Activation Domains of CCAAT Enhancer Binding Protein
: Regions Required for Native Activity and Prostaglandin E2-Dependent Transactivation of Insulin-Like Growth Factor I Gene Expression in Rat Osteoblasts
Changhua Ji,
Weizhong Chang,
Michael Centrella and
Thomas L. McCarthy
Department of Surgery, Plastic Surgery Section, Yale University School of Medicine, New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: Thomas L. McCarthy, Ph.D., Department of Surgery, Plastic Surgery Section, Yale University School of Medicine, P.O. Box 208041, New Haven, Connecticut 06520-8041. E-mail: thomas.mccarthy{at}yale.edu.
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ABSTRACT
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In osteoblasts, hormones such as prostaglandin E2 that activate protein kinase A increase the translocation of transcription factor CCAAT/enhancer binding protein
(C/EBP
) from the cytoplasm to the nucleus where it rapidly induces IGF-I gene expression. In this study, we identified activation and suppression domains in C/EBP
using native and heterologous gene promoter assay systems. We demonstrated functional interactions between C/EBP
and trans-gene-expressed cAMP response element binding protein-binding protein, and showed that the ability of C/EBP
to promote gene expression was suppressed by viral protein E1A, which blocks the activity of native cAMP response element binding protein-binding protein. Site-directed mutations at serines 62 or 191 within C/EBP
reduced its basal transcriptional activity, whereas mutation at serine 191 suppressed the stimulatory effect of prostaglandin E2 on C/EBP
function as well as its DNA binding potential. These results are consistent with the location of serine 191 in the DNA binding domain of C/EBP
. Our studies provide the first evidence for regions of C/EBP
that are important for basal and for hormone-induced transcriptional activity, and for its interactions with other enhancers and suppressers of gene expression.
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INTRODUCTION
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THE CCAAT/ENHANCER BINDING proteins (C/EBPs) comprise a family of six known basic leucine zipper transcription factors that regulate a diversity of genes, including those associated with tissue differentiation, the acute phase response, and many viruses (1, 2). For example, expression of the adipocyte phenotype in 3T3-L1 cells coincides with an orderly transition of C/EBP isoform synthesis, progressing from C/EBP
to C/EBPß, to a point where terminal differentiation requires C/EBP
expression. Homozygous gene deletion studies in mice further revealed that C/EBP
and C/EBPß double-knockout and C/EBP
single-knockout mice tend to die perinatally with defects in lipid storage, carbohydrate metabolism, and thermal regulation (3, 4, 5). Similarly, a C/EBP isoform cascade results in C/EBPß and C/EBP
expression by differentiated liver cells (6, 7). In contrast to cells from these tissues and to situations associated with the acute phase response to infection and inflammation, differentiated osteoblasts express high levels of C/EBP
in a constitutive manner (8, 9). In this context, we showed that transcription factor Runx2 (previously termed AML-3, Cbfa1, or PEBP2
A1), which is essential for development of the osteoblast phenotype, maintains and regulates C/EBP
expression in native and in prostaglandin E2 (PGE2)-activated osteoblasts (9).
C/EBP
is a strong transcriptional activator. It accounts largely for protein kinase A (PKA)-dependent-activation of IGF-I gene expression in primary cultures of rat osteoblasts (8, 10). A single, high-affinity cis-acting element for C/EBP
occurs in the transcribed, noncoding exon 1 of the rat IGF-I gene (+202 to +209), and mutation or deletion of this element alone eliminates PKA-dependent induction of IGF-I in osteoblasts (11). Locally produced IGF-I is a key growth factor in longitudinal bone growth and the anabolic response to hormones such as PTH and PGE2 that activate PKA, and are themselves principal activators of bone remodeling (12, 13). Therefore, IGF-I and C/EBP
appear to be important and perhaps essential mediators of hormone-activated bone formation.
Whereas earlier studies identified several functional domains within the C/EBP
, C/EBPß, C/EBP
, and C/EBP
isoforms, little is known about activation and repression domains in C/EBP
(14, 15, 16, 17). Among the C/EBP gene family members, considerable sequence homology exists in the carboxyl-terminal DNA binding (basic region) and dimerization (leucine zipper) domains. Although two-thirds of the amino-terminal region of the six principal C/EBP family members diverge in primary structure, several conserved motifs emerge from sequence analysis. In this study we defined activation and repression domains in C/EBP
and identified critical amino acids that independently regulate C/EBP
-dependent transactivation of the IGF-I gene promoter in unstimulated and PGE2-induced osteoblasts.
