help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0452
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wiper-Bergeron, N.
Right arrow Articles by Lee, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wiper-Bergeron, N.
Right arrow Articles by Lee, J. M.
Molecular Endocrinology 21 (9): 2124-2135
Copyright © 2007 by The Endocrine Society

CCAAT/Enhancer Binding Protein ß Abrogates Retinoic Acid-Induced Osteoblast Differentiation via Repression of Runx2 Transcription

Nadine Wiper-Bergeron, Catherine St-Louis and Jonathan M. Lee

Department of Cellular and Molecular Medicine (N.W.-B., C.S-L.) and Department of Biochemistry, Microbiology and Immunology (N.W.-B., J.M.L.), University of Ottawa, Ottawa, Ontario, Canada K1H 8M5

Address all correspondence and requests for reprints to: Dr. Nadine Wiper-Bergeron, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. E-mail: Nadine.WiperBergeron{at}uottawa.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Runx2/CBFA1/AML3 is a master regulator of the osteoblast lineage and has been shown to directly control the transcription of numerous osteoblast-specific genes including alkaline phosphatase, osteopontin, and type I collagen. In its absence, ossification does not occur during development resulting in animals with cartilaginous skeletons and no osteoblasts. In humans, loss of one copy of Runx2 causes cleidocranial dysplasia characterized by malformations of the facial and cranial bones and the clavicle. Despite its important role in osteoblast biology, relatively little is known about the transcriptional regulation of the Runx2 gene. In the present study, we show that CCAAT/enhancer binding protein ß (C/EBPß) is a negative regulator of Runx2 expression and acts by directly binding a C/EBP element located at –591/–576 within the osteoblast-specific Runx2 P1 promoter. Ectopic expression of C/EBPß in C3H10T1/2 cells causes a reduction in Runx2 expression concomitant with a decrease in osteogenic potential during all-trans retinoic acid (ATRA)-induced differentiation. In nondifferentiating cells, C/EBPß can be found occupying the C/EBP negative response element within the Runx2 P1 promoter. ATRA, the effects of which are mediated by retinoic acid receptor {alpha} and {gamma} in C3H10T1/2 cells, stimulates the dissociation of C/EBPß from this element and promotes Runx2 expression. Thus, ATRA initiates osteoblastic differentiation of C3H10T1/2 cells, at least in part, by triggering the dissociation of C/EBPß from the Runx2 promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MESENCHYMAL PRECURSOR CELLS have the capability to differentiate into different lineages (osteoblast, chondrocyte, adipocyte, myoblast) in response to hormone and growth factor signals. The transcriptional cascades controlling osteoblast differentiation are the subject of intense study, but it is known that the expression of Runx2 (CBFA1/AML3) is absolutely required for differentiation into the osteoblast lineage (1, 2). Runx2 is a transcription factor that drives the expression of osteoblast specific genes, including alkaline phosphatase, osteocalcin, osteopontin, and collagen type 1 (3, 4, 5, 6). Ectopic Runx2 expression is sufficient to induce osteoblastic differentiation of fibroblasts and the transdifferentiation of myocytes (7, 8). Genetic ablation of Runx2 in mice results in the complete absence of osteoblasts, resulting in a cartilaginous skeleton (1, 2). In postnatal life, Runx2 function is required for the maintenance of bone mass and osteoblast function (9). In humans, heterozygous mutations in the Runx2 locus is associated with cleidocranial dysplasia, a disease characterized by short stature, delayed closure of the fontanels, craniofacial malformations, and the absence or malformation of the collar bones (10).

