Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

CCAAT/Enhancer-Binding Protein Beta

  • Herman E. Popeijus
  • Sophie E. van der Krieken
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101550

Synonyms

Historical Background

The nuclear factor for IL-6 expression (NF-IL6) was discovered as a member of the CCAAT-enhancer binding proteins (C/EBP) in 1990, from which it derived its current name: C/EBP-β (Akira et al. 1990). C/EBP-β was first found to bind to an interleukin 1 (IL1) responsive element necessary for IL-6 expression (Akira et al. 1990). In general, C/EBP-β is thought to be an important transcription factor involved in immune responses, hematopoiesis, endoplasmic reticulum stress (ER-stress), adipogenesis, central nervous system (CNS) diseases, gluconeogenesis, liver regeneration, and embryogenesis. The C/EBP protein family consists of six members: C/EBP-α, -β, -δ, -ε, -γ, and -ζ (Gene names CEBPA, CEBPB, CEBPG, CEBPD, CEBPE, and CEBPZ, respectively). Each member belongs to the basic-leucine zipper (bZIP) class of transcription factors (Fig. 1). C/EBP-ζ is the only exception that does not contain the basic region. Furthermore, all members of the C/EBP family possess a DNA binding domain (DBD) and are thought to bind to the CCAAT boxes, though for C/EBP-β this box resembles more a consensus of TT(G/A)CGCAA (Pulido-Salgado et al. 2015). Using their zipper domains, C/EBPs are thought to form homodimers thereby initiating transcription (Fig. 2a, b). In addition, C/EBP-β can form a heterodimers with activating transcription factor 4 and 6 (ATF4 and 6) and can bind to a nonclassical C/EBP-β binding site, CRE/ATF (Fig. 2c) (Podust et al. 2001; Kalvakolanu and Gade 2012). Also hetero-dimerization with other bZIP family members was reported (Tsukada et al. 2011). The C/EBP-β gene encodes potentially for three protein isoforms. These isoforms are liver-enriched transcriptional activator protein star (LAP*; isoform a), liver-enriched transcriptional activator protein (LAP; isoform b), and liver-enriched transcriptional inhibitory protein (LIP; isoform c). In humans, LAP*, LAP, and LIP are, respectively, 345, 322, and 147 amino acids in length (Fig. 3). The LAP* isoform is slightly larger (23 amino acids) than LAP. LAP was discovered first together with LIP. It is thought that these three isoforms arise from the same mRNA but use an alternative start codon through leaky ribosomal scanning instead of proteolytic cleavage, alternative internal ribosomal binding sites, or alternative splicing (Xiong et al. 2001). The human full-length C/EBP-β protein contains four transactivation domains, several phosphorylation sites, and acetylation sites. All isoforms share the same leucine zipper (LZ) and DNA binding domain (DBD) at their c-terminus. The LAP* and LAP proteins are nearly the same though the LAP* contains a few extra amino acids at its n-terminus. LIP, however, lacks the transactivation domains and only contains het DBD and the LZ (Figs. 3 and 4). For a more extensive description of the other family members of C/EBP and the structure of their isoforms, we refer to an earlier review (Tsukada et al. 2011).
CCAAT/Enhancer-Binding Protein Beta, Fig. 1

Schematic representation of the bZIP domain of the C/EBP protein family dimerization. The alpha helix leucine zipper (LZ) is colored yellow, the DNA binding domain (DBD) is depicted in blue. The dotted lines represent the DNA double helix. At the ends of the protein, the free amino-terminus (N) and carboxyl-terminus (C) are indicated. The consensus DNA binding site is presented below the figure

CCAAT/Enhancer-Binding Protein Beta, Fig. 2

Homo dimerization of LAP*/LAP (a), homodimerization with the inactive form LIP (b), and hetero dimerization of C/EBP-β with ATF (c)

CCAAT/Enhancer-Binding Protein Beta, Fig. 3

Amino acid sequence of human C/EBP-β protein LAP* (NCBI Reference Sequence: NP_005185.2). The first amino acid methionine (m) of Liver- enriched activator protein star (LAP*), Liver- enriched activator protein (LAP) and Liver-enriched inhibitory protein (LIP) is indicated in blue. The sequence in front of the scissors within the LAP* sequence is not translated into protein for the indicated isoform (LAP and LIP). The Transactivation domains (TAD1–4) are depicted in red, the DNA binding domain (DBD) in blue and the Leucine zipper in yellow. The “p” indicates sites for phosphorylation and “a” indicates the possible sites for acetylation. Fig. 4 Schematic representation of the Liver- enriched activator protein star (LAP*; isoform a), Liver- enriched activator protein (LAP; isoform b) and Liver-enriched inhibitory protein (LIP; isoform c). The Transactivation domains (TAD1–4) are depicted in red, the DNA binding domain (DBD) in blue and the Leucine zipper in yellow. The “p” indicates sites for phosphorylation and “a” indicates the possible sites for acetylation. At the ends of the protein, the free amino-terminus (N) and carboxyl-terminus (C) are indicated

CCAAT/Enhancer-Binding Protein Beta, Fig. 4

Schematic representation of the Liver- enriched activator protein star (LAP*; isoform a), Liver- enriched activator protein (LAP; isoform b) and Liver-enriched inhibitory protein (LIP; isoform c). The Transactivation domains (TAD1-4) are depicted in red, the DNA binding domain (DBD) in blue and the Leucine zipper in yellow. The “p” indicates sites for phosphorylation and “a” indicates the possible sites for acetylation. At the ends of the protein, the free amino-terminus (N) and carboxyl-terminus (C) are indicated

Regulation of CEBP-β

The intron-less C/EBP-β mRNA is expressed in many human tissues including the liver, lung, intestine, pancreas, spleen, adipose tissue, kidney, and myelomonocytic cells (Su et al. 2004) (Fig. 5). The promoter of the C/EBP-β gene is located at chromosome 20, position q13.1 (Genebank accession number NM_005194.3) and contains a TATA box and several binding sites for transcription factors to modulate its expression. These include the binding places for among others transcription factor C/EBP-β, signal transducer and activator of transcription 3 (STAT3), specificity protein 1 (Sp1), members of the cAMP responsive element binding protein (CREB)/(ATF) family, early growth response 2 (EGR2), Fos-related antigen 2 (Fra-2), sterol-regulatory element-binding protein 1c (SREBP1c), myoblastosis transcription factor (Myb), nuclear factor-κB (NF-κB), and retinoic acid receptor α (RARα) (Huber et al. 2012; Pulido-Salgado et al. 2015; van der Krieken et al. 2015). C/EBP-β is regulated in a rather complex yet not fully understood way. First, its expression can be induced by various extracellular molecules like hormones, cytokines, inflammatory factors, and polyamides. An excellent overview of currently known co-repressors, co-activators, and non-bZIP transcription factors is given by Pulido-Salgado et al. (2015). In addition to this transcriptional regulation, C/EBP-β has a relative rapid turnover of approximately 50±10 minutes. C/EBP-β activity and protein turnover can be modulated by acetylation, phosphorylation, and binding to other transcription factors. In addition, ubiquitination via Mdm2 is described to target C/EBP-β degradation by the proteasome (Fu et al. 2015). For further reading see Tsukada et al. (2011), Huber et al. (2012), Pulido-Salgado et al. (2015), and references herein. Interestingly, the cellular myeloblastosis protein (cMyb), a proto-oncogene transcription factor, binds to the LZ domain of C/EBP-β and thereby can increases transcriptional activity (Tahirov et al. 2002). As all isoforms contain the LZ domain, this might be another way LIP can induce transcriptional activity although generally it is thought to inhibit transcription. In this light, C/EBP-β might be involved in hematopoiesis as this is one of the crucial roles of cMyb (Oh and Reddy 1999).
CCAAT/Enhancer-Binding Protein Beta, Fig. 5

Expression profile of C/EBP-β based on the GeneAtlas U133A dataset (Su et al. 2004). Relative expression and standard deviations are shown including the median, 3 times the median and 10 times the median (3 × M and 10 × M)

C/EBP-β Growth and Development

During embryogenesis, the C/EBP protein family and especially C/EBP-β is involved in the expression of placenta related genes in the human trophoblast (Bamberger et al. 2004). By direct protein interaction of C/EBP-β with erythroblastosis virus E26 oncogene homologue 2 (Ets-2), trophoblast-specific genes are regulated (Chakrabarty and Roberts 2007).

Besides its involvement in proper embryogenesis, C/EBP-β plays an important role in the development of mesenchymal cells. The mesenchymal cells comprise, after differentiation, mainly of the following cell types: osteoblasts, chondrocytes, adipocytes, fibroblasts, and myoblasts. To form these cells from the mesenchymal stem cells, each cell type governs a unique transcription factor pattern. The main transcription factors in osteoblasts are Runt-related transcription factor 2 (Runx2) and Transcription factor specificity protein (Sp7), in chondrocytes it is SRY-box 9 (Sox9), and in adipocytes they are C/EBP family members and together with Peroxisome proliferator-activated receptor gamma (PPARγ) transcription factors play a role (Smink and Leutz 2012). In preadipocytes, C/EBP-β activity is necessary to start the differentiation of the pre-adipocyte to differentiate to adipocyte. C/EBP-β is activated upon increased levels of polyamines that inhibit mRNA expression of C/EBP homologous protein (CHOP). CHOP functions as a dominant-negative inhibitor by binding to C/EBP-β and other CEBP family members. Following the inhibition of CHOP, constitutive C/EBP-β activation is able to induce mitotic clonal expansion and to activate the expression of both PPARγ and C/EBP-α (Brenner et al. 2015) (Fig. 6).
CCAAT/Enhancer-Binding Protein Beta, Fig. 6