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RESULTS
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Activation and Repression Domains in C/EBP
To study functional domains of C/EBP
independently of its ability to dimerize and bind to native regulatory DNA sequences, we used a mammalian one-hybrid assay system. Functional domains of C/EBP
were first localized by preparing eukaryotic expression constructs that encode fragments of C/EBP
fused carboxyl to a Gal4 DNA binding domain (DBD) (designated M1
). This vector also contains a nuclear localization signal that facilitates the analysis independently of constraints that retain C/EBP
in the cytoplasm in uninduced osteoblasts (29). Cells were transiently transfected with a C/EBP
fusion plasmid construct in combination with the 5X Gal4-Luc reporter plasmid. In this way, the C/EBP
fragment supplies transcriptional regulatory domains that potentially control reporter gene expression. Initial analysis identified a strong activation domain between amino acids 59 and 76 of C/EBP
, and maximal activity by the first 102 amino acids. This construct induced high-level reporter gene expression, essentially equivalent to a plasmid containing the Gal4 DBD fused to the activation domain of herpes simplex viral protein 16 (MVP16; Fig. 1A
). C/EBP
protein fragments longer in sequence than the 102 amino-terminal amino acids showed reduced reporter expression, consistent with the presence of repression domains. These differences did not relate to variations in expression efficiency, since similar levels of each C/EBP
fragment were detected by Western blot analysis in transfected cell nuclear extracts (Fig. 1B
).
To identify the activation domain of C/EBP
more precisely, constructs were generated by PCR to span the region between amino acids 59 and 102. Whereas virtually no transcriptional activity was induced with any fragment up to amino acid 63, the next five amino acids imparted a large increase in C/EBP
function. Small increments in activity occurred by addition of various amino acid fragments up to position 102 (Fig. 2A
), but only fragments encoding 193 or 1102 were significantly greater in activity by comparison to M1
168. Internal deletions were then used to verify the region that spanned this strong activation domain. Deletion of amino acids 2340 in M1
122/41102 had no effect, whereas loss of amino acids 7793 in M1
176/94102 reduced activity only marginally. Deletion of amino acids 4168 in M1
140/69102 or 5268 in M1
151/69102 produced far greater losses in reporter expression, while near basal activity was detected by elimination of amino acids 5276 and 6076 in constructs M1
151/77102 and M1
159/77102 (Fig. 2B
). Together, these results identify an important domain between amino acids 4176 that contribute the majority of C/EBP
activation, with a critical function for amino acids 6068.
C/EBP
fusion constructs spanning amino acids 102180 confirmed the presence of an endogenous suppression domain between amino acids 103 and 116 (Fig. 3A
). When fragments from this region were examined for their ability to regulate heterologous gene expression by fusion to the DBD/VP16 fusion protein, suppression within this context required a slightly larger protein fragment that included amino acids 117130 (Fig. 3B
). Therefore, a minimal repressor domain occurs in this region of C/EBP
. However, it is more active within the context of C/EBP
itself, perhaps from structural constraints that occur with the amino-terminal portion of endogenous C/EBP
molecule, but not with the heterologous recombinant DBD/VP16 fusion protein.
Interactions between C/EBP
and cAMP Response Element Binding Protein (CREB)-Binding Protein (CBP)
Earlier studies demonstrated a functional relationship between C/EBPß and the large multivalent coactivator protein termed CBP (30, 31). Whereas considerable sequence homology exists between C/EBPß and C/EBP
, functional interaction between C/EBP
and CBP has not yet been demonstrated. To do so, cells were cotransfected with expression plasmids encoding either full-length C/EBP
or its amino-terminal 102-amino acid fragment each fused to Gal4 DBD, in combination with CBP and the 5X GAL4-luciferase reporter. CBP significantly increased C/EBP
-dependent reporter gene expression in a dose-dependent way. The 3- to 4-fold stimulatory effect of CBP on C/EBP
is similar to or greater than that previously reported for C/EBPß (30). Slightly more CBP expression was required to activate the C/EBP
truncation fragment M1
1102. It is important to note that M1
1102, which eliminates the carboxyl-terminal repressor domain, has approximately 30-fold higher basal activity by comparison to full-length M1
. Even within this context, however, M1
1102 activity was enhanced by approximately 2-fold (Fig. 4A
). Interactions with CBP did not occur with the repressor, basic region/DBD, or leucine zipper domains (data not shown).