Expression of Runx2 is controlled by two promoters, designated P1 and P2, and alternative splicing events result in three protein products (4, 11, 12, 13, 14). Of these, only Runx2 produced from the P1 promoter is osteoblast specific (15). The P1 promoter is complex, with numerous putative DNA binding motifs for transcription factors including Runx2 itself (14). Indeed, studies have shown that Runx2 is able to autoregulate its own transcription, and likely assumes this role in the mature osteoblast (15). In addition, an osteoblast-specific enhancer at –415/–375 binds both NF-1 and the AP-I family of transcription factors (16). In nonosteogenic cell lines, the element is occupied by NF1A, which blocks transcription by steric hindrance, whereas in osteogenenic cell lines the site is occupied by FosB (a member of the AP-1 family of transcription factors expressed specifically in osteoblasts), which acts to activate the P1 promoter. Smad3 has also been shown to negatively regulate the Runx2 promoter, attenuating Runx2 expression and thereby the osteoblast differentiation of mouse caIB 2T3 cells (6). Despite these findings, it remains unclear how the expression of Runx2 is initiated during development or in precursor cells which do not already express Runx2.

Members of the CCAAT/enhancer binding protein (C/EBP) family are predicted to bind the Runx2 promoter based on sequence analysis (14). C/EBPs are members of the bzip family of transcription factors and are involved in many cellular differentiation processes including adipogenesis, liver regeneration, and mammary gland development (17, 18, 19). C/EBPß, C/EBP{delta}, and C/EBP{alpha} have been shown to be involved in mesenchymal differentiation, in particular in the stimulation of adipocyte differentiation (19, 20). C/EBPß is an early activator of differentiation programs and commits cells to differentiation by increasing the expression of transcription factors required for terminal differentiation (20, 21). As a consequence, the transcriptional activity of C/EBPß is tightly regulated and is controlled by posttranslational modification, alterations in subcellular localization, and sequestration away from DNA substrates (22, 23, 24, 25, 26). Although C/EBPß promoter interaction is required for transactivation, the binding of C/EBPß to DNA does not necessarily correlate with activation of target gene transcription (27, 28). Rather, the transcriptional activity of C/EBPß is further modulated by interactions with coregulatory molecules such as p300/cAMP response element binding protein-binding protein (CBP), general control of amino acid 5 (GCN5)/p300 CBP-associated factor (PCAF), members of the Swi/Snf family, and a histone deacetylase 1 (HDAC1)-containing corepressor complex (26, 27, 29, 30). In addition, C/EBPß has been shown to occupy the promoters of several cyclin D1 target genes, where it acts to prevent transcription until cyclin D1 levels increase (28). In this regard, C/EBPß can act as a transcriptional switch, whose transcriptional output is dependent upon the integration of extra- and intracellular signals.

In the present study, we investigated the role of C/EBPß in the initiation of osteogenesis and Runx2 transcription in pluripotent C3H10T1/2 cells. We demonstrate that exogenous C/EBPß expression decreases alkaline phosphatase activity and Runx2 expression in differentiating mesenchymal cells. C/EBPß can bind a C/EBP response element located at –591/–576 in the P1 promoter and can repress Runx2 transcription directly. All-trans retinoic acid (ATRA), which stimulates C3H10T1/2 cells to differentiate into osteoblasts, triggers the displacement of C/EBPß from the Runx2 promoter, resulting in enhanced expression of Runx2 and other Runx2-dependent osteoblast genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ectopic C/EBPß Inhibits ATRA-Induced Osteoblast Differentiation of C3H10T1/2 Cells
To evaluate the effect of exogenous C/EBPß on osteoblast differentiation, we transduced C3H10T1/2 cells with either a retrovirus encoding C/EBPß or with empty virus (pLXSN). We subsequently induced differentiation of these cells using ATRA, which has been shown previously to stimulate the expression of alkaline phosphatase and Runx2 and to induce matrix mineralization in these cells (31). Treatment of C3H10T1/2 cells with standard osteogenic medium containing ascorbic acid, ß-glycerophosphate, and dexamethasone fails to induce significant osteoblastogenesis but results instead in robust adipogenesis and is therefore not a suitable approach for this particular study (see supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Six days after ATRA addition, we assessed differentiation by visualizing alkaline phosphatase (ALP) expression both histochemically and via an in vitro enzymatic assay (Fig. 1AGo, B); this time point has been previously identified as the peak in ALP activity during differentiation of these cells (31). ATRA-treated cultures expressing ectopic C/EBPß demonstrated a 50% reduction in alkaline phosphatase activity relative to cells transduced with empty virus. Western blot analysis revealed that, whereas Runx2 expression was strongly up-regulated in cells transduced with empty virus (pLXSN) 6 d after ATRA treatment, the expression of Runx2 in cells overexpressing C/EBPß was abrogated (Fig. 1CGo). In addition, expression of osteopontin, a Runx2 target gene, was also compromised, whereas C/EBPß expression was increased approximately 2-fold (1.74 ± 0.39) in retrovirally transduced cultures (Fig. 1CGo). Together, these results suggest that overexpression of C/EBPß inhibits early ATRA-induced osteoblast differentiation events.


