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Molecular Endocrinology, doi:10.1210/me.2002-0235
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Molecular Endocrinology 17 (9): 1834-1843
Copyright © 2003 by The Endocrine Society

Activation Domains of CCAAT Enhancer Binding Protein {delta}: 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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In osteoblasts, hormones such as prostaglandin E2 that activate protein kinase A increase the translocation of transcription factor CCAAT/enhancer binding protein {delta} (C/EBP{delta}) 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{delta} using native and heterologous gene promoter assay systems. We demonstrated functional interactions between C/EBP{delta} and trans-gene-expressed cAMP response element binding protein-binding protein, and showed that the ability of C/EBP{delta} 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{delta} reduced its basal transcriptional activity, whereas mutation at serine 191 suppressed the stimulatory effect of prostaglandin E2 on C/EBP{delta} 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{delta}. Our studies provide the first evidence for regions of C/EBP{delta} that are important for basal and for hormone-induced transcriptional activity, and for its interactions with other enhancers and suppressers of gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{delta} to C/EBPß, to a point where terminal differentiation requires C/EBP{alpha} expression. Homozygous gene deletion studies in mice further revealed that C/EBP{delta} and C/EBPß double-knockout and C/EBP{alpha} 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{alpha} 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{delta} in a constitutive manner (8, 9). In this context, we showed that transcription factor Runx2 (previously termed AML-3, Cbfa1, or PEBP2{alpha}A1), which is essential for development of the osteoblast phenotype, maintains and regulates C/EBP{delta} expression in native and in prostaglandin E2 (PGE2)-activated osteoblasts (9).

C/EBP{delta} 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{delta} 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{delta} appear to be important and perhaps essential mediators of hormone-activated bone formation.

Whereas earlier studies identified several functional domains within the C/EBP{alpha}, C/EBPß, C/EBP{epsilon}, and C/EBP{gamma} isoforms, little is known about activation and repression domains in C/EBP{delta} (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{delta} and identified critical amino acids that independently regulate C/EBP{delta}-dependent transactivation of the IGF-I gene promoter in unstimulated and PGE2-induced osteoblasts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation and Repression Domains in C/EBP{delta}
To study functional domains of C/EBP{delta} 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{delta} were first localized by preparing eukaryotic expression constructs that encode fragments of C/EBP{delta} fused carboxyl to a Gal4 DNA binding domain (DBD) (designated M1{delta}). This vector also contains a nuclear localization signal that facilitates the analysis independently of constraints that retain C/EBP{delta} in the cytoplasm in uninduced osteoblasts (29). Cells were transiently transfected with a C/EBP{delta} fusion plasmid construct in combination with the 5X Gal4-Luc reporter plasmid. In this way, the C/EBP{delta} 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{delta}, 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. 1AGo). C/EBP{delta} 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{delta} fragment were detected by Western blot analysis in transfected cell nuclear extracts (Fig. 1BGo).



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  		Fig. 1. Initial Mapping of Domains within C/EBP{delta}

In panel A, a deletion nest of Gal4 DBD-C/EBP{delta} (M1{delta}) fusion proteins was created by restriction digestion or PCR with C/EBP{delta} cDNA and expression plasmid M1 containing the Gal4 DBD. Osteoblasts were cotransfected for 48 h with 250 ng/well of 5X Gal4 reporter plasmid and 50 ng/well of the Gal4 DBD-C/EBP{delta} expression constructs indicated. Basal effects by the C/EBP{delta} 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. In panel B, nuclear extract from transfected cells was examined by Western blot with anti-Gal4DBD antiserum to assess relative expression of the Gal4 DBD-C/EBP{delta} fusion proteins. Similar results were found in duplicate experiments.

 
To identify the activation domain of C/EBP{delta} 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{delta} function. Small increments in activity occurred by addition of various amino acid fragments up to position 102 (Fig. 2AGo), but only fragments encoding 1–93 or 1–102 were significantly greater in activity by comparison to M1{delta}1–68. Internal deletions were then used to verify the region that spanned this strong activation domain. Deletion of amino acids 23–40 in M1{delta}1–22/41–102 had no effect, whereas loss of amino acids 77–93 in M1{delta}1–76/94–102 reduced activity only marginally. Deletion of amino acids 41–68 in M1{delta}1–40/69–102 or 52–68 in M1{delta}1–51/69–102 produced far greater losses in reporter expression, while near basal activity was detected by elimination of amino acids 52–76 and 60–76 in constructs M1{delta}1–51/77–102 and M1{delta}1–59/77–102 (Fig. 2BGo). Together, these results identify an important domain between amino acids 41–76 that contribute the majority of C/EBP{delta} activation, with a critical function for amino acids 60–68.



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  		Fig. 2. Fine Mapping of the C/EBP{delta} Activation Domain

In panel A, osteoblasts were cotransfected with a deletion nest of Gal4 DBD-C/EBP{delta} fusion plasmids encoding C/EBP{delta} up to amino acid 102 and 5X Gal4 reporter plasmid. In panel B, osteoblasts were cotransfected with a deletion nest of Gal4 DBD-C/EBP{delta} fusion plasmids with various internal deletions introduced within C/EBP{delta} 1–102 and 5X Gal4 reporter plasmid. Basal effects by the C/EBP{delta} 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. *, Statistically significant increase in gene expression compared with M1{delta}1–68, P < 0.05.

 
C/EBP{delta} fusion constructs spanning amino acids 102–180 confirmed the presence of an endogenous suppression domain between amino acids 103 and 116 (Fig. 3AGo). 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 117–130 (Fig. 3BGo). Therefore, a minimal repressor domain occurs in this region of C/EBP{delta}. However, it is more active within the context of C/EBP{delta} itself, perhaps from structural constraints that occur with the amino-terminal portion of endogenous C/EBP{delta} molecule, but not with the heterologous recombinant DBD/VP16 fusion protein.



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  		Fig. 3. A Repression Domain within the Central Region of C/EBP{delta}

In panel A, osteoblasts were cotransfected with a deletion nest of Gal4 DBD-C/EBP{delta} fusion plasmids encoding C/EBP{delta} fragments up to amino acid 180 and 5X Gal4 reporter plasmid. In panel B, osteoblasts were cotransfected with a nest of internal fragments of C/EBP{delta} between amino acids 102 and 149 fused to the activation domain of herpes virus protein 16 (VP16) and Gal4 DBD, and 5X Gal4 reporter plasmid. Basal effects by the C/EBP{delta} 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. In panel C, nuclear extract from transfected cells was examined by Western blot with anti-VP16 antiserum to assess relative expression of the Gal4 DBD-MVP16 C/EBP{delta} fusion proteins. Similar results were found in duplicate experiments.

 
Interactions between C/EBP{delta} 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{delta}, functional interaction between C/EBP{delta} and CBP has not yet been demonstrated. To do so, cells were cotransfected with expression plasmids encoding either full-length C/EBP{delta} 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{delta}-dependent reporter gene expression in a dose-dependent way. The 3- to 4-fold stimulatory effect of CBP on C/EBP{delta} is similar to or greater than that previously reported for C/EBPß (30). Slightly more CBP expression was required to activate the C/EBP{delta} truncation fragment M1{delta}1–102. It is important to note that M1{delta}1–102, which eliminates the carboxyl-terminal repressor domain, has approximately 30-fold higher basal activity by comparison to full-length M1{delta}. Even within this context, however, M1{delta}1–102 activity was enhanced by approximately 2-fold (Fig. 4AGo). 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{delta} and CBP

In panel A, osteoblasts were cotransfected with a Gal4 DBD-C/EBP{delta} fusion plasmid encoding full-length C/EBP{delta} (M1{delta}, on the left) or C/EBP{delta} amino acids 1–102 (M1{delta}1–102, on the right), increasing amounts of expression plasmid encoding CBP, and 5X Gal4 reporter plasmid. In panel B, osteoblasts were cotransfected with M1{delta} 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{delta} 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{delta} + CBP in panel B, P < 0.05. In panel C, nuclear extract from COS-7 cells, cotransfected to express C/EBP{delta} 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{delta}.

 
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{delta} 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{delta}-dependent gene transactivation, but E1Am was significantly less effective (Fig. 4BGo). Nuclear extract from COS-7 cells cotransfected to expressed CBP and C/EBP{delta}, collected with antibody to CBP and probed by Western blot analysis with antibody to C/EBP{delta}, revealed coprecipitation of C/EBP{delta}, consistent with a physical interaction between these proteins (Fig. 4CGo).

PGE2-Sensitive Domains in C/EBP{delta}
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{delta}, 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{delta} 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{delta} activation in this way, point mutations were introduced into full-length C/EBP{delta} 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 41–76, and on potential kinase substrates that are conserved among C/EBP{alpha}, C/EBPß, and C/EBP{delta} at amino acids 191 and 208 (14, 15, 16). In uninduced cells, overexpression of native C/EBP{delta} (M1{delta}1–268) significantly enhanced IGF-I promoter activity, as we reported previously (8, 10). C/EBP{delta} 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. 5AGo). In cells stimulated with PGE2, only the serine-to-alanine substitution at ser191 effectively suppressed C/EBP{delta} activity (Fig. 5BGo). Each native or mutated construct produced nearly equivalent protein, as assessed by Western blot analysis (Fig. 5CGo). 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{delta} binding to DNA (Fig. 5Go, C and D). Notably, ser191 resides in the DBD of C/EBP{delta}.



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  		Fig. 5. Amino Acid Domains in C/EBP{delta} Required for Basal and PGE2-Dependent Transactivation of the IGF-I Gene Promoter

Osteoblasts were cotransfected with parental vector, Gal4 DBD-C/EBP{delta} fusion plasmids encoding full-length C/EBP{delta} (M1{delta}1–268), or M1{delta}1–268 with serine- or tyrosine-to-alanine substitutions at the locations indicated, and IGF-I promoter reporter plasmid IGF-I711b. In panel A cells were treated with vehicle and in panel B, cells were treated for 6 h with 1 µM PGE2. 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 effect by mutation compared with M1{delta}1–268, P < 0.05. In panel C, nuclear extract from untreated cells was examined by Western blot with anti-GAL4 DBD antiserum. Similar results were found in duplicate experiments. In panel D, nuclear extract from untreated cells was combined with 32P-labeled oligonucleotide probe HS3D encoding the C/EBP binding site in exon 1 of the IGF-I gene. Similar results were found in duplicate experiments.

 
Recombinant mutated C/EBP{delta} 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{delta} 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{delta} activity in control or PGE2-stimulated cells (Fig. 6Go). While the activity of C/EBP{delta} 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{delta} DBD. Therefore, native C/EBP{delta}-dependent gene promoter activity may require changes in conformation or phosphorylation status that do not occur in these mutated C/EBP{delta}s.



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  		Fig. 6. Amino Acid Domains in C/EBP{delta} Required for Basal and PGE2-Dependent Transactivation of the 5X Gal4 DBD Reporter Plasmid

Osteoblasts were cotransfected with parental vector, Gal4 DBD-C/EBP{delta} fusion plasmids encoding full-length C/EBP{delta} (M1{delta}1–268), or M1{delta}1–268 with serine- or tyrosine-to-alanine substitutions at the locations indicated, and 5X Gal4 reporter plasmid. Cells were treated for 6 h with vehicle (control) or 1 µM PGE2 as indicated. Data are means ± SE from a minimum of nine samples from three separate experiments, corrected for protein content and relative transfection efficiency.

 
Finally, ser67 resides just within the core of the potent activation domain of C/EBP{delta} that we defined in Figs. 1Go and 2Go. Unlike C/EBP{delta} with a mutation at ser191, when C/EBP{delta} 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{delta} (Fig. 7Go).



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  		Fig. 7. Mutation of ser (67) within the Potent C/EBP{delta} Activation Domain Does Not Reduce Gene Transactivation Potential

Osteoblasts were cotransfected with parental vector (M1), Gal4 DBD-C/EBP{delta} fusion plasmids encoding full-length C/EBP{delta} (M1{delta}1–268), or M1{delta}1–268 with serine-to-alanine substitutions at the locations indicated, in combination with IGF-I promoter reporter plasmid IGF-I711b (top panel), or in combination with 5X Gal4 reporter plasmid (bottom panel). Cells were treated for 6 h with vehicle (-) or 1 µM PGE2 (+). Data are means ± SE from six to nine samples from three separate experiments, corrected for protein content and relative transfection efficiency. * Indicates a statistically significant effect by mutation compared with M1{delta}1–268, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, C/EBPß, C/EBP{epsilon}, and C/EBP{gamma} have been previously identified, but much less is currently known about activation and repression domains within C/EBP{delta}. In this study, we identified activation and repression domains within C/EBP{delta} in several ways, using primary cultures of rat osteoblasts that are extremely sensitive to changes in C/EBP{delta} expression and activation. A strong transcriptional activation domain occurs within amino acids 41–76, 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{delta} 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{delta} 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{alpha} 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{epsilon} 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{delta} and C/EBP{epsilon} 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{delta} 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{delta} with CBP indicates that this association is both physical and functional. Moreover, viral protein E1A successfully competed with both C/EBP{delta} 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{delta} activity. The steroid receptor coactivator 1 can enhance transcription induced by some steroid hormone receptors such as estrogen receptor-{alpha}. 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{delta}. This suggests a potential mechanism for the counteracting effects of estrogen on PKA-activated IGF-I expression in osteoblasts (25).

The C/EBP{delta} activation domain fused to Gal4 DBD (M1{delta} 1–102) by itself potently activates transcription, comparable to viral protein VP16. However, full-length C/EBP{delta} promotes far less gene expression in the Gal4 DBD-dependent one-hybrid assay by comparison to M1{delta} 1–102. This may occur from secondary or tertiary structural restrictions of full-length C/EBP{delta} that limit its strong activation potential. The activation domain of C/EBP{delta} may be unmasked by dimerization or by phosphorylation, which themselves may be codependent events. Furthermore, we showed here that C/EBP{delta} has a downstream repression domain similar to other C/EBP isoforms. When fused directly to VP16, this region of C/EBP{delta} partially repressed the potent activation domain of VP16, suggesting that it acts, at least in part, independently of the C/EBP{delta} activation domain.

Potential phosphorylation sites that could regulate DNA binding or gene activation are present in several C/EBPs. Those found in C/EBP{delta} 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{delta} 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{delta} 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{delta}, comprising nuclear-localization domains and DBDs. A decrease in C/EBP{delta}-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{delta} 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{delta} ala191 or other mutated or wild-type C/EBP{delta} fusion proteins.

Rat C/EBP{delta} 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{delta} 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{delta} 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{delta} (29). C/EBP{delta} 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{delta} 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{delta} and the Runt homology domain of Runx2 also appears to limit C/EBP{delta} promoter activity, establishing an efficient inhibitory feed-back loop in C/EBP{delta} expression when sufficiently high C/EBP{delta} levels are achieved.

In summary, C/EBP{delta}, 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{delta} span amino acids 41–102. In addition, ser191, which occurs within a PKA-sensitive domain in C/EBP{delta}, appears essential for the stimulatory effect of PGE2 on IGF-I. Ser191 also locates within the DBD of C/EBP{delta}. Mutation at this site reduced C/EBP{delta} binding to its regulatory element, as well as basal and PGE2-induced IGF-I gene promoter activity. However, the C/EBP{delta}/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{delta} 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{delta} to DNA and, in this way, enhances new IGF-I gene transcription.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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{delta} 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{delta} 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{delta}. 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{alpha} 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 50–75 ng of Gal4 DBD-fused C/EBP{delta} 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 50–75% 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{delta} 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 [{alpha}-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 (0.1–0.2 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{delta}. 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{delta} antibody.

Reagents
All chemical reagents were from Sigma Chemical Co. Anti-C/EBP{delta}, 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.


   FOOTNOTES
 
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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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