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Fig. 4. Functional Interaction between C/EBP and CBP
In panel A, osteoblasts were cotransfected with a Gal4 DBD-C/EBP fusion plasmid encoding full-length C/EBP (M1 , on the left) or C/EBP amino acids 1102 (M1 1102, on the right), increasing amounts of expression plasmid encoding CBP, and 5X Gal4 reporter plasmid. In panel B, osteoblasts were cotransfected with M1 and 50 ng/well of expression plasmids encoding CBP and/or native viral protein E1A, or dominant negative mutated E1Am, and 5X GAL4 reporter plasmid. Basal effects by the C/EBP expression constructs were assessed in untreated cultures. Data are means ± SE from a minimum of nine samples from three separate experiments, corrected for protein content and relative transfection efficiency. * Indicates a statistically significant increase in gene expression compared with no CBP in panel A, or to no E1A or CBP coexpression in panel B. ** Indicates a statistically significant difference compared with M1 + CBP in panel B, P < 0.05. In panel C, nuclear extract from COS-7 cells, cotransfected to express C/EBP and CBP and treated for 4 h with forskolin to activate PKA, was incubated with nonimmune mouse IgG (left lane) or monoclonal antibody to CBP (right lane) and collected with protein G-agarose. Samples were fractionated by SDS-PAGE, electroblotted, and probed with antibody to C/EBP .
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The viral protein E1A binds to a domain of CBP that also has been reported to bind C/EBPß. In this way, E1A disrupts a functional association between CBP and C/EBPß or other transcription factors and thereby selectively enhances viral protein expression. Indeed, expression of native E1A suppressed basal gene transactivation by the C/EBP
fusion protein, whereas mutant E1A (E1Am), which has reduced ability to bind CBP, was ineffective. Moreover, native E1A also fully suppressed the stimulatory effect of CBP on C/EBP
-dependent gene transactivation, but E1Am was significantly less effective (Fig. 4B
). Nuclear extract from COS-7 cells cotransfected to expressed CBP and C/EBP
, collected with antibody to CBP and probed by Western blot analysis with antibody to C/EBP
, revealed coprecipitation of C/EBP
, consistent with a physical interaction between these proteins (Fig. 4C
).
PGE2-Sensitive Domains in C/EBP
Earlier studies in rat osteoblasts showed that a single C/EBP binding site at nucleotides +202 to +209 within exon 1 of the rat IGF-I gene confers a PKA-dependent stimulatory effect by PGE2 on IGF-I gene promoter activity (11). Furthermore, rat osteoblasts constitutively express C/EBP
, although it remains in the cytoplasm from which it translocates to the nucleus after activation by PGE2 (8). The identity and specificity of C/EBP
binding by EMSA were previously demonstrated with homologous and mutated DNA oligomers and by antibody supershift studies (8, 10, 11). To assess amino acids that may account for C/EBP
activation in this way, point mutations were introduced into full-length C/EBP
cloned within the M1 expression construct. Recombinant constructs were then tested for gene-promoting activity in combination with the rat IGF-I promoter 1 reporter plasmid IGF-I711b-Luc. Potentially important sites for mutation were chosen based on the transcription activation domain between amino acids 4176, and on potential kinase substrates that are conserved among C/EBP
, C/EBPß, and C/EBP
at amino acids 191 and 208 (14, 15, 16). In uninduced cells, overexpression of native C/EBP
(M1
1268) significantly enhanced IGF-I promoter activity, as we reported previously (8, 10). C/EBP
constructs containing serine-to-alanine substitutions at ser 62 or ser191 exhibited a significant approximately 50% decrease in their ability to drive the IGF-I gene promoter. In contrast, substitution of tyr64 to ala64 had no significant effect on basal gene promoter activity (Fig. 5A
). In cells stimulated with PGE2, only the serine-to-alanine substitution at ser191 effectively suppressed C/EBP
activity (Fig. 5B
). Each native or mutated construct produced nearly equivalent protein, as assessed by Western blot analysis (Fig. 5C
). However, by EMSA with oligonucleotide containing the C/EBP-binding element from the rat IGF-I promoter, only the serine-to-alanine substitution at ser191 reduced C/EBP
binding to DNA (Fig. 5
, C and D). Notably, ser191 resides in the DBD of C/EBP
.
Recombinant mutated C/EBP
fusion proteins were tested in parallel cultures for their ability to regulate gene expression by the heterologous 5X Gal4-Luc promoter/reporter. This assay does not require DNA binding by C/EBP
through its own DBD, allowing an independent assessment of the importance of these amino acid substitutions. In contrast to their inhibitory effects on IGF-I gene promoter activity, mutations at ser62 or ser191 did not suppress C/EBP
activity in control or PGE2-stimulated cells (Fig. 6
). While the activity of C/EBP
with a native gene promoter is disrupted significantly by these mutations, similar effects do not occur in situations that bypass the need for a functional C/EBP
DBD. Therefore, native C/EBP
-dependent gene promoter activity may require changes in conformation or phosphorylation status that do not occur in these mutated C/EBP
s.
Finally, ser67 resides just within the core of the potent activation domain of C/EBP
that we defined in Figs. 1
and 2
. Unlike C/EBP
with a mutation at ser191, when C/EBP
with a ser67-to-ala67 mutation was transfected into osteoblasts, its effect on basal or PGE2-stimulated IGF-I promoter (1711b) or the heterologous 5X Gal4-Luc promoter/reporter was not reduced by comparison to native C/EBP
(Fig. 7
).
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DISCUSSION
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C/EBP transcription factor family members contain similar carboxyl-terminal DBD, nuclear localization (basic region), and dimerization (leucine zipper region) domains. In contrast, the amino-terminal transcriptional regulatory domains of the C/EBPs diverge considerably, although sequence analysis of these regions reveals several conserved, potential phosphorylation motifs. Functional domains within C/EBP
, C/EBPß, C/EBP
, and C/EBP
have been previously identified, but much less is currently known about activation and repression domains within C/EBP
. In this study, we identified activation and repression domains within C/EBP
in several ways, using primary cultures of rat osteoblasts that are extremely sensitive to changes in C/EBP
expression and activation. A strong transcriptional activation domain occurs within amino acids 4176, and a somewhat broad and less potent domain occurs beyond this region through amino acid 102. It was not possible, however, to identify the true boundaries of the activation domain, since little or no activity was detected when partial or incomplete domains were examined. Consistent with several C/EBP isoforms, C/EBP
also has a repressor domain that can modify its transcription potential in the context of unactivated cells. Data from two different analyses show a functional repressor region of C/EBP
between amino acids 116 and 130.
In several C/EBP isoforms, the activation and repressor domains are independent of the basic DBD and nuclear localization motifs, and the carboxyl-terminal leucine zipper, where dimerization with other bZIP transcription factors occurs. For example, C/EBP
has two activation domains separated by a central attenuator domain (14), and C/EBPß has three small activation domains that precede two repressor domains (15). C/EBP
has a distinct activation domain in its amino terminus, and a repressor domain adjacent to the basic region of the protein (16), and therefore, C/EBP
and C/EBP
appear to share a more similar structural organization.
C/EBPß binds to the transcriptional integrator molecule CBP, which potentiates gene transcription. We found that C/EBP
activity increased in cells transfected to overexpress CBP, and that this occurred to a similar or greater extent than was earlier reported for C/EBPß (30). Coimmunoprecipitation of C/EBP
with CBP indicates that this association is both physical and functional. Moreover, viral protein E1A successfully competed with both C/EBP
and C/EBPß (Ref.30 , present study, and other data not shown) for coactivation by CBP. This suggests that these C/EBPs contain common domains that bind to the COOH-terminal region of CBP where E1A associates. Furthermore, other factors that also bind CBP in this region may compete with and suppress C/EBPß or C/EBP
activity. The steroid receptor coactivator 1 can enhance transcription induced by some steroid hormone receptors such as estrogen receptor-
. The function of steroid receptor coactivator 1 is also inhibited by the viral protein E1A (32, 33), indicating that it too may share a CBP-binding domain with C/EBPß and C/EBP
. This suggests a potential mechanism for the counteracting effects of estrogen on PKA-activated IGF-I expression in osteoblasts (25).
The C/EBP
activation domain fused to Gal4 DBD (M1
1102) by itself potently activates transcription, comparable to viral protein VP16. However, full-length C/EBP
promotes far less gene expression in the Gal4 DBD-dependent one-hybrid assay by comparison to M1
1102. This may occur from secondary or tertiary structural restrictions of full-length C/EBP
that limit its strong activation potential. The activation domain of C/EBP
may be unmasked by dimerization or by phosphorylation, which themselves may be codependent events. Furthermore, we showed here that C/EBP
has a downstream repression domain similar to other C/EBP isoforms. When fused directly to VP16, this region of C/EBP
partially repressed the potent activation domain of VP16, suggesting that it acts, at least in part, independently of the C/EBP
activation domain.
Potential phosphorylation sites that could regulate DNA binding or gene activation are present in several C/EBPs. Those found in C/EBP
occur within motifs that may be sensitive to various kinases. These include PKA (arg-x-ser-x, at ser191 and ser208, PKC (lys/arg1-3-x0-2-ser/thr-x0-2-arg/lys1-3, at ser208 and ser241, ras-dependent MAPK (pro-x-ser/thr-pro, at thr19, thr106, thr156, ser160, and ser256, and CaM kinase or Ca2+/calmodulin-dependent protein kinase (arg-x-x-ser/thr-x, at ser113). We earlier reported an essential role for PKA with regard to the stimulatory effect of C/EBP
on IGF-I gene expression in osteoblasts, and we therefore focused on potential PKA-sensitive sites in this study (24). The basal activity of full-length C/EBP
was reduced by ser-to-ala substitutions at amino acids 62 and 191. Whereas ser191 falls within a PKA-sensitive motif, ser62 does not reside within a previously characterized protein kinase target sequence. A similar motif encompassing ser240 is thought to permit PKA-dependent phosphorylation of C/EBPß (34). Ser191 is conserved in its relative location among the known C/EBP isoforms. Importantly, ser191 resides within the basic region of C/EBP
, comprising nuclear-localization domains and DBDs. A decrease in C/EBP
-dependent activation of the IGF-I promoter by mutation at this site is consistent with reduced binding to its cognate DNA-binding element in the IGF-I gene. The presence of an exogenous nuclear-localization domain from the Gal4 DBD did not rescue PGE2-induced activation of the IGF-I promoter by M1-C/EBP
ala191, consistent with the likelihood that reduced DNA binding, rather than nuclear translocation, significantly compromises promoter transactivaton. Addition of an epitope tag, like the GAL4 DBD, may modify protein-to-protein or protein-to-DNA interactions, and influence transactivation or binding to cognate elements, as in an EMSA. However, no generalized interference was observed among multiple GAL4-DBD fusion constructs, reducing concern for allosteric effects that the tag may have engendered to M1-C/EBP
ala191 or other mutated or wild-type C/EBP
fusion proteins.
Rat C/EBP
contains 20 serines, but only ser191 and ser208 are conserved among the various C/EBPs. Moreover, ser67 resides within the potent transactivation domain of C/EBP
that we defined in this study, but mutation at this site also failed to reduce basal or PKA-stimulated gene transactivation. Nevertheless, other serines, alone or in combination, may regulate C/EBP
activity in the basal state or in response to select kinase activators in osteoblasts or alternate cell systems. Studies to address additional potential phosphorylation sites will be an important goal for further analyses.
In osteoblasts, activation of PKA increases cytoplasmic-to-nuclear translocation of C/EBP
(29). C/EBP
expression initially appeared limited to the acute-phase response, or to an early phase of tissue differentiation. However, we recently reported that the C/EBP
gene promoter contains a regulatory element that is sensitive to the osteoblast-restricted transcription factor Runx2, which itself is activated by PGE2 (9). A physical interaction between the DBDs and leucine-zipper domains of C/EBP
and the Runt homology domain of Runx2 also appears to limit C/EBP
promoter activity, establishing an efficient inhibitory feed-back loop in C/EBP
expression when sufficiently high C/EBP
levels are achieved.
In summary, C/EBP
, a key regulator of IGF-I expression in osteoblasts, rapidly translocates to the nucleus and activates gene expression after PGE2 treatment in a PKA-dependent way. Potent transcriptional activation domains within the amino terminus of C/EBP
span amino acids 41102. In addition, ser191, which occurs within a PKA-sensitive domain in C/EBP
, appears essential for the stimulatory effect of PGE2 on IGF-I. Ser191 also locates within the DBD of C/EBP
. Mutation at this site reduced C/EBP
binding to its regulatory element, as well as basal and PGE2-induced IGF-I gene promoter activity. However, the C/EBP
/Gal4 fusion protein containing this mutation readily translocated to the nucleus and enhanced gene expression when DNA binding occurred through the Gal4 DBD sequence rather than through the C/EBP
DBD, demonstrating that this site is not itself essential for gene transactivation. Therefore, the stimulatory PKA-dependent effect of PGE2 appears to be required for both nuclear translocation and binding of native C/EBP
to DNA and, in this way, enhances new IGF-I gene transcription.
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MATERIALS AND METHODS
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Cell Cultures
Cells were prepared from parietal bones of 22-d-old Sprague Dawley rat fetuses (Charles River Breeding Laboratories, Raleigh, NC) by methods approved by the Yale Animal Care and Use Committee. Sutures were eliminated by dissection, and cells were released by five sequential 20-min collagenase digestions, as described previously (18). Cells from the last three digestions exhibit biochemical characteristics associated with differentiated osteoblasts, including high levels of PTH receptors and type I collagen synthesis, and a rise in osteocalcin expression in response to 1,25-(OH)2D3. Histochemical staining demonstrates that approximately 80% of the cells express alkaline phosphatase (McCarthy, T. L., and M. Centrella, unpublished data). By these criteria, differential sensitivity to TGF-ß, bone morphogenetic protein-2, various PGs, and the ability to form mineralized nodules in vitro (19, 20, 21), osteoblast-enriched cultures are easily distinguished from less differentiated periosteal cells. Cells pooled from the last three digestions were plated at 4800 per cm2 in DMEM containing 20 mM HEPES (pH 7.2), 100 µg/ml ascorbic acid, penicillin, and streptomycin (Life Technologies, Gaithersburg, MD), and 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO). Treatments were in serum-free medium.
Plasmids
Transfection plasmids for mammalian one-hybrid gene transactivation studies were obtained from Dr. Ivan Sadowski (University of British Columbia) and Dr. Richard Maurer (Oregon Health Sciences University) (22, 23). Full-length or fragments of C/EBP
were subcloned in frame with the Gal4 DBD peptide sequence within an simian virus 40 promoter-driven expression plasmid by standard restriction digestion or PCR methods. The 5X GAL4-E1b-Luciferase promoter/reporter construct (5X GAL4-Luc) was used as a detection system. In this assay, reporter gene expression requires interaction between the trans-acting Gal4-DBD fused to potential activation domain region(s) of C/EBP
and the cis-acting Gal4 DNA binding element upstream of the luciferase gene. Some transcription repression studies used a positive control expression vector containing Gal4 DBD and the herpes simplex virus protein VP16 activation domain (DBD/VP16) fused in frame to fragments of C/EBP
. Expression constructs encoding CBP and wild-type or mutant E1A viral protein were kindly provided by Drs. R.H. Goodman and Xin-Hua Feng (Oregon Health Sciences University and Baylor College of Medicine, respectively). Single amino acid substitutions were introduced with the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) and verified by sequence analysis. The rat IGF-I promoter construct, IGF-I711b-Luc, was used previously to characterize PKA-dependent activation of the IGF-I gene promoter in rat osteoblasts (8, 10, 11, 24, 25). Plasmids were propagated in Escherichia coli strain DH5
with ampicillin selection, and were prepared with a QIAGEN Midiprep Kit (QIAGEN, Chatsworth, CA).
Transfections
Cells were transfected with TransIT LT1 (PanVera Corp., Madison, WI; 0.03%) using 250 ng of 5X GAL4-E1b-Luciferase reporter plasmid DNA, and 5075 ng of Gal4 DBD-fused C/EBP
recombinant plasmid DNA per 4.8-cm2 culture (11, 24, 26). In some instances cells were also transfected with a vector encoding the ß-galactosidase gene under control of the simian virus 40 promoter to assess relative gene expression. Cultures at 5075% confluence were changed to serum-depleted medium and exposed to plasmids plus transfection reagent for 16 h. Cultures were then supplemented to achieve a final concentration of 5% fetal bovine serum. After 48 h, the cells were rinsed twice with PBS and harvested for analysis. In some instances, cells were rinsed and treated with vehicle or PGE2 as indicated in the various figures. For reporter gene analysis, cultures were detergent lysed, nuclei were removed by centrifugation, and enzyme activity in supernatants was measured with commercial reagents to assess luciferase or ß-galactosidase expression. Data were corrected for protein content (27). COS-7 cells were used to produce recombinant C/EBP
and CBP by transient transfection.
Nuclear Extracts
Cells were rinsed, harvested, and lysed in a hypotonic buffer containing 10 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, phosphatase inhibitors (1 mM sodium orthovanadate and 10 mM sodium fluoride), protease inhibitors (0.5 mM phenylmethyl sulfonylfluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, and 2 µg/ml aprotinin), and 1% Triton X-100. Nuclei were collected, resuspended in hypertonic buffer containing 0.42 M NaCl, 0.2 mM Na2EDTA, 25% glycerol, and the phosphatase and protease inhibitors indicated above. Soluble nuclear proteins released after 30 min of incubation were collected by centrifugation at 12,000 x g for 5 min (8, 11, 24, 26, 28).
EMSA
Radiolabeled double-stranded DNA probes were prepared with complementary oligonucleotides (Life Technologies, Inc., Gaithersburg, MD), followed by fill-in of single-stranded overhangs with dCTP, dGTP, dTTP, and [
-32P]dATP using Klenow fragment of DNA polymerase I. The DNA probe contained the C/EBP binding sequence (underlined) designated HS3D from the IGF-I gene promoter (5'-GAGCAGATAGAGCCTGCGCAATCGAAATAAAGTC-3'). Five to 10 µg of nuclear protein were preincubated with 2 µg of poly(dI:dC), without or with unlabeled specific or nonspecific competitor DNA in 60 mM KCl, 25 mM HEPES (pH 7.6), 7.5% glycerol, 0.1 mM EDTA, and 5 mM dithiothreitol, and 0.025% BSA. Samples were supplemented with 5 x 104 cpm of DNA probe (0.10.2 ng) for 30 min and fractionated through a preelectrophoresed 5% nondenaturing polyacrylamide gel. Dried gels were exposed to x-ray film at -75 C with an intensifying screen (24, 25, 26).
Western Immunoblot Analysis
Nuclear extracts were fractionated on a 15% polyacrylamide-sodium dodecyl sulfate gel and electroblotted onto Immobilon-P membranes (Millipore Corp., Bedford, MA) along with prestained molecular weight markers. Filters were air dried, hydrated, and blocked in 5% fat-free powdered milk in standard Tris-buffered saline with 0.05% Tween 20 before probing with specific antibodies. Antibody binding was visualized with secondary antibody linked to horseradish peroxidase and chemiluminescence amplification (26).
Immunoprecipitation
Nuclear extract was prepared from COS-7 cells cotransfected to express CBP and C/EBP
. Nuclear protein (100 µg) was diluted 10-fold, incubated for 16 h at 4 C with 4 µg of either monoclonal antibody to CBP or nonimmune mouse IgG, and collected by coprecipitation with protein G-agarose (Roche, Indianapolis, IN) and centrifugation. The precipitates were washed five times with PBS, fractionated on a 10% polyacrylamide-sodium dodecyl sulfate gel, electroblotted with prestained molecular weight markers, and probed by Western immunoblot analysis with rabbit anti-C/EBP
antibody.
Reagents
All chemical reagents were from Sigma Chemical Co. Anti-C/EBP
, anti-GAL4 DBD, and anti-CBP antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-VP16 antibody was from CLONTECH Laboratories, Inc. (Palo Alto, CA).
Statistical Analysis
Data were assessed by one-way ANOVA, with Kruskal-Wallis or Bonferonni methods for post hoc analysis, using SigmaStat software. Statistical differences were assumed by P < 0.05.
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FOOTNOTES
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This work was supported by NIH Grant DK56310 and the Arthritis Foundation.
C.J. and W.C. each contributed comparable levels of effort to this study and both should be considered primary authors.
Abbreviations: CBP, cAMP response element binding protein (CREB)-binding protein; C/EBP, CCAAT enhancer binding protein; DBD, DNA-binding domain; PGE2, prostaglandin E2; PKA, protein kinase A.
Received for publication July 5, 2002.
Accepted for publication May 27, 2003.
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REFERENCES
|
---|
- Wedel A, Ziegler-Heitbrock HW 1995 The C/EBP family of transcription factors. Immunobiology 193:171185[Medline]
- Lekstrom-Himes J, Xanthopoulos KG 1998 Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273:2854528548[Abstract/Free Full Text]
- Yeh WC, Cao Z, Classon M, McKnight SL 1995 Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev 9:168181[Abstract]
- Tanaka T, Yoshida N, Kishimoto T, Akira S 1997 Defective adipocyte differentiation in mice lacking the C/EBPß and/or C/EBP
gene. EMBO J 16:74327443[Abstract/Free Full Text]
- Darlington GJ, Ross SE, MacDougald OA 1998 The role of C/EBP genes in adipocyte differentiation. J Biol Chem 273:3005730060[Free Full Text]
- Diehl AM, Michaelson P, Yang SQ 1994 Selective induction of CCAAT/enhancer binding protein isoforms occurs during rat liver development. Gastroenterology 106:16251637[Medline]
- Diehl AM 1998 Roles of CCAAT/enhancer-binding proteins in regulation of liver regenerative growth. J Biol Chem 273:3084330846[Abstract/Free Full Text]
- Umayahara Y, Ji C, Centrella M, Rotwein P, McCarthy TL 1997 CCAAT/enhancer-binding protein
activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone. J Biol Chem 272:3179331800[Abstract/Free Full Text]
- McCarthy TL, Ji C, Chen Y, Kim KK, Imagawa M, Ito Y, Centrella M 2000 Runt domain factor (Runx)-dependent effects on CCAAT/enhancer-binding protein
expression and activity in osteoblasts. J Biol Chem 275:2174621753[Abstract/Free Full Text]
- Umayahara Y, Billiard J, Ji C, Centrella M, McCarthy TL, Rotwein P 1999 CCAAT/enhancer-binding protein
is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts. J Biol Chem 274:1060910617[Abstract/Free Full Text]
- Thomas MJ, Umayahara Y, Shu H, Centrella M, Rotwein P, McCarthy TL 1996 Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts. J Biol Chem 271:2183521841[Abstract/Free Full Text]
- McCarthy TL, Centrella M, Canalis E 1989 Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone. Endocrinology 124:12471253[Abstract]
- McCarthy TL, Centrella M, Raisz LG, Canalis E 1991 Prostaglandin E2 stimulates insulin-like growth factor I synthesis in osteoblast-enriched cultures from fetal rat bone. Endocrinology 128:28952900[Abstract]
- Nerlov C, Ziff EB 1994 Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-
on the serum albumin promoter. Genes Dev 8:350362[Abstract]
- Friedman AD, McKnight SL 1990 Identification of two polypeptide segments of CCAAT/enhancer-binding protein required for transcriptional activation of the serum albumin gene. Genes Dev 4:14161426[Abstract]
- Williamson EA, Xu HN, Gombart AF, Verbeek W, Chumakov AM, Friedman AD, Koeffler HP 1998 Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein
. J Biol Chem 273:1479614804[Abstract/Free Full Text]
- Cooper C, Henderson A, Artandi S, Avitahl N, Calame K 1995 Ig/EBP (C/EBP
) is a transdominant negative inhibitor of C/EBP family transcriptional activators. Nucleic Acids Res 23:43714377[Abstract]
- McCarthy TL, Centrella M, Canalis E 1988 Further biochemical and molecular characterization of primary rat parietal bone cell cultures. J Bone Miner Res 3:401408[Medline]
- Centrella M, Casinghino S, McCarthy TL 1994 Differential actions of prostaglandins in separate cell populations from fetal rat bone. Endocrinology 135:16111620[Abstract]
- Centrella M, Casinghino S, Kim J, Pham T, Rosen V, Wozney J, McCarthy TL 1995 Independent changes in type I and type II receptors for transforming growth factor ß induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype. Mol Cell Biol 15:32733281[Abstract]
- Centrella M, Casinghino S, Gundberg C, McCarthy TL, Wozney J, Rosen V 1996 Changes in bone morphogenetic protein sensitivity relative to differentiation in fetal rat bone cell cultures. Ann NY Acad Sci 785:224226[Medline]
- Sadowski I 1998 Plasmids for one- and two-hybrid analysis in mammalian cells. Anal Biochem 256:245247[CrossRef][Medline]
- Sun P, Maurer RA 1995 An inactivating point mutation demonstrates that interaction of cAMP response element binding protein (CREB) with the CREB binding protein is not sufficient for transcriptional activation. J Biol Chem 270:70417044[Abstract/Free Full Text]
- McCarthy TL, Thomas MJ, Centrella M, Rotwein P 1995 Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element. Endocrinology 136:39013908[Abstract]
- McCarthy TL, Ji C, Shu H, Casinghino S, Crothers K, Rotwein P, Centrella M 1997 17ß-Estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures. J Biol Chem 272:1813218139[Abstract/Free Full Text]
- McCarthy TL, Ji C, Chen Y, Kim K, Centrella M 2000 Time- and dose-related interactions between glucocorticoid and cyclic adenosine 3',5'-monophosphate on CCAAT/enhancer-binding protein-dependent insulin-like growth factor I expression by osteoblasts. Endocrinology 141:127137[Abstract/Free Full Text]
- Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254[CrossRef][Medline]
- Lee KA, Bindereif A, Green MR 1988 A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene Anal Tech 5:2231[CrossRef][Medline]
- Billiard J, Umayahara Y, Wiren K, Centrella M, McCarthy TL, Rotwein P 2001 Regulated nuclear-cytoplasmic localization of CCAAT/enhancer-binding protein
in osteoblasts. J Biol Chem 276:1535415361[Abstract/Free Full Text]
- Mink S, Haenig B, Klempnauer KH 1997 Interaction and functional collaboration of p300 and C/EBPß. Mol Cell Biol 17:66096617[Abstract]
- Guo S, Cichy SB, He X, Yang Q, Ragland M, Ghosh AK, Johnson PF, Unterman TG 2001 Insulin suppresses transactivation by CAAT/enhancer-binding proteins ß (C/EBPß). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major activation domain of C/EBPß. J Biol Chem 276:85168523[Abstract/Free Full Text]
- Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B, Kurokawa R, Brown M 1996 p300 is a component of an estrogen receptor coactivator complex. Proc Natl Acad Sci USA 93:1154011545[Abstract/Free Full Text]
- Klotz DM, Hewitt SC, Korach KS, Diaugustine RP 2000 Activation of a uterine insulin-like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor-
. Endocrinology 141:34303439[Abstract/Free Full Text]
- Trautwein C, Caelles C, van der Geer P, Hunter T, Karin M, Chojkier M 1993 Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain. Nature 364:544547[CrossRef][Medline]