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 1. Overexpression of C/EBPß in C3H10T1/2 Cells Inhibits Early ATRA-Mediated Osteoblast Differentiation

A, ALP staining of differentiating C3H10T1/2 cultures retrovirally transduced with empty virus (pLXSN) or to express C/EBPß. Confluent C3H10T1/2 cultures were induced to differentiate with 10–6 M ATRA and 6 d after induction were fixed and stained for alkaline phosphatase. Images are representative of three independent experiments performed in duplicate. B, C3H10T1/2 cells retrovirally transduced and induced to differentiate as in panel A were lysed after 6 d and ALP activity was measured by colorimetric assay. ALP activity was corrected for protein levels using a standard Bradford assay. C, Western analysis of osteoblast marker expression in ATRA induced C3H10T1/2 cells retrovirally transduced as in panel A. Whole cell lysates were prepared from 10–6 M ATRA stimulated C3H10T1/2 cultures 6 d after induction to differentiate and the expression of Runx2, osteopontin (OPN), C/EBPß and actin were analyzed. D, Alizarin red staining for calcium deposition on C3H10T1/2 cultures retrovirally transduced and induced to differentiate as in panel A. Staining was performed 27 d after induction to differentiate. Images are representative of three independent experiments. E, Semiquantitative analysis of late osteoblast markers by RT-PCR performed 27 d after induction to differentiate. Expression of collagen Ia1 (colIa1), osteocalcin (OC), and Runx2 were analyzed and standardized to glyceraldehyde-3-phosphate (GAPDH) message. Data are shown relative to pLXSN-infected controls.

 
To determine whether the early repression of Runx2 expression by C/EBPß impacted on the efficiency of late osteoblast differentiation, we repeated the differentiation experiment but allowed cultures to differentiate for 27 d in the presence of ATRA and assessed matrix mineralization and late osteoblast marker expression. Whereas cultures infected with empty virus stained robustly with Alizarin red, cultures overexpressing C/EBPß consistently stained less intensely, suggesting that the deposition of calcium onto the extracellular matrix was abrogated by C/EBPß overexpression (Fig. 1DGo). Analysis of late osteoblastic marker expression (osteocalcin and collagen type I) by semiquantitative RT-PCR revealed that there were no significant differences between the C/EBPß-overexpressing and control cultures (Fig. 1EGo). Similarly, Runx2 expression at this late time point was equivalent in both test and control cells, suggesting that the low levels of Runx2 expression seen in early stages of differentiation may over time accumulate to levels that are sufficient in long-term culture to stimulate not only the production of Runx2 but its target genes as well. The defect in matrix mineralization may reflect a lag in the expression of osteoblast markers in C/EBPß-overexpressing cells.

