Biological Role of the CCAAT/Enhancer-binding Protein Family of Transcription Factors*

CCAAT/enhancer-binding proteins (C/EBPs) comprise a family of transcription factors that are critical for normal cellular differentiation and function in a variety of tissues. The prototypic C/EBP is a modular protein, consisting of an activation domain, a dimerization bZIP region, and a DNA-binding domain. All family members share the highly conserved dimerization domain, required for DNA binding, by which they form homo- and heterodimers with other family members. C/EBPs are least conserved in their activation domains and vary from strong activators to dominant negative repressors. The pleiotropic effects of C/EBPs are in part because of tissue- and stage-specific expression. Dimerization of different C/EBP proteins precisely modulates transcriptional activity of target genes. Recent work with mice deficient in specific C/EBPs underscores the effects of these factors in tissue development, function, and response to injury.

trostatic interactions between amino acids along the dimerization interface determine the specificity of dimer formation among C/EBP family members as well as with transcription factors of the NF-B and Fos/Jun families (2). C/EBP dimerization is a prerequisite to DNA binding (4). DNA binding specificity, however, is determined by the DNA contact surface, the "basic" region of approximately 20 amino acids, upstream of the leucine zipper, specifically by three amino acids lying along the protein-DNA interface (1,5). Domains responsible for transcriptional activation and/or repression are located in the N-terminal end of the protein.
In this review, C/EBP genes are designated C/EBP␣, -␤, -␥, -␦, -⑀, and -as proposed by Cao et al. (6); however, Table I lists alternative nomenclature. C/EBP␣ was the first member cloned (7)(8)(9)(10)(11)(12). Expression patterns of C/EBP␣ mRNA are similar in the mouse and human with measurable levels in liver, adipose, intestine, lung, adrenal gland, peripheral blood mononuclear cells, and placenta (8,12). In liver and adipose, highest levels of C/EBP␣ mRNA are detected only in differentiated tissue (8,12). Autoregulation of C/EBP␣ mRNA occurs by different mechanisms in the mouse and in humans. The murine C/EBP␣ promoter directly binds C/EBP␣ within 200 base pairs of the transcriptional start resulting in 3-fold activation (9). Autoregulation of the human C/EBP␣ promoter occurs by C/EBP␣-induced binding of USF, a ubiquitously expressed transcription factor, to its upstream site within the C/EBP␣ promoter (13).
Two isoforms of C/EBP␣ are generated from its mRNA by a ribosomal scanning mechanism (14,15). The full-length protein is 42 kDa and contains three transactivation domains (TEI-III) (16 -18). TEI and TEII mediate cooperative binding of C/EBP␣ to TBP (TATA box-binding protein) and TFIIB, two components of the RNA polymerase II basal transcriptional apparatus (17). TEIII contains a negative regulatory subdomain (16).
A fraction of ribosomes ignore the first two AUG codons and initiate translation at the third AUG, 351 nucleotides downstream of the first AUG (14,15). This shorter 30-kDa protein retains its dimerization and DNA-binding domains; however, it possesses an altered transactivation potential compared with the 42-kDa isoform (14,15).
The human, mouse, and rat genes for C/EBP␤ have been cloned (6, 19 -23). Constitutive expression of C/EBP␤ is highest in liver, intestine, lung, and adipose; however, in the mouse, it is also detectable in kidney, heart, and spleen by Northern analysis (6). Stimulation with lipopolysaccharide (LPS), IL-6, IL-1, dexamethasone, and glucagon strongly induces C/EBP␤ expression, suggesting a role in the mediation of the inflammatory response (20, 24 -26).
Like C/EBP␣, two C/EBP␤ isoforms are generated from a single mRNA by a leaky ribosomal scanning mechanism. The full-length 32-kDa protein, also termed LAP, encodes for the conserved activation domains found in other C/EBP proteins, as well as two regulatory domains, RD1 and RD2, which confer DNA binding inhibition in a cell type-specific manner (27). The truncated protein, LIP, translated from the third, in-frame AUG, possesses only the DNA-binding and leucine zipper domains (22,28). Heterodimerization of the truncated isoform with the full-length C/EBP␤ (LAP) attenuates transcriptional activity in substoichiometric amounts, suggesting a dominant negative mechanism of transcriptional regulation (28).
C/EBP␤ was originally identified as a mediator of IL-6 signaling, binding to IL-6-responsive elements in the promoters of acute phase response genes TNF, IL-8, and G-CSF (20,22). Signal transduction of the acute phase response by IL-1 and LPS also induces C/EBP␤ transcription (20,25). TNF␣ promotes nuclear localization of C/EBP␤ and C/EBP␦ in response to inflammatory stress (29). Cytokine stimulation further increases C/EBP␤ transcriptional activity by enhanced DNA binding (22). Post-transcriptional modifications of C/EBP␤ by protein kinases in the signal transduction pathway of C/EBP␤ appear to activate transcription (30,31).
C/EBP␥ is a short, intronless gene, whose mRNA is ubiquitously expressed with highest levels found in non-differentiated, progenitor cells (19,32,33). The 16.4-kDa encoded protein possesses a leucine zipper dimerization domain and DNA-binding region; however, it lacks transcriptional transactivating elements (33). Heterodimerization with C/EBP␣ and C/EBP␤ attenuates transcriptional activation of target genes, suggesting dominant negative regulation of C/EBP transactivation in undifferentiated, non-induced cells (33).
C/EBP␦ is an intronless gene (6, 34 -38). Constitutive expression of C/EBP␦ is detected in intestines, adipose, and lung, with high levels of expression in all tissues following LPS stimulation (6,25,36). The 269-amino acid protein encodes a leucine zipper dimerization domain and DNA-binding region, readily forming heterodimers with C/EBP␣ and C/EBP␤ (6). Transactivating efficiency of C/EBP␦ is comparable with that of C/EBP␣ and C/EBP␤ (6). The DNA-binding region of C/EBP␦ differs from C/EBP␣ in that it contains 2 proline and 4 glycine residues, which may interrupt the predicted ␣-helical structure (6). Diminished DNA binding affinity of the C/EBP␦ basic domain compared with C/EBP␣ and C/EBP␤ is likely the result of sequence divergence (6).
C/EBP⑀ was originally identified from a rat genomic library, but the start site could not be determined and no expression was detected (38). Subsequently, the full-length C/EBP⑀ gene was cloned (39,40). Human C/EBP⑀ contains two intronic sequences and five in-frame AUG initiation sites, three of which satisfy the Kozak context (41). Four mRNA isoforms, expressed primarily in myeloid and lymphoid cells, are generated by the use of alternative promoters combined with differential splicing (41). The highest level of expression is detected in promyelocyte and late myeloblastlike cell lines (39,42). Further, induction of C/EBP⑀ mRNA with retinoids promotes granulocytic differentiation of promyelocyte line NB4 (42,43). The four C/EBP⑀ mRNA isoforms translate into three proteins possessing identical leucine zipper domains and variably truncated activation domains, with differing transcriptional activities (40,41).
C/EBP, which is induced by DNA damage, was originally cloned in hamster and named growth arrest and DNA damageinducible gene (gadd153) (44). Spanning 5 kilobases, it consists of four exons and is expressed ubiquitously (45). Like other C/EBP proteins, C/EBP possesses a leucine zipper dimerization domain and DNA-binding region (45). C/EBP readily heterodimerizes with other C/EBPs; however, the presence of two prolines in the DNAbinding region disrupts its helical structure and prevents dimer binding to the cognate DNA enhancer element (45). C/EBP functions as a dominant negative inhibitor of C/EBP transcriptional activation by preventing heterodimer binding of C/EBP␣ and C/EBP␤ to classic C/EBP enhancer sequences (45).

C/EBP-deficient Animal Models
Hepatic Phenotypes-Coordinate expression of specific C/EBP isoforms is essential for normal hepatic synthetic activity and response to injury; however, C/EBP␣ is the predominant nuclear signal regulating terminal hepatocyte differentiation and function. Elimination of C/EBP␣ in targeted mouse knockout models results in profound derangement of liver structure and function (Table I). C/EBP␣ Ϫ/Ϫ mice have disturbed hepatic architecture with acinar formation, resembling proliferative or pseudoglandular hepatocellular carcinoma (46,47). c-Myc and c-Jun RNAs are induced consistent with a proliferative liver (46). Metabolic derangements are pronounced with an impairment of hepatic glycogen storage, and the majority of mice die soon after birth because of hypoglycemia (46,47). Known target genes of C/EBP␣ have decreased expression at birth, including albumin, glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase (47). Low level expression of phosphoenolpyruvate carboxykinase and perinatal lethality is also seen in a subset of C/EBP␤ Ϫ/Ϫ mice, suggesting involvement of the C/EBP␤ isoform in gluconeogenic pathways (48).