Effect of C/EBP-β signaling in myoblast and adipocytes differ

In mice myoblasts, C/EBP-β is reported to be an important factor to control differentiation being an inhibitor of myogenesis (Marchildon et al. 2012). For instance, C/EBP-β knockout myoblasts differentiated faster and more robust which resulted in muscle fiber hypertrophy. Subsequent overexpression of C/EBP-β in these myoblast inhibited differentiation. These results are in line with the finding that C/EBP-β inhibits myoblast differentiation in cancer cachexia, while loss of C/EBP-β restores this (Marchildon et al. 2015). Furthermore, C/EBP-β stimulation by cyclic adenosine monophosphate phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) results in more myoblast clonal expansion due to inhibited differentiation which continues following drug removal (Lala-Tabbert et al. 2016). In the normal situation, Mouse double minute 2 homolog (Mdm2) is needed to ubiquitinate C/EBP-β and thereby it triggers degradation of C/EBP-β, leading to removal of the differentiation blockade to start the differentiation (Fu et al. 2015) (Fig. 6). These insights in the role of C/EBP-β in myoblast differentiation might therefore be used in further research to aid help people who are suffering of cancer cachexia or muscle disease. Cancer cachexia is thought to result from muscle weight loss that is not replenished by new differentiated myoblast because of increased myoblast cell stress and thereby increased C/EBP-β that subsequent prevents differentiation (Marchildon et al. 2015). In muscle diseases like Duchenne muscular dystrophy (DMD), transplantation of enough myoblast is a problem as the introduced myoblast differentiates too fast. In this case, temporarily inducing C/EBP-β might provide a solution that allows clonal expansion to generate first a substantial number of myoblasts (Lala-Tabbert et al. 2016).

C/EBP-β is also involved in processes related to the central nervous system, as C/EBP- β is important for memory formation and the outgrowth of neurites. Furthermore, C/EBP-β plays a role in inflammatory responses in the microglial cell. In these cells, overexpression of the dominant negative isoform LIP inhibits inflammation-related markers like Nitric oxide synthase 2 (NOS2) and tumor necrosis factor alpha (TNFα). In addition, C/EBP-β knockout mice were significantly less responsive to lipopolysaccharide (LPS) induced inflammation. Therefore, C/EBP-β is thought to possess pro-inflammatory properties. For further reading on the role of C/EBP-β within the nervous system, we refer to Pulido-Salgado et al. (2015) and references herein.

C/EBP-β in Relation to Endoplasmic Reticulum Stress and Inflammation

C/EBP-β induces acute phase proteins like c-reactive protein, glycoprotein, and various cytokines including interleucin-6 (IL-6). These products regulated by C/EBP-β are involved in various inflammatory processes including the regulation of macrophages activation and polarization (Juhas et al. 2015). C/EBP-β is induced during endoplasmic reticulum stress (ER-stress) which is also linked to inflammation (van der Krieken et al. 2015). A nice example is described by Bai et al. (2016), where LPS-induced ER-stress is clearly coupled to a C/EBP-β response. In addition, ER-stress also induces CHOP that might counter the effect of increased C/EBP-β. Furthermore, ATF6 is increased during ER-stress, as ATF6 is a dimeric partner of C/EBP-β; they together are able to alter the cells transcriptional profile during ER-stress. Prolonged ER-stress ultimately leads to cell cycle arrest and may finally result in apoptosis. Taking into account the known roles of C/EBP-β in myoblast and adiposity differentiation, changed levels of C/EBP-β in combination with increased levels of CHOP as often seen during ER-stress are likely to alter differentiation and clonal expansion. In addition, often the LIP/LAP ratio is discussed as important parameter that determines the final outcome (to start differentiation or continue clonal expansion) as it is supposed to reflect the balance between C/EBP-β activation and inactivation (van der Krieken et al. 2015).

Summary

C/EBP-β is an important regulator of many biologic processes. Currently, the role of the C/EBP- β gene in mesenchymal cell development and differentiation is most extensively investigated. Nowadays, research on the effect of C/EBP-β and its activation status is focused on the relative amount of LIP and LAP protein that is produced. It is this ratio that should reflect activity or inactivity. However, the use of this ratio alone neglects the many possible other factors that are of influence on C/EBP-β activity, such as posttranslational modifications and proteins like CHOP. Furthermore, the effects of the separate C/EBP- β isoforms are difficult to investigate. Since the products are all derived from a single mRNA transcript, RNA interference would knock out all C/EBP-β isoforms simultaneously. To allow more insight in the mechanisms that the three isoforms are part of, one could overexpress the different isoforms separately, although this might trigger homeostatic mechanisms that might partly counter the overexpression. Altogether, to elucidate the exact mechanism by which C/EBP-β isoforms exert their action, more basic research directed to the three isoforms and their dimerizing partners is warranted.

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Herman E. Popeijus
    • 1
  • Sophie E. van der Krieken
    • 1
  1. 1.Department of Human BiologyMaastricht UniversityMaastrichtThe Netherlands