C/EBPß Overexpression Represses 5-Azacytidine-Induced Osteoblast Differentiation of C3H10T1/2 Cells
Due to massive demethylation of DNA, treatment with 5-azacytidine stimulates mesenchymal precursors to differentiate into all possible fates, in this case osteoblasts, adipocytes, chondrocytes and myoblasts. After 27 d of differentiation in the presence of 5-azacytidine, we performed histological staining and RT-PCR to evaluate osteoblastogenesis in our cultures. Although empty virus control C3H10T1/2 cells differentiated efficiently into osteoblasts, as evidenced by Alizarin Red staining, cells overexpressing C/EBPß displayed a significant number of adipocytes (evidenced by Oil red O staining) with a concomitant reduction in Alizarin Red staining relative to controls (Fig. 2AGo). RT-PCR analysis of adipocyte and osteoblast markers revealed that, whereas there was no statistical difference in PPAR{gamma} expression (an adipocyte marker) upon 5-azacytidine-induced differentiation, expression of both Runx2 and osteocalcin were significantly reduced in the C/EBPß expressing cells (Fig. 2Go, B and C). Thus, even in the absence of ATRA stimulation, C/EBPß overexpression reduced the efficiency of osteoblastogenesis in C3H10T1/2 cells stimulated with 5-azacytidine.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 2. C/EBPß Expression Inhibits 5-Azacytidine-Induced Osteoblast Differentiation

A, C3H10T1/2 cells expressing C/EBPß or transduced with empty virus (pLXSN) were induced to differentiate with a 48-h treatment with 5-Azacytidine (AzaC) and cultured for 27 d. Cells were fixed and stained with Alizarin red or Oil red O. Images are representative of three independent experiments. B, Semiquantitative analysis of osteoblastic gene expression by RT-PCR from cells treated as in panel A. PPAR{gamma} is an adipocyte marker and glyceraldehyde-3-phosphate (GAPDH) is a loading control. C, Quantification of RT-PCR results from panel B.

 
C/EBPß Overexpression Decreases Osteoblastogenesis of Mesenchymal Precursor Cells But Not Preosteoblasts
To determine whether the repression of early osteoblast differentiation by ectopically expressed C/EBPß was a property of the mesenchymal stem cell line, we used retroviral transduction to overexpress C/EBPß in MC3T3-E1 preosteoblast cells. These cells are already committed to the osteoblast lineage and differentiate efficiently and rapidly into osteoblasts after treatment with an induction cocktail containing ascorbic acid, ß-glycerophosphate and the synthetic glucocorticoid dexamethasone. Twenty-four hours after treatment with this cocktail, we measured the induction of alkaline phosphate using an in vitro enzymatic assay. Similar to what was observed in C3H10T1/2 cells, ALP activity was significantly reduced in cells overexpressing C/EBPß when compared with empty virus controls (Fig. 3AGo). Cultures allowed to differentiate for 3 wk were stained with Alizarin red to assess calcium deposition in the extracellular matrix. In contrast to C3H10T1/2 cells, there were no differences in Alizarin red staining of C/EBPß-overexpressing MC3T3-E1 cells and control cultures (Fig. 3BGo). In addition, Runx2 expression, as assessed by Western blot, was also equivalent between the test and control cultures (Fig. 3CGo), suggesting that C/EBPß, although impacting on ALP activity, was unable to repress osteoblastic differentiation of preosteoblasts.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 3. C/EBPß Overexpression Does Not Repress Osteoblastic Differentiation in MC3T3-E1 Preosteoblasts

A, MC3T3-E1 cells, transduced with retrovirus to express C/EBPß or with empty virus (pLXSN), were induced to differentiate with dexamethasone (DEX), ascorbic acid, and ß-glycerophosphate (AB) for 24 h, after which cells were harvested for ALP activity. ALP activity was standardized for cellular protein content. B, Alizarin red staining of cultures transduced and induced to differentiate as in panel A. Three weeks after induction, cells were fixed and stained. C, Western analysis of Runx2 and C/EBPß protein levels in MC3T3-E1 cultures differentiated as in panel B. Actin is a loading control.

 
C/EBPß Represses Transcription from the Osteoblast-Specific Runx2 P1 Promoter
Because ectopic C/EBPß affects the expression of Runx2 and its target genes (ALP, osteopontin) in C3H10T1/2 cells, we hypothesized that C/EBPß may be acting to interfere with transcription from the Runx2 locus. The osteoblast-specific Runx2 P1 promoter contains putative DNA binding elements for several transcription factors including two potential C/EBP motifs located at –45/–35 and at –591/–576, designated site I and site II, respectively (Fig. 4AGo). To evaluate the potential role of these two elements in the control of Runx2 transcription, we transiently transfected NIH 3T3 cells with a –976 /+111 fragment of the Runx2 P1 promoter cloned upstream of a luciferase reporter gene (16). NIH 3T3 cells do not express significant endogenous C/EBPß (Fig. 4BGo). Cotransfection of the P1 reporter construct and a C/EBPß expression vector resulted in a 50% decrease in reporter transcription relative to controls, suggesting that C/EBPß acts as a negative regulator of Runx2 transcription (Fig. 4CGo). In contrast, ectopic C/EBPß stimulated transcription from the C/EBP{alpha} promoter 2-fold, consistent with previously published results (Fig. 4DGo) (27). In the pluripotent cell line, C3H10T1/2 ectopic expression of C/EBPß did not repress transcription from the Runx2 P1 promoter (Fig. 4EGo). However, C3H10T1/2 cells express readily detectable levels of C/EBPß protein, suggesting that the Runx2 P1 promoter is already repressed by endogenous C/EBPß in these cells (Fig. 4Go, B and E). Together, these results suggest that C/EBPß acts as a transcriptional repressor of the Runx2 P1 promoter.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 4. C/EBPß Is a Repressor of Runx2 Transcription

A, Schematic representation of the mouse –976/+111 Runx2 P1 promoter reporter construct. Putative C/EBP sites and their locations are indicated. B, Western analysis of C/EBPß protein levels in NIH 3T3 and C3H10T1/2 cells. Actin is used as a loading control. C, Transient transcription assay. NIH 3T3 cells were transfected with the Runx2 reporter construct and C/EBPß expression vector as indicated. Standard luciferase assay was performed on extracts prepared 48 h after transfection. Luciferase activity was corrected for transfection efficiency using a cotransfected pRSV-ßgal construct. Data are represented as a percent of the activity of cells not expressing ectopic C/EBPß. D, Transient transcription assay as in panel C using the –350/+7 C/EBP{alpha} reporter construct. E, Transient transcription experiment as in panel C performed in C3H10T1/2 cells.

 
C/EBPß Can Bind the Runx2 P1 Promoter in Vitro and in Vivo
To determine whether the repressive effects of C/EBPß on Runx2 P1 promoter activity were mediated through direct interaction with the cognate promoter sequences, we performed chromatin immunoprecipitation (ChIP) in C3H10T1/2 cells. As shown in Fig. 5AGo, the Runx2 P1 promoter coprecipitated with endogenous C/EBPß indicating that C/EBPß does indeed occupy the promoter in these cells. Control experiments with a type matched nonspecific antibody did not precipitate the Runx2 promoter, demonstrating the specificity of this interaction.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. C/EBPß Interacts with the Runx2 Promoter

A, ChIP of the endogenous mouse Runx2 promoter with anti-C/EBPß antibody performed in C3H10T1/2 cells. A type matched nonspecific antibody (NS) was used as a negative control. Inputs represent approximately 25% of the material used for immunoprecipitation. B, ABCD assay to assess binding of C/EBPß from C3H10T1/2 extracts to biotinylated double-stranded DNA oligos encoding the two putative C/EBP sites (sites I and II). An oligo from an area of the Runx2 P1 promoter not predicted to bind C/EBP factors (NS) was used as a negative control. Inputs represent 10% of the whole cell extract used for binding. C, ABCD assay to assess binding of C/EBPß to biotinylated double-stranded DNA oligos encoding the site II or the site II mutant (siteIImt). D, Transient transcription assay using the Runx2 promoter reporter construct (wt) or reporter constructs with mutations in the C/EBP motifs performed in C3H10T1/2 cells.

 
Because the two potential C/EBP DNA binding motifs within the P1 promoter are in close proximity to one another (<600 bp), it was not possible to determine the relative occupancy of site I vs. site II using ChIP analyses. We therefore performed an ABCD (avidin-biotin conjugated DNA) assay, which assesses the interaction of endogenous proteins with biotinylated double-stranded DNA oligonucleotides containing the motif(s) of interest. Interaction with the DNA oligonucleotide is measured by Western analysis after the precipitation of the DNA fragment with streptavidin-coated beads. Incubation of DNA oligonucleotides containing either site I or site II or a region of the promoter containing no putative C/EBP elements (–363/–337) with C3H10T1/2 whole cell lysates revealed that C/EPBß can bind to site II but not site I or the control oligonucleotide (Fig. 5BGo). Thus, the C/EBP element located at –591/–576 may act as a negative regulatory element within the Runx2 P1 promoter.

To determine whether the interaction of C/EBPß with the Runx2 promoter impacts on promoter activity, we mutated site II to abolish C/EBPß binding. Loss of binding to the mutated oligonucleotide was verified by ABCD assay (Fig. 5CGo). When transiently transfected into C3H10T1/2 cells, the site II mutant demonstrated a 9-fold increase in basal transcription from the Runx2 promoter when compared with the wild-type promoter (Fig. 5DGo). In contrast, mutation of site I, which does not bind C/EBPß, had no effect on basal transcription from the P1 sequences (Fig. 5DGo). These results are consistent with the observation that C/EBPß represses Runx2 promoter activity by direct interaction with site II.

Repression of Transcription from the Runx2 Promoter by C/EBPß Is Independent of Deacetylase Activity
C/EBPß has been shown to interact with many coregulatory molecules including a corepressor complex which restrains its transcriptional potential through HDAC1 recruitment to promoter elements (26, 27, 29, 30). To evaluate a potential role for deacetylases in C/EBPß-mediated repression of the Runx2 P1 promoter, we repeated the transient transcription experiment in NIH 3T3 cells and included treatment with dexamethasone, a synthetic glucocorticoid that stimulates the titration of C/EBPß-associated HDAC1 via the 26S proteasome, and with trichostatin A, a histone deacetylase inhibitor (27). Treatments with dexamethasone or trichostatin A failed to reverse C/EBPß-mediated repression of the Runx2 promoter, suggesting that repression of Runx2 transcription by C/EBPß occurs through a class I and II HDAC-independent mechanism (Fig. 6Go).


Figure 6
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 6. Repression of cbfa1 Transcription by C/EBPß Is Independent of Deacetylase Activity

Transient transcription assay using the wild-type Runx2 promoter and ectopic C/EBPß in NIH 3T3 cells. Twenty-four hours after transfection, cells were treated with vehicle, 10–6 M dexamethasone (DEX) or 160 nM trichostatin A (TSA) as indicated.

 
ATRA Triggers the Displacement of C/EBPß from the Runx2 P1 Promoter
Given that pharmacological inhibition of deacetylase activity was unable to reverse C/EBPß-mediated repression of the Runx2 P1 promoter, we considered the possibility that occupancy of the promoter by C/EBPß may block access of positive acting transcription factors necessary for Runx2 expression. Indeed, this type of mechanism has been described for many promoters, including repression of C/EBP{alpha} transcription by CUP (32). Because ATRA stimulates the expression of Runx2 and the early osteoblastic differentiation of C3H10T1/2 cells, we investigated the occupancy of the P1 promoter by C/EBPß after ATRA treatment (31). We performed ChIP analysis in C3H10T1/2 cells treated with vehicle or ATRA for 48 h. As observed previously, immunoprecipitated C/EBPß efficiently coprecipitated the Runx2 promoter indicating occupancy (Fig. 7AGo). Treatment with ATRA resulted in a 75% decrease in C/EBPß occupancy of the Runx2 promoter (Fig. 7Go, A and B). This loss of C/EBPß occupancy could not be attributed to changes in C/EBPß expression because its protein levels were stable during early differentiation (Fig. 7CGo). In addition, reduction of promoter occupancy was not observed after a 24 h ATRA treatