Autoregulation of the human C/EBP- promoter could be experimentally abolished by deletion of the USF binding site, CACGTG. Expression of human C/EBP- after transfection did not stimulate USF binding. These studies suggest a mechanism whereby tissue-specific autoregulation can be achieved via a transacting factor that is expressed in all cell types. Thus, direct binding of the C/EBP- protein to the promoter of the C/EBP- gene is not required for autoregulation (Timchenko et al., 1995). Studies with the Xenopus laevis C/EBP- promoter showed that, in contrast to the human promoter and in common with the murine gene, the xenopus C/EBP- promoter was subject to direct autoregulation (Kockar et al., 2001).
3. Thyroid Hormone Positively Regulates C/EBP- Expression.
A genomic clone containing 1171 bp of the 5'-flanking region of the rat C/EBP- gene was shown to be an active promoter in MB492 cells, which is an immortalized brown adipocyte cell line that expresses the endogenous C/EBP- gene in a thyroid hormone (T3)-dependent manner. This genomic clone and the MB492 cell line were used for deletion, mutagenesis, and gel mobility shift experiments to further characterize the rat C/EBP- promoter. Results from these experiments showed that the TRE-1 element (-602/-589) of the rat C/EBP- promoter possesses a ER2-type response element that represents a functional T3 response element regulated by thyroid hormone and that thyroid hormone is a factor that positively regulates C/EBP- gene expression in a direct fashion (Menendez-Hurtado et al., 2000). Furthermore, several peptide regions within the transactivation domain of C/EBP- could be identified that enhance the ability of thyroid hormone to stimulate gene transcription (Jurado et al., 2002). Therefore, it can be assumed that C/EBP- expression is enhanced by thyroid hormone 2-fold: directly via a T3 response element in the C/EBP- promoter and indirectly by interaction with the transactivation domain of C/EBP- leading to an enhanced C/EBP- autoregulatory loop.
4. Possible Regulatory Function of Alternative C/EBP- Translation Products.
C/EBP- mRNA is translated into two major proteins, p42-C/EBP- and p30-C/EBP-, that differ in their content of N-terminal amino acid sequences (Lin et al., 1993; Ossipow et al., 1993). p30-C/EBP is an alternative translation product initiated at the third in-frame AUG (methionine) codon of the C/EBP- message (Lin et al., 1993; Ossipow et al., 1993). It was demonstrated that the small 5'ORF is crucial to the leaky scanning mechanism of ribosomes causing a fraction of them to ignore the first C/EBP- AUG codon and to start at internal AUGs (Calkhoven et al., 1994). The full-length p42-C/EBP- acts as a transactivator in the liver, whereas the N-terminally truncated p30-C/EBP- lacks transcription activation potential (Ossipow et al., 1993; Calkhoven et al., 1994). Unlike p42-C/EBP-, which inhibits cell proliferation, p30-C/EBP- is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP- contains an amino acid sequence necessary for antimitotic activity. Furthermore, during differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42-C/EBP-/p30-C/EBP- ratio changes, raising the possibility of a regulatory role (Lin et al., 1993). The impact of C/EBP- on cell cycle control as well as its role in development are discussed in the following paragraphs in greater detail.
5. The Transactivation Domains of C/EBP-.
The N-terminal portion of C/EBP- contains three distinct domains. The first domain (amino acids 1 to 107 = TE I) appears to be a highly potent transactivator. The second domain (amino acids 107 to 170 = TE II) does not appear to exhibit either activation or repression activity. The third domain (amino acids 171 to 245 = TE III) is a relatively weaker transactivator with a striking proline-rich motif (Pei and Shih, 1991). The C/EBP- transactivation domain thus contains three transactivation elements (TEs) that synergistically activate transcription in mammalian cells. Two of these elements, TE-I and -II, cooperatively mediate in vitro binding of C/EBP- to TBP and TFIIB, two essential components of the RNA polymerase II basal transcriptional apparatus. The TBP and TFIIB binding elements of C/EBP- coincide and require amino acid motifs conserved between the activating members of the C/EBP family. These same motifs are necessary for the transcription activation function of TE-I and -II in both yeast and mammalian cells. This indicates that this modularity is conserved in eukaryote evolution. It was suggested that domains of TBP and TFIIB that interact with activating surfaces are functionally similar and may be structurally related. This supports the idea that the same amino acid motifs in an activator carry out multiple functions during the initiation process (Nerlov and Ziff, 1995).
6. RFC140 Acts as a Coactivator for C/EBP-.
Western blotting experiments showed that the bZIP domain of C/EBP- interacts with the DNA-binding region of RFC140, a large subunit of the replication factor C complex from rat liver nuclear extracts. Overexpression of RFC140 in mammalian cells increased the transactivation activity of C/EBP- on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBP- and RFC140 proteins with the cognate DNA element was detected in vitro. The specific interaction between C/EBP- and RFC140 was detected in the terminal differentiation of 3T3-L1 preadipocytes to adipocytes. The synergistic transcription effect of these two proteins increased the promoter activity and protein expression of peroxisome proliferator-activated receptor-, which is a main regulator of adipocyte differentiation. These results demonstrate that the specific transcription factor C/EBP- and the general DNA replication factor RFC140 interact functionally and physically (Hong et al., 2001). Noticeably, the levels of the general replication factor obviously modulate the functional activity of the specific transcription factor C/EBP- as a coactivator.
7. CAAT Displacement Protein as a Competitive Repressor for C/EBP--Mediated Transactivation.
The nuclear matrix protein CAAT displacement protein (CDP) was discovered as a competitive repressor for CCAAT binding factors in experiments on gene regulation of the sperm histone H2B-1 of the sea urchin Psammechinus miliaris by Barberis et al. (1987). In blastula and gastrula embryo extracts, CDP binds with high affinity to sequences overlapping the proximal CCAAT element of the sperm histone H2B-1 promoter, thus preventing the DNA interaction of the CCAAT-binding factor in the embryo where the sperm H2B gene is not expressed (Barberis et al., 1987).
Investigations on liver-specific gene regulation of the human cholesterol 7-hydroxylase CYP7A1 gene revealed a transcriptional repressor within the first intron of CYP7A1in transient transfection experiments with HepG2 cells. In the first intron, three DNase I hypersensitivity sites were found with five binding sites for the nuclear matrix protein CDP (Antes et al., 2000). Further, a matrix attachment site was found throughout the entirety of intron 1 of the CYP7A1 gene. Gel retardation experiments and cell transfection studies provided evidence for the repression mechanism by CDP. It was shown that repression of the human CYP7A1 gene is mediated by the matrix attachment site-bound repressor CDP, involves displacement of two hepatic transcriptional activators, HNF-1 and C/EBP-, from their binding sites within intron 1 of the CYP7A1 gene, and thus represses transactivation mediated by these two activators (Antes et al., 2000). The observation that CDP is abundant in undifferentiated cells and is down-regulated in differentiated epithelial cells (Ai et al., 1999) might indicate that the availability of CDP for its sites within intron 1 of the CYP7A1 gene is high early in liver development and thus binding of the liver-specific transcription factors may be blocked (Antes et al., 2000). Later in development, when hepatocytes are further differentiated and when CDP expression may be down-regulated, binding by HNF-1 and C/EBP- may occur at a time when CYP7A1 functions are needed metabolically.
8. Translational Inhibition of C/EBP- Expression by Calreticulin.
The process of "quality control" in the endoplasmic reticulum involves a variety of mechanisms that collectively ensure that only correctly folded, assembled, and modified proteins are transported along the secretory pathway. In contrast, non-native proteins are retained and eventually targeted for degradation. Recent work provides structural insights into the process of glycoprotein folding in the endoplasmic reticulum involving the lectin chaperone calreticulin (for review, see Cabral et al., 2001; Ellgaard and Helenius, 2001). Calreticulin is a calcium-binding protein of the endoplasmic reticulum that has been found to function as a nuclear export factor for a large family of nuclear receptors. Atypical nuclear export pathways may thus exist that regulate the compartmentalization and activity of a distinct set of transcription factors (DeFranco, 2001).
It could be demonstrated that calreticulin interacts with GCN repeats within C/EBP- and C/EBP- mRNAs. GCN repeats within these mRNAs form stable SL structures. The interaction of calreticulin with SL structures of C/EBP- and C/EBP- mRNAs leads to the inhibition of translation of C/EBP proteins in vitro and in vivo. Deletions or mutations abolishing the formation of SL structures within C/EBP- and C/EBP- mRNAs lead to a failure of calreticulin to inhibit translation of C/EBP proteins. Calreticulin-dependent inhibition of C/EBP- is sufficient to block the growth-inhibitory activity of C/EBP-. This finding further defines the molecular mechanism for post-transcriptional regulation of the C/EBP- and C/EBP- proteins (Timchenko et al., 2002).
C. C/EBP- (Formerly Also Named Liver-Activating Protein, Interleukin 6DBP, CRP2, or Nuclear Factor Interleukin 6)
C/EBP- is a 32-kDa protein that shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP-. C/EBP- and C/EBP- can interact in vitro to form heterodimers that bind to DNA with the same specificity as the respective homodimers (Descombes et al., 1990; Poli et al., 1990).
1. C/EBP- Isoforms Are the Result of Alternative Translation Initiation.
Both C/EBP- and C/EBP- are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons (Ossipow et al., 1993; Welm et al., 1999). C/EBP- has been reported to produce four isoforms: full-length 38-kDa C/EBP-, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform (Ossipow et al., 1993; Welm et al., 1999). The production of multiple proteins from a single strand of mRNA is not only shared between different C/EBP family members but also appears to be a conserved mechanism in vertebrate evolution (Ossipow et al., 1993; Calkhoven et al., 1994; Welm et al., 1999).
The C/EBP- mRNA has four in-frame AUGs and an internal out-of-frame AUG associated with a small ORF. Initiation of translation at the in-frame AUGs forms 40-kDa (AUG-1), 35-kDa (AUG-2), 20-kDa (AUG-3), and 8.5-kDa (AUG-4) isoforms (Xiong et al., 2001).
It is important to understand the regulation and the production of various C/EBP- isoforms as they display different transcriptional capabilities. For instance, the truncated C/EBP- isoform, LIP, is up-regulated in rat livers after partial hepatectomy via an alternative translation mechanism (Burgess-Beusse et al., 1999; Timchenko et al., 1999a; Welm et al., 1999). A number of mechanisms have been elucidated that lead to differential C/EBP- isoform production, as detailed below.
2. Liver-Specific Proteosomal Regulation of C/EBP- Isoforms by C/EBP-.
Using an in vitro translation system, it was found that LIP was produced by two mechanisms: alternative translation and a proteolytic cleavage of full-length C/EBP-. In C/EBP- knockout mice (C/EBP--/-), the regulation of C/EBP- proteosomal degradation was impaired (Burgess-Beusse et al., 1999; Welm et al., 1999) (see also Fig. 5). Induction of C/EBP- in cultured cells leads to induced cleavage of C/EBP- to generate the LIP isoform. The cleavage activity in mouse liver extracts is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBP--/- animals. The lack of cleavage activity in the livers of C/EBP--/- mice correlates with the decreased levels of LIP in the livers of these animals (Burgess-Beusse et al., 1999; Welm et al., 1999).
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3. Translational Regulation of C/EBP- Isoforms by CUGBP1.
CUG repeat binding protein, CUGBP1, interacts with the 5' region of C/EBP- mRNA and regulates translation of C/EBP- isoforms. Two binding sites for CUGBP1 are located side by side between the first and second AUG codons of C/EBP- mRNA. One binding site is observed in- and out-of-frame short ORF that has been shown previously to regulate initiation of translation from different AUG codons of C/EBP- mRNA. Analysis of cytoplasmic and polysomal proteins from rat liver after partial hepatectomy evidenced that CUGBP1 is associated with polysomes to translate low-molecular-weight isoforms of C/EBP-. The binding activity of CUGBP1 to the 5' region of C/EBP- mRNA shows increased association with these polysomal fractions after partial hepatectomy. Addition of CUGBP1 into a cell-free translation system leads to increased translation of low-molecular-weight isoforms of C/EBP-. These data therefore demonstrate that CUGBP1 protein is an important component for the regulation of initiation from different AUG codons of C/EBP- mRNA (Timchenko et al., 1999a).
4. Translational Inhibition of C/EBP- Expression by Calreticulin.
As described above for C/EBP-, calreticulin is also able to interact with GCN repeats that form stable SL structures within C/EBP- mRNAs, leading to an inhibition of C/EBP- translation in vitro and in vivo (Timchenko et al., 2002).
5. Regulation of the C/EBP- Promoter.
Within the 5'-regulatory region of mouse C/EBP-, three protein factor-binding motifs, named UF1 (-376 to -352), UF2 (-254 to -223), and UF3 (-220 to -190), as well as two Sp1 motifs (-309 to -277 and -264 to -241) were identified by DNase I footprinting assays using nuclear extracts from lipopolysaccharide (LPS)-stimulated or unstimulated livers. Biochemical analysis confirmed that C/EBP- binds to the UF1 and UF2 sites, implying autoregulation of C/EBP- during the acute phase response (Chang et al., 1995).
Deletion analysis of the 5'-flanking region of rat C/EBP-, located upstream of the start site of transcription in the C/EBP- gene, demonstrated that a small region in close proximity to the TATA box (bp -121 to -71) is important in maintaining high levels of transcription of luciferase reporter gene constructs. Electromobility shift experiments identified two sites that are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein demonstrated CREB binding to both sites in the C/EBP- promoter with an affinity similar to that with the CREB consensus sequence. Moreover, transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and C/EBP- inducibility through CREB. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and C/EBP- mRNA transcription during liver regeneration (Niehof et al., 1997). As several pathways control CREB phosphorylation, these results provide evidence for the transcriptional regulation of C/EBP- via CREB under different physiological conditions.
6. Tissue-Specific Autoregulation of C/EBP- Transcription.
The two cAMP responsive elements within the rat C/EBP- promoter between nucleotides -121 and -71 play an important role in C/EBP- autoregulation as demonstrated by deletion analysis and luciferase reporter gene experiments. Gel shift experiments using oligonucleotides with overlapping point mutations aided in the identification of the GCAATGA-sequence (-site) adjacent to and partially overlapping the first CRE-like site as the core motif for C/EBP- binding. Further analysis of a mutated -site in reporter gene experiments showed the functional relevance of this site for autoregulation. The composite C/EBP--CRE-element in the promoter enabled synergistic activation of transcription by C/EBP- and the CREB pathway in a cell type-specific manner. In HepG2 hepatoma cells, NF-B increased autoregulation and could therefore mediate enhanced activation during inflammatory responses (Niehof et al., 2001a). These results demonstrated that the assembly of the three binding sites within the C/EBP- promoter and thus the interaction between C/EBP- and members of the CREB or NF-B family allows the specific control of C/EBP- gene transcription in different physiologic contexts such as inflammation or the acute phase response.
7. Negative Regulatory Domains of C/EBP-.
It could be demonstrated that C/EBP- contains a negative regulatory region composed of two elements, called RD1 and RD2. Deletions of RD2 relieve the inhibition of C/EBP- activity in intestinal endocrine L cells, but do not affect C/EBP- function in HepG2 hepatoma cells. These deletions also increase the DNA binding activity of C/EBP- approximately 3-fold, suggesting that RD2-mediated repression of DNA binding activity is responsible for C/EBP- inhibition in L cells. The adjacent RD1 element functions independently of RD2 and modulates the C/EBP- activation domain, which was shown to be composed of three subdomains that are conserved within the C/EBP protein family. RD1 does not affect cell type specificity, but it inhibits the transactivation potential of GAL4-C/EBP- hybrid proteins by 50-fold. These findings suggest that C/EBP- assumes a tightly folded conformation in which the DNA binding and activation domains are masked by interactions with the regulatory domain, and that to function efficiently in HepG2 cells, the protein must undergo an activation step. It was proposed that relief of inhibition conferred by the regulatory domains also accounts for C/EBP- activation in response to extracellular signals (Williams et al., 1995). It was suggested that phosphorylation plays a unique role to derepress rather than to enhance the transactivation domain as a novel mechanism to regulate gene expression by C/EBP- (Kowenz-Leutz et al., 1994).
8. Post-translational Phosphorylation of C/EBP-.
It could be demonstrated that post-translational site-specific phosphorylation of C/EBP- is an essential mechanism in the regulation of C/EBP--dependent gene regulation (Mahoney et al., 1992; Trautwein et al., 1993, 1994; Buck et al., 1999). Phosphorylation of C/EBP- at Ser299 and Ser277 by protein kinase C or by M-kinase resulted in an attenuation of binding to a 32P-labeled CCAAT oligodeoxynucleotide in vitro (Mahoney et al., 1992). Phosphorylation of C/EBP- by cAMP-dependent protein kinase A or by protein kinase C at Ser240 within the DNA-binding domain resulted in an inhibition of DNA binding (Trautwein et al., 1994), whereas phosphorylation at Ser105 by protein kinase C within the activation domain of C/EBP- enhances its transcriptional efficacy (Trautwein et al., 1993). To identify the region that is important for gene activation by phosphorylation of Ser105 of C/EBP-, a series of C/EBP- mutants were constructed, and domain swapping experiments with the DNA-binding domain of GAL4 were performed. These experiments point to an acidic region located between amino acids 21 and 105 of C/EBP- that activates genes independently of the DNA-binding domain and the leucine zipper of C/EBP-. Computer-assisted predictions revealed two regions, a helical and a hydrophobic region in the transactivation domain, that could be important in mediating the direct interaction with the basal machinery. Site-directed mutagenesis of acidic residues in both regions demonstrates that the hydrophobic region located between amino acids 85 and 95 is the likely motif for the interaction with the basal machinery. These results demonstrated that a hydrophobic region in the acidic transactivation domain of C/EBP- seems to be relevant in mediating gene activation of C/EBP--dependent genes (Trautwein et al., 1995).
9. Phosphorylation of C/EBP- Ser239 leads to C/EBP- Nuclear Export.
Tumor necrosis- (TNF-) treatment of primary mouse hepatocytes or TNF- overexpression in a mouse model of cachexia induces oxidative stress, nitric oxide synthase expression, and phosphorylation of C/EBP- on Ser239 within the nuclear localization signal, thus inducing its nuclear export, which inhibits transcription from the albumin gene. Similar molecular abnormalities were found in the liver of patients with cancer-related cachexia. (Buck et al., 2001b). The cytoplasmic localization and association of phosphorylated C/EBP- Ser239 with CRM1 (exportin-1) in TNF--treated hepatocytes were shown to be inhibited by leptomycin B, a blocker of CRM1 activity. Hepatic cells expressing the nonphosphorylatable C/EBP- alanine mutant were refractory to the inhibitory effects of TNF- on albumin transcription since the mutant remained localized to the nucleus. Treatment of TNF- mice with antioxidants or nitric oxide synthase inhibitors prevented phosphorylation of C/EBP- on Ser239 and its nuclear export and rescued the abnormal albumin gene expression in these animals (Buck et al., 2001b).
10. Phosphorylation of C/EBP- Thr217 by p90 Ribosomal S Kinase.
The hepatotoxin CCl4 was shown to be able to induce p90 ribosomal S kinase (RSK) leading to phosphorylation of C/EBP- on Thr217 with consecutive stellate cell proliferation in normal mice. In contrast, CCl4 treatment of C/EBP- knockout mice (C/EBP--/-) or transgenic mice carrying the C/EBP--Ala217 mutant (a dominant-negative nonphosphorylatable mutant) led to stellate cell apoptosis. It could be demonstrated that the association of C/EBP- phosphorylated at Thr217 with the procaspases 1 and 8 inhibited their activation and thus created a functional XEXD caspase inhibitory box in vivo and in vitro, which might be critical for cell survival </