Hepatocyte proliferation following partial hepatectomy is accompanied by profound changes in C/EBP expression patterns. C/EBP␣ mRNA decreases following partial hepatectomy whereas C/EBP␦ mRNA increases (49 -51). C/EBP␤ mRNA levels rise following partial hepatectomy, as well as sham surgery, in keeping with its role as an inflammatory/injury response mediator (50). C/EBP␣:C/ EBP␤ heterodimers are replaced by increased amounts of C/EBP␤ homodimers during the early G 1 period after partial hepatectomy (52,53). The necessary down-regulation of C/EBP␣ expression during liver regeneration may be mediated by the increased binding of C/EBP␤ homodimers to the C/EBP␣ promoter, normally transactivated by a:b heterodimers in the non-proliferative state (53).
The duality of C/EBP␣ function in mediating cell cycle arrest and hepatic metabolism is clearly demonstrated in the C/EBP␣ knockout mouse. C/EBP␣ functions similarly in adipose tissue, inducing adipocyte differentiation and mediating transcription of adiposespecific genes.
Adipose Phenotype-Adipocytes grown in tissue culture and in animal models lacking C/EBP␣ fail to accumulate lipids. Uncoupling protein is responsible for uncoupled mitochondrial respiration and heat generation and is a marker for differentiation of brown adipose tissue. C/EBP␣-deficient mice have minimal levels of uncoupling protein expression at 2 h postpartum, which increases to 60% that of control mice by 32 h postpartum (47). Gene transcription of fatty acid synthase, GLUT4, and 422/aP2 is unaltered in white adipose tissue of the C/EBP␣-deficient mouse, which is inconsistent with transcriptional data from 3T3-L1 cell lines (47, 54 -56). Redundant transcriptional elements operating in the animal model may regulate the fatty acid synthesis pathway, compensating for the lack of C/EBP␣. Mice deficient for both C/EBP␤ and C/EBP␦ expire perinatally, similar to C/EBP␣ knockout mice (57). C/EBP␤:C/EBP␦ double knockout mice did not accumulate lipid droplets in brown adipose tissue and had significantly reduced epidydimal fat pads in surviving adults (57). Despite these defects, C/EBP␣ and PPAR␥ expression was normal, suggesting that C/EBP␣ and PPAR␥ are not sufficient for adipocyte differentiation in the absence of C/EBP␤ and C/EBP␦ (57).
Preadipocyte differentiation into functional adipocytes results from a highly regulated cascade of C/EBP isoform expression. Dexamethasone-and methylisobutylxanthine-stimulated 3T3-L1 preadipocytes express high levels of C/EBP␤ and C/EBP␦. These factors diminish during the late phase of differentiation concordant with the appearance of high levels of C/EBP␣ (6,58). Ectopic expression of C/EBP␣ in 3T3-L1 cells arrests mitotic growth (59). Likewise, abrogation of C/EBP␣ expression, either by antisense interactions or hydrocortisone administration, prevents terminal adipocyte differentiation (14,60).
Transient modulation of C/EBP levels in response to insulin and dexamethasone suggests a dynamic role in adipocyte metabolism (65). Induction of C/EBP␤ and C/EBP␦ occurs within 1 h of insulin stimulation, resulting in a 20-fold increase of transcription factor levels by 4 h (65). Insulin treatment also decreased DNA binding of C/EBP␣ while increasing nuclear C/EBP␤ and C/EBP␦ binding (65). Insulin also induces rapid dephosphorylation of C/EBP␣ and represses C/EBP␣ expression, modulating adipocyte gene transcription (e.g. GLUT4) (65,66). Another gene target of C/EBP␣, the obese gene (67,68), may be similarly regulated. Likewise, dexamethasone rapidly induces C/EBP␦ levels, reciprocally repressing C/EBP␣ expression (69).
CHOP regulates stress-inducible growth arrest in adipose tissue. Late in adipogenesis and during conditions of nutrient deprivation CHOP mRNA transcription is enhanced (45,70,71). CHOP attenuates C/EBP␣ and C/EBP␤ activity by forming non-DNA binding heterodimers, and if expressed early in the adipogenesis program, will inhibit differentiation (45,70). Induction of CHOP in adipocytes by cellular stress blocks G 1 to S phase progression resulting in growth arrest (72).
The oncogenic variant of CHOP is found exclusively in myxoid liposarcomas (73). Chromosomal translocation of t(12;16)(q13;p11) fuses CHOP to an RNA-binding protein, which possesses strong homology to protein expressed in Ewing's sarcoma (74). TLS-CHOP (translocated in liposarcoma-CHOP) fails to cause cell grow arrest and interferes with normal CHOP activity (72).
Hematopoietic Phenotypes-Profound abnormalities of the hematopoietic system are seen in C/EBP␣-, C/EBP␤-, and C/EBP⑀deficient mice. Mice deficient in C/EBP␣ display an early block in the maturation of granulocytes (75). Peripheral blood and bone marrow smears show only myeloblastic cells of the myeloid lineage (75). The G-CSF receptor message is undetectable in these cells, suggesting a loss of G-CSF signal-directed maturation (75). In transient assays, C/EBP␣ contributes to tissue-specific expression of G-CSF and GM-CSF receptors (76 -78) and neutrophil elastase (79,80). Evidence suggests that C/EBP␣ plays an early, pivotal role in the granulocyte lineage.
C/EBP␤-deficient mice are highly susceptible to Candida albicans, Listeria monocytogenes, and Salmonella typhi (81,82). Lethality from these pathogens may be in part because of macrophage defects and escape of phagocytosed bacteria from the phagosome to the cytoplasm (82). Low IL-12 levels and depressed delayed-type hypersensitivity, consistent with an impaired Th1 immune response, are seen in these mice (81). Elevated IL-6 levels, reported by one group, in C/EBP␤-deficient mice coincide with splenomegaly, peripheral lymphadenopathy, plasmacytosis, and extramedullary hematopoiesis, as seen in Castleman's disease in humans (81).
In B cells, C/EBP␥ is the predominant isoform in early cells, decreasing with cellular maturity (83). C/EBP␤ becomes highly expressed in mature B cells and with LPS stimulation (83). Consistent with this observation, C/EBP sites are activators in mature B cells but not in early cells, suggesting that C/EBP␤ and C/EBP␥ play reciprocal roles (83).
Mice nullizygous for C/EBP⑀ survive only 2-5 months after birth (84). Frequently, these mice succumb to tissue effacement by immature granulocytes; however, 60% of mice typed have a systemic infection with Pseudomonas aeruginosa at time of death (84). C/EBP⑀-deficient mice generate atypical hyposegmented granulocytes that are functionally defective, lacking an oxidative burst (84). Additionally, derangements in cytokine signaling are evidenced by low levels of mRNAs for interferon-␥, IL-2, IL-4, IL-12p40, and TNF-␣ (84). These results suggest that C/EBP⑀ acts temporally downstream of C/EBP␣ in granulopoiesis, blocking the last steps in terminal differentiation of mature segmented granulocytes.
Other Systems-C/EBPs role in the function of other organ systems is only beginning to be elucidated. A significant percentage of C/EBP␣-deficient mice succumb to respiratory defects soon after birth (46). Histologic examination of C/EBP␣-deficient lungs shows hyperproliferation of type 2 pneumocytes (46). C/EBP␣ expression is temporally correlated with the appearance of surfactant A protein and is not present in A549 cells, a cell line that does not express surfactant proteins (85).
Normal ovarian physiology is dependent upon both C/EBP␣ and C/EBP␤. Rat ovarian follicles express C/EBP␣ in a cell-, time-, and hormonally specific manner (86). Attenuation of C/EBP␣ expression results in decreased responsiveness to exogenous gonadotropins and decreased ovulation rate (86). Additionally, attenuation of C/EBP␣ expression is associated with elevated expression of proto-oncogene c-myc (86). C/EBP␤ mediates signal transduction of luteinizing hormone and is essential for the formation of corpora lutea (87). C/EBP␤-deficient mice fail to down-regulate expression of prostaglandin endoperoxidase synthase 2 and p450 aromatase in response to luteinizing hormone and are sterile (87).

Conclusions
C/EBPs act as pivotal regulators of cellular differentiation, terminal function, and response to inflammatory insult. Their extensive involvement in hepatic, adipose, and hematopoietic systems suggests the certainty of C/EBPs role in other tissues and systems. As potent mediators of gene expression, C/EBPs may be the future of some gene therapies or offer a deeper understanding of the forces driving oncogenesis. We are only beginning to understand the intricate pathways that transduce cell surface receptor signaling to gene transcription and subsequent protein activation. Future work with animal models deficient in multiple C/EBP isoforms will further elucidate these complex pathways.