Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, NCI-Frederick, Frederick, MD 21702-1201, USA
e-mail: johnsopf{at}ncifcrf.gov
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Summary |
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Key words: C/EBP, Cell cycle, Proliferation arrest
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Introduction |
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C/EBP proteins are a family of basic region leucine zipper (bZIP) transcription factors that includes six members, C/EBP, C/EBPß, C/EBP
, C/EBP
, C/EBP
and C/EBP
(genetic nomenclature CEBPA, CEBPB, CEBPG, CEBPD, CEBPE and CEBPZ, respectively), with related sequences and functions (Fig. 1A). Except for C/EBP
, which lacks a canonical basic region, each protein contains similar basic region and leucine zipper sequences at its C-terminus, which mediate DNA binding and dimerization, respectively. C/EBPs bind to palindromic DNA sites as homo- or heterodimers (Fig. 1B). The N-terminal portion of each protein contains effector domains that mediate transcriptional activation, repression and autoregulatory functions. C/EBP
, owing to its small size (
20 kDa), lacks any known functional domains outside the bZIP region. The C/EBP
and C/EBPß transcripts also encode internally initiated translation products termed p30 and liver inhibitory protein (LIP), respectively, which retain the C-terminal DNA-binding domain (Descombes and Schibler, 1991
; Lin et al., 1993
; Ossipow et al., 1993
). These proteins lack the major N-terminal transactivation domain and can dominantly inhibit the activating C/EBP isoforms.
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Initial evidence for antiproliferative functions of C/EBP![]() |
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Phenotypes of C/EBP![]() |
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Terminal differentiation of granulocytes is also dramatically impaired in the absence of C/EBP (Zhang et al., 1997
). C/EBP
/ embryos exhibit a lack of mature neutrophils and eosinophils and increased numbers of blasts in blood and fetal liver (FL). This maturation defect results in part from reduced transcription of the gene encoding granulocyte colony-stimulating factor receptor (G-CSFR), whose promoter is a known target of C/EBP
(Smith et al., 1996
). However, the granulopoietic defects in C/EBP
/ mice are more severe than those of animals lacking the G-CSFR gene (Liu et al., 1996
), suggesting that there are additional functions and targets for C/EBP
in the regulation of terminal granulocytic differentiation. This conclusion is further supported by experiments in which dominant-negative C/EBP and the G-CSFR were co-expressed in a myeloid cell line (Wang and Friedman, 2002
).
C/EBP/ FL also lacks mature macrophages and macrophage progenitors (Heath et al., 2004
). In addition, there are fewer bipotential granulocyte/macrophage precursors in C/EBP
/ FL but normal numbers of multipotent cells. Mutant FL cells transplanted into wild-type irradiated recipients fail to generate macrophages but give rise to very large, hyperproliferative spleen colonies, and the host mice develop myelodysplastic syndrome. This indicates that C/EBP
functions not only during terminal differentiation but also at an early stage of myelopoiesis. Interestingly, C/EBP
/ FL cells grown in liquid culture with hematopoietic growth factors such as interleukin (IL)-3 show unrestrained mitotic growth and increased self-renewal, whereas wild-type cells undergo terminal myeloid differentiation and cease to proliferate after a few weeks. The C/EBP
/ cells behave like an immortalized cell line and can be cultured indefinitely, although the cells remain dependent on hematopoietic growth factors for proliferation and survival (Heath et al., 2004
). Thus, the existing data point to a critical role for C/EBP
in maturation of granulocytes and macrophages and its involvement in control of cell-cycle exit during terminal differentiation.
Recent experiments in which the CEBPA gene was inducibly deleted in adult mice demonstrate that C/EBP is required for the common myeloid progenitor cell (CMP) to give rise to the more committed granulocyte/monocyte progenitor (GMP) (Zhang et al., 2004
). In addition, deletion of C/EBP
leads to increased numbers of blast cells and enhances the competitive repopulation of hematopoietic stem cells (HSCs) relative to wild-type cells in transplantation experiments. The increased self-renewal potential of C/EBP
/ HSCs may, at least in part, be due to elevated expression of the polycomb gene Bmi-1. The latter two studies therefore reveal that C/EBP
functions much earlier in hematopoiesis than previously thought, providing a partial brake on proliferation even in stem cells and progenitor cells.
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C/EBP![]() |
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The expression of truncated or other mutant C/EBP polypeptides probably facilitates AML development by imposing a differentiation block and disrupting normal cell-cycle exit. The hyper-proliferating mutant cells may be at increased risk of acquiring other oncogenic mutations that are necessary for transformation and leukemogenesis. Whether CEBPA mutations initiate leukemogenesis or arise during later stages of cancer development is unclear, and further experiments using animal models will be necessary to answer this question.
The studies of Pabst et al. lead to two important conclusions: first, C/EBP has a role in regulating differentiation of human myeloid cells, as in mice; second, loss of C/EBP
function can contribute to human cancers. Experiments showing that reinstating C/EBP
expression in AML cells suppresses their proliferation in vitro and tumorigenicity in vivo support these conclusions (Truong et al., 2003
). Although CEBPA mutations have not been reported for other cancers, C/EBP
expression is known to be downregulated in lung tumors (Halmos et al., 2002
), hepatocarcinomas (Friedman et al., 1989
; Xu et al., 1994
) and squamous cell carcinomas (Oh and Smart, 1998
; Shim et al., 2005
). In addition, the oncogenic fusion proteins BCR-ABL and AML1-MDS1-EVI1 (AME), which are synthesized as a consequence of chromosomal translocations associated with myeloid leukemias, inhibit C/EBP
translation (Helbling et al., 2004
; Perrotti et al., 2002
), and AML-ETO blocks CEBPA transcription (Cilloni et al., 2003
; Pabst et al., 2001a
). Collectively, these observations indicate that `dialing down' C/EBP
activity by one of several mechanisms is necessary for the decreased differentiation and increased proliferative capacity of cancer cells, specifically those in which C/EBP
controls differentiation of the normal tissue. Hence, therapeutic compounds selected for their ability to reactivate C/EBP
expression (thus inducing cell-cycle arrest and differentiation) might be effective agents for treating cancers such as AML (Tenen, 2003
).
Recently, Yoon and Smart identified another antiproliferative function of C/EBP involving DNA-damage-induced growth arrest in epidermal keratinocytes. Exposure of keratinocytes to ultraviolet B (UVB) radiation causes DNA damage and activates a p53-dependent G1 checkpoint that arrests cell-cycle progression during DNA repair. C/EBP
expression is strongly induced by UVB irradiation in cultured keratinocytes and mouse skin (Yoon and Smart, 2004
). Knocking down C/EBP
by RNA interference (RNAi) greatly reduces UVB-induced cell-cycle arrest; furthermore, C/EBP
induction is impaired in p53-mutant cells, and p53 inducibly associates with the CEBPA promoter in chromatin immunoprecipitation (ChIP) assays. These results suggest that CEBPA is a direct transcriptional target of p53 in keratinocytes and that C/EBP
functions as a critical effector of the p53-dependent growth arrest response. Thus, in addition to regulating terminal differentiation, C/EBP
can be induced by stress signals that inhibit cell proliferation during DNA repair, which could contribute to its role in tumor suppression. Whether C/EBP
has a widespread function in DNA repair responses or whether this is mainly restricted to epidermal keratinocytes, which are particularly susceptible to UVB-induced DNA damage, remains to be established.
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Mechanisms of C/EBP![]() |
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Later studies argue against a significant role for p21 (Muller et al., 1999). Leutz and coworkers discovered that the HPV16 E7 oncoprotein disrupts the ability of C/EBP
to induce cell-cycle exit but affects neither its transcriptional activation function nor its differentiation-inducing activity in pre-adipocytes (Muller et al., 1999
). These observations reveal that the growth arrest and differentiation functions of C/EBP
are separable activities. The authors also found that C/EBP
can induce cell-cycle exit in p21-deficient mouse embryo fibroblasts (MEFs), and that E7 blocks C/EBP
-induced growth arrest in p21-null cells. Thus, at least in fibroblasts, p21 is dispensable for the anti-mitotic activity of C/EBP
. The normal responses to C/EBP
in p21-deficient cells are difficult to reconcile with an important role for a C/EBP
-p21 mechanism in proliferation arrest.
Inhibition of cyclin-dependent kinase (CDK) activity
Subsequent work has implicated CDK2 and CDK4 as targets of C/EBP inhibition (Harris et al., 2001
; Wang et al., 2001
). Both play key roles in cell-cycle progression in G1-phase by phosphorylating specific substrates, the most important of which is the retinoblastoma (pRB) tumor suppressor. Phosphorylation inactivates pRB, causing its release from E2F transcription factors and de-repression of S-phase genes that are either required for DNA replication or regulate subsequent cell-cycle events. C/EBP
binds CDK2 and CDK4 in vitro and inhibits their ability to phosphorylate substrates such as histone H1. Moreover, deletion of the CDK-interacting sequences abrogates C/EBP
-induced growth arrest in transfected cells.
Wang et al. (Wang et al., 2001) mapped the CDK2/CDK4-binding region on C/EBP
to a sequence spanning residues 175-187; this group also observed that C/EBP
disrupts the association of CDKs with cyclins. By contrast, Harris et al. localized the CDK2-interacting region to residues 119-160 (Harris et al., 2001
), a segment that also appears to be involved in p21 binding. Harris et al. suggested that C/EBP
stabilizes the CDK2-p21 inhibitory complex, whereas Wang et al. found that the activity of free CDK2/CDK4 can be inhibited by C/EBP
(Wang et al., 2001
). The latter group also detected C/EBP
-CDK2/CDK4 interactions in cells by co-immunoprecipitation but did not observe p21 in this complex. In addition, they showed that over-expression of CDK2 overcomes C/EBP
-mediated proliferation arrest in HT1 fibrosarcoma cells. Clearly, these two reports contain discrepancies with respect to the CDK-binding domains identified and the precise target for inhibition by C/EBP
(CDKs alone or p21-CDK2 complexes), but there is no obvious explanation for the conflicting results.
The inhibition of CDK2/CDK4 clearly does not require DNA binding, and this conclusion is supported by the observation that DNA-binding-defective C/EBP mutants still slow cell-cycle progression (Wang et al., 2001
; Wang et al., 2003
). Nevertheless, the underlying evidence is based on overexpression studies, which raises the possibility of experimental artifacts. High levels of C/EBP
in the cell could sequester and inactivate an interacting protein(s), which might not occur under physiological conditions. Therefore, a more definitive test of the CDK inhibition mechanism would be to use a gene-knockin approach to examine the effects of CDK-binding site mutations in vivo to circumvent spurious results resulting from supraphysiological levels or inappropriate timing of C/EBP
expression. Indeed, a C/EBP
mutant lacking the CDK-interaction region apparently produces no overt phenotype when integrated into the mouse germline (Nerlov, 2004
).
Regulation of RB-E2F complexes
The RB family of proteins (pRB, p107 and p30) plays a key role in controlling cell proliferation and suppressing tumorigenesis. RBs have overlapping as well as distinct functions that depend in part on the cellular context, and they are also differentially expressed during the cell cycle (Classon and Harlow, 2002). RB proteins function mainly through their interactions with E2F transcription factors, forming complexes that repress transcription of S-phase genes. p107 and p130 preferentially associate with E2F4 and E2F5, whereas pRB can bind to all the E2Fs (Classon and Harlow, 2002
). Analysis of cells lacking various combinations of RB genes reveals that all three proteins contribute proliferative constraints in primary fibroblasts (Dannenberg et al., 2000
; Sage et al., 2000
).
Darlington and coworkers have proposed that C/EBP directly modulates RB-E2F complexes involved in regulating cell-cycle progression. They first showed that C/EBP
disrupts E2F-p107 complexes that are present during S-phase and are associated with proliferating cells (Timchenko et al., 1999a
). In wild-type day 18 mouse embryos, E2F-p107 complexes in liver that are present at earlier stages disappear, and these are also absent in newborn animals. However, in C/EBP
/ mice, E2F-p107 complexes remain detectable until birth. The addition of purified C/EBP
to liver nuclear extracts disrupts p107-containing E2F complexes, and a synthetic peptide corresponding to a small region of the C/EBP
transactivation domain that shares sequence similarity with E2F also displays this activity. C/EBP
might thus block the association between E2F and p107. In a second study, Darlington and coworkers found that ectopic C/EBP
expression in 3T3-L1 pre-adipocytes increases the abundance of E2F-p130 complexes (Timchenko et al., 1999b
). They postulated that this response involves the previously reported induction/stabilization of p21, which inhibits CDK activity.
From these studies, the authors suggested that C/EBP alters the pattern of RB-E2F associations in a manner that promotes cell-cycle withdrawal (i.e. reduced numbers of p107 complexes and increased numbers of p130 complexes). However, given current information, it seems unlikely that E2F-p107 stimulates growth or that disruption of this complex would decrease cell proliferation. Indeed, the opposite might be expected given the growth properties of cells lacking p107 and other RB-family members, which reveal antiproliferative functions for p107 (Dannenberg et al., 2000
; Sage et al., 2000
). To date, the E2F-p107 disruption mechanism for C/EBP
-mediated growth inhibition remains unconfirmed, although the idea that C/EBP
directly or indirectly promotes the formation of growth-inhibiting E2F-RB complexes or enhances their activity remains a viable hypothesis.
Inhibition of E2F-mediated transcription
C/EBP might also suppress cell proliferation through interactions with free E2F, as opposed to regulating RB-E2F complexes. Using an inducible C/EBP
system, Slomiany et al. (Slomiany et al., 2000
) showed both that C/EBP
inhibits proliferation of murine fibroblast lines and that C/EBP
is present in a complex that binds to E2F sites in genes such as dihydrofolate reductase (DHFR) and E2F-1 that are upregulated during the G1-S transition. However, this binding appears to be indirect, since purified recombinant C/EBP
does not bind to E2F site probes. C/EBP
also represses transcription from reporter constructs containing the DHFR or E2F-1 promoters or an artificial E2F-driven promoter. A similar study demonstrated that C/EBP
can repress transcription from the Myc promoter, which also contains an E2F-binding element, and it was suggested again that C/EBP
acts indirectly through the E2F site (Johansen et al., 2001
). C/EBP
might thus associate with E2F complexes and convert them into repressors capable of inhibiting S-phase gene transcription. In this model (as well as in the p21 and CDK2/CDK4 inhibition mechanisms), the ability of C/EBP
to bind DNA is irrelevant to its growth arrest functions.
Porse et al. (Porse et al., 2001) have provided further support for the E2F co-repression model, delineating C/EBP
sequences required for repression of E2F-driven transcription. Their analysis identified transactivation element I (TE-I) at the N-terminus as a critical inhibitory determinant. They also investigated basic region residues that are predicted to face away from DNA (on the basis of molecular modeling using crystal structures for the bZIP proteins Fos, Jun and GCN4), reasoning that these sequences might mediate protein-protein interactions that are important for C/EBP
activity. Two basic region mutants (BRMs), BRM-5 (Y285A) and BRM-2 (I294A/R297A), show decreased repression of E2F-driven transcription compared with wild-type C/EBP
. These also exhibit diminished antiproliferative activity in NIH 3T3 cells and reduced ability to induce differentiation of pre-adipocytes. A gene-knockin approach revealed that mice homozygous for BRM-2, BRM-5, or a third mutant (BRM-1) that shows unimpaired activity in cell-based assays, survive to adulthood and have normal levels of hepatic glycogen and gluconeogenic enzymes. However, the BRM-2- and BRM-5-knockin animals display significant reductions in white adipose tissue and an absence of differentiated neutrophils, whereas other hematopoietic lineages develop normally. EMSA analysis of liver nuclear proteins using a C/EBP site probe showed detectable C/EBP
DNA-binding activity in all genotypes. However, a C/EBP
-containing complex present in wild-type extracts that binds to an E2F site from the DHFR promoter is not observed in extracts from the BRM-2 and BRM-5 mutants. The basic region residues mutated in BRM-2 and BRM-5 thus might mediate binding of C/EBP
to E2F and this association could control the differentiation and cell-cycle-exit functions of C/EBP
. These results support the findings of Slomiany et al. and Johansen et al., which suggest that C/EBP
inhibits expression of E2F-regulated genes through direct interactions with E2F transcription factors (Slomiany et al., 2000
; Johansen et al., 2001
).
Other observations raise questions about this seemingly straightforward model. In the crystal structure of the C/EBP bZIP domain bound to DNA (Miller et al., 2003
), the DNA-contacting residues differ in several respects from those predicted by the molecular modeling of Porse et al. (Porse et al., 2001
). In particular, Tyr285 (the residue mutated to alanine in BRM-5) makes critical contacts with other basic region residues that stabilize the DNA-protein interface (Fig. 4). Tyr285 also makes a phosphate contact with the DNA backbone. Importantly, the Y285A (BRM-5) protein exhibits strongly decreased affinity for C/EBP sites in vitro and significantly reduced ability to transactivate a C/EBP reporter construct (Miller et al., 2003
). BRM-5 is thus a hypomorphic allele and the mutant protein has intrinsically lower DNA-binding activity than wild-type C/EBP
. The phenotypes of BRM-5 mice could therefore be explained by impaired interaction of C/EBP
with target promoters to which it binds directly.
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The BRM mutations might have effects on DNA binding that are not apparent from in vitro binding assays, especially if the major C/EBP complexes detected in cell extracts are heterodimers containing other C/EBP proteins, such as C/EBP
(Parkin et al., 2002
). In such cases, one subunit would have normal binding affinity, partially overcoming the effect of the mutation on DNA binding. However, C/EBP
homodimers (which may have critical biological roles) would exhibit more severely impaired binding. Moreover, the fact that in vitro binding assays are generally performed under conditions of excess DNA probe might mask minor but important differences in DNA-binding affinities. In view of these potential complications, it will be instructive to determine whether the in vivo binding of BRM mutant proteins to chromatin targets is diminished (e.g. using ChIP assays).
No definitive evidence for C/EBP-E2F complexes bound to E2F sites via the E2F moiety has been reported to date. Biochemical characterization of such a complex, including identification of the E2F-family members involved, would greatly strengthen the model, as would further mutational studies of the E2F and C/EBP
proteins to identify sequences that mediate their interaction. Also, it is possible that C/EBP
-binding sites are present in some S-phase gene promoters and these might mediate the antiproliferative effects of C/EBPs. We recently identified a C/EBP site in the murine DHFR promoter juxtaposed to the E2F element that is required for transcriptional repression by C/EBPß in reporter assays (T. Sebastian, S. Thomas, J. Sage and P.F.J., unpublished). Further investigation should reveal whether other S-phase genes also contain previously unrecognized C/EBP-binding sites.
The C/EBP-E2F interaction model predicts that RB proteins are dispensable for C/EBP
-induced proliferation arrest, whereas current dogma holds that RB-E2F complexes are the ultimate effectors of most, if not all, cellular pathways that regulate G1 arrest (Sage et al., 2000
). Recent observations from our laboratory indicate that cell-cycle arrest induced by C/EBP
or C/EBPß in primary fibroblasts requires RB-E2F complexes. MEFs lacking all three pocket proteins (i.e. triple-knockout, or TKO, cells) do not undergo growth arrest following ectopic expression of either C/EBP protein, whereas wild-type cells cease to proliferate (T. Sebastian, S. Thomas, J. Sage and P.F.J., unpublished). This is consistent with earlier studies showing that C/EBPß physically and functionally interacts with pRB (Chen et al., 1996a
; Chen et al., 1996b
). Moreover, expression of a dominant-negative E2F-1 protein (i.e. lacking transactivation and RB-binding sequences) in wild-type MEFs similarly disrupts C/EBP-induced proliferation arrest. Interestingly, in cells lacking RB or E2F function, over-expression of C/EBP stimulates, rather than inhibits, cell proliferation. These observations demonstrate a clear requirement for RB proteins and E2F in C/EBP-induced cell-cycle arrest and, as such, raise questions about the C/EBP-E2F co-repressor model, at least in terms of an RB-independent mechanism. It is also possible that the RB-E2F-dependent C/EBP
effector pathway is cell specific and that different mechanisms of C/EBP
-mediated cell-cycle arrest operate in non-fibroblastic cells.
Auxiliary factors
Additional clues as to the mechanism of C/EBP-induced growth arrest have come from studies to examine cellular pathways required for cell-cycle exit. A combination of biochemical experiments and genetic studies indicates that the SWI/SNF chromatin-remodeling complex (Eberharter and Becker, 2004
) is necessary for C/EBP
-regulated cell-cycle exit. Pedersen et al. (Pedersen et al., 2001
) found that the TE-III region of C/EBP
mediates docking with the SWI/SNF complex and that SWI/SNF binding is necessary for C/EBP
to induce adipocyte differentiation and to activate expression of adipocyte- or myeloid-specific target genes. Subsequently, Iakova et al. (Iakova et al., 2003
) reported that livers of old animals contain a high-molecular-weight C/EBP
complex that contains E2F4, Rb and the Brm (Brahma) subunit of the SWI/SNF complex. This complex is less abundant in livers of young mice and its appearance seems to correlate with the impaired regeneration of aging liver tissue following partial hepatectomy. The C/EBP
-E2F4-Rb-Brm complex is associated with the Myc promoter in vivo and may confer a stable form of repression on Myc and other growth-promoting genes that is extremely difficult to reverse. This might prevent hepatocytes in older animals from undergoing proliferation and regeneration following liver damage. Irreversible cell-cycle arrest is a defining feature of senescent cells, whose numbers increase during aging. Thus, it is tempting to speculate that C/EBP
plays an important role in establishing the senescent state in cells such as hepatocytes.
Muller et al. (Muller et al., 2004) expressed C/EBP
in human cell lines that lack endogenous Brm. These lines cannot undergo C/EBP
-induced cell-cycle exit; however, expression of Brm restores this ability. In addition, siRNA-induced ablation of endogenous Brm in a SWI/SNF-positive cell line blocks the antiproliferative effects of C/EBP
. These results, together with the previous studies, establish a clear requirement for the SWI/SNF chromatin-remodeling activity in C/EBP
-dependent cell-cycle arrest. The involvement of chromatin-remodeling activity tends to support a transcriptional mechanism for C/EBP
action. The antiproliferative activity of pRB likewise involves SWI/SNF function (Muchardt and Yaniv, 2001
), lending further credence to the idea that C/EBP
acts in combination with RB proteins to impose G1 arrest.
Signaling to C/EBP
Given its strong antiproliferative activity, C/EBP is likely to be regulated dynamically in cells. Wang et al. (Wang et al., 2004
) have observed that C/EBP
levels remain relatively high in proliferating hepatocytes following partial hepatectomy, as well as in liver tumor cells, indicating that the anti-mitotic effects of C/EBP
might be suppressed under certain conditions. A putative phosphorylation site in murine C/EBP
, Ser193 (Fig. 2), appears to be essential for its growth arrest activity. Mutational analysis showed that this residue is required for C/EBP
to bind to CDK2/CDK4 and also to Brm. Signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces dephosphorylation of Ser193, probably through increased nuclear accumulation of the PP2A phosphatase. Ser193 dephosphorylation thus disengages two major C/EBP
growth arrest effector pathways. A mutant containing the S193A substitution still activates transcription in reporter assays, again suggesting that the growth arrest and transcriptional activation functions of C/EBP
are separable activities. At first glance, these results appear to contradict the findings showing that SWI/SNF binding is required for C/EBP
to induce transcription of endogenous differentiation-specific genes. However, transcriptional activation of chromatin-embedded genes is likely to require chromatin remodeling, whereas this may be dispensable for transactivation in transient reporter assays. It will be informative to test whether the S193A mutant can induce differentiation in adipocytes and myeloid cells.
Previous work showed that insulin-mediated activation of the PI3K-Akt pathway in adipocytes induces C/EBP dephosphorylation on Thr222 and Thr226, substrates for glycogen synthase kinase 3 (GSK3) (Ross et al., 1999
). GSK3 activity is suppressed by engagement of the insulin-PI3K-Akt pathway, which also activates the PP1 and PP2A phosphatases. Both reduced GSK3 activity and enhanced phosphatase activity may therefore contribute to C/EBP
dephosphorylation; however, the functional consequences of phosphorylation/dephosphorylation on Thr222 and Thr226 are unknown. The possibility of cross-talk between the GSK3 sites and Ser193 is attractive, especially since the PI3K/Akt pathway impinges on both targets, but this idea lacks experimental evidence. Since the kinase that modifies Ser193 is unknown, identification of this protein will be an important advance in our understanding of the pathways that modulate C/EBP
activity.
Because PI3K-Akt signaling is associated with hepatocyte proliferation during liver regeneration and occurs in hepatoma cells, Wang et al. (Wang et al., 2004) have suggested that C/EBP
deactivation is a major target of this pathway in liver tissue. If this is the case, strategies aimed at reactivating C/EBP
by disrupting this post-translational inhibitory mechanism could be another promising avenue for cancer therapy.
Another phosphoacceptor in C/EBP, Ser21, is a direct substrate of ERK1/2 (Ross et al., 2004
). Phosphorylation of Ser21 blocks the ability of C/EBP
to induce granulopoiesis in bipotential myeloid progenitor cell lines but does not affect monocyte differentiation or adipogenesis. This modification also appears to induce a conformational shift in C/EBP
dimers. The effect of Ser21 phosphorylation on cell proliferation has not been described, and it will be interesting to determine its role, if any, in cell-cycle arrest and to elucidate the molecular mechanism by which it affects lineage-specific cellular differentiation.
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Antiproliferative activities of other C/EBP-family members |
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C/EBPß
Forced expression of C/EBPß in HepG2 hepatocarcinoma cells arrests the cells at or near the G1-S boundary (Buck et al., 1994). This effect requires both the bZIP domain and the N-terminal transactivation sequences, and is not reproduced by the transcriptionally inert LIP isoform. Moreover, C/EBPß-knockout mice display a lymphoproliferative disorder, which suggests that C/EBPß inhibits expansion of the lymphoid cell compartment (Screpanti et al., 1995
). Several observations also indicate an antiproliferative function for C/EBPß in epidermal keratinocytes: (1) expression of C/EBPß in BALB/MK2 keratinocytes inhibits colony formation; (2) mice lacking C/EBPß exhibit mild epidermal hyperplasia; and (3) keratinocytes derived from C/EBPß mutant mice display partially defective Ca2+-induced growth arrest in vitro (Zhu et al., 1999
). Expression of the differentiation markers keratin 1 (K1) and K10 is also reduced in these cells, indicating that C/EBPß regulates proliferation arrest as well as differentiation-specific genes. Thus, C/EBPß contributes to growth arrest and differentiation of keratinocytes, which is similar to the dual functions of C/EBP
in terminally differentiating cells.
Ectopic expression of C/EBPß in primary fibroblasts induces cell-cycle exit through a mechanism requiring RB-E2F activity (T. Sebastian, S. Thomas, J. Sage and P.F.J., unpublished). C/EBPß-arrested fibroblasts exhibit morphological features that are indicative of senescent cells. A similar senescent state is also provoked by over-expression of RasV12 and other oncogenes in primary, non-immortalized cells (Lin and Lowe, 2001; Serrano et al., 1997
). RasV12-expressing primary cells enter an irreversible G1 arrest that requires induction of the p16INK4a-RB and p19ARF-p53 tumor suppressor pathways. This oncogene-induced, `premature' senescence is thought, like apoptosis, to provide a barrier to neoplastic transformation and cancer. Interestingly, RasV12-expressing C/EBPß/ MEFs fail to senesce and instead continue to proliferate, although (in contrast to p19ARF- or p53-deficient cells) they are not fully transformed (T. Sebastian, S. Thomas, J. Sage and P.F.J., unpublished). C/EBPß seems to function in concert with RB-E2F to arrest cells at the G1-S boundary, probably by repressing S-phase genes. Thus, C/EBPß is an important regulator of cell-cycle exit/senescence induced by RasV12 in primary cells. Post-translational activation of C/EBPß by Ras-stimulated kinases (Hanlon and Sealy, 1999
; Mo et al., 2004
; Nakajima et al., 1993
; Shuman et al., 2004
) may be one pathway by which oncogenic RasV12 signaling is linked to premature senescence.
Growth arrest induced by C/EBPß is highly context specific, because in several cases C/EBPß displays growth-promoting activity. For example, mammary epithelial cells (MECs) from C/EBPß/ female mice have a proliferation defect that leads to impaired ductal morphogenesis and a failure to lactate (Robinson et al., 1998; Seagroves et al., 1998
). Conversely, ectopic C/EBPß expression in a human MEC cell line (MCF10A) induces hyper-proliferation and the cells acquire a partially transformed phenotype (Bundy and Sealy, 2003
). A growth-stimulatory effect of C/EBPß is also observed in macrophage tumor cells (Wessells et al., 2004
). Interestingly, deletion of the CEBPB gene renders mice totally resistant to carcinogen-induced skin tumor development (Zhu et al., 2002
), and low levels of C/EBPß expression enhance, rather than inhibit, RasV12-induced focus formation in NIH 3T3 cells (Shuman et al., 2004
; Zhu et al., 2002
). A data-mining approach has also associated C/EBPß expression with cyclin-D1-dependent tumors (Lamb et al., 2003
). Hence, C/EBPß can function as a pro-oncogenic transcription factor that promotes proliferation and/or survival of some tumor cells. How it stimulates mitotic growth and why it elicits completely opposite effects on proliferation in different cellular contexts are intriguing questions for future investigation.
C/EBP
DeWille and coworkers have described a role for C/EBP in G0 arrest of MECs. Proliferating HC11 or COMMA D mammary cells express low levels of C/EBP
, but when they undergo G0 arrest in response to serum withdrawal, C/EBP
mRNA and protein levels increase significantly (O'Rourke et al., 1997
; O'Rourke et al., 1999
). The effect is specific to G0 MECs, since C/EBP
levels do not increase in other cells, nor is induction observed when MECs arrest at other stages of the cell cycle. Expression of C/EBP
antisense RNA inhibits endogenous C/EBP
expression and prevents the cells from entering G0 upon serum withdrawal. Further experiments have implicated the transcription factor STAT3 in activating CEBPD gene transcription in response to low levels of serum (Hutt et al., 2000
). A cytokine or related factor, acting in an autocrine manner, might therefore control CEBPD induction by activating Jak/STAT3 signaling. Indeed, oncostatin M induces growth arrest in MECs by a C/EBP
-dependent mechanism (Hutt and DeWille, 2002
).
C/EBP also regulates proliferation of MECs in vivo. C/EBP
is induced during mouse mammary gland involution, which is the period following the end of lactation when extensive tissue remodeling and regression occur (Gigliotti and DeWille, 1998
). Female C/EBP
-knockout mice reproduce and lactate normally (Sterneck et al., 1998
); however, nulliparous mutant animals display increased mammary ductal growth and branching, and show a higher epithelial bromodeoxyuridine (BrdU) labeling index than wild-type controls (Gigliotti et al., 2003
). Involution and associated cellular apoptosis was not affected, although other work shows that these responses are delayed in C/EBP
/ animals (E. Sterneck, personal communication). The different results might reflect the fact that C/EBP
/ mice of different strain backgrounds were used in the two studies. The involvement of C/EBP
in control of MEC proliferation raises the possibility that C/EBP
functions as a tumor suppressor. It will therefore be interesting to determine whether tumor-associated C/EBP
mutations exist and contribute to the transformed phenotype of cancer cells.
C/EBP
C/EBP is expressed exclusively in hematopoietic cells and their progenitors. C/EBP
-null mice are viable but lack functional neutrophils and eosinophils, and eventually develop myelodysplasia (Verbeek et al., 2001
; Yamanaka et al., 1997
). Conversely, forced expression of C/EBP
induces differentiation of promyelocytic leukemia cells (Lekstrom-Himes, 2001
; Truong et al., 2003
). These findings indicate a role for C/EBP
in differentiation and proliferation arrest of myeloid progenitors. The antiproliferative activity of C/EBP
might involve E2F-RB, since interactions with E2F1 and pRB have been observed and C/EBP
can repress transcription of E2F targets such as Myc (Gery et al., 2004
). C/EBP
thus mimics many of the functions of C/EBP
and may provide a second pathway by which terminal granulocytic differentiation is implemented (Zhang et al., 2002
).
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Conclusions and perspectives |
---|
With the importance of C/EBP-family members in the control of cell proliferation now well established, the next challenge is to acquire a more comprehensive description of C/EBP targets and biological activities. The availability of increasingly sophisticated tools to identify target genes, binding sites in chromosomal DNA, protein-protein interactions and protein modifications, combined with powerful genetic and computational methods, should soon provide significant advances in our knowledge of how C/EBP proteins regulate cell proliferation and differentiation. Thus, we can anticipate the eventual characterization of `regulons' for each C/EBP-family member in different cell types and cellular states (e.g. proliferating versus arrested, normal versus neoplastic), as well as networks of protein interactions and regulatory pathways that control their activities. This information should yield answers to several important questions. Do different C/EBP-family members regulate distinct sets of target genes in the same cell and, if so, how is this specificity achieved? What combinatorial relationships exist between C/EBPs and other transcription factors to establish cell-specific transcriptional programs? What assemblies of co-activators or co-repressors are involved in determining positive or negative regulation of target genes? How do C/EBP proteins contribute to disease states such as cancer and inflammation? The challenge ahead for researchers in the field is to adopt these new research tools and technologies, and also to develop cooperative networks so that the large, complex data sets generated from these experiments can be shared and thus used to maximum effect.
![]() |
Acknowledgments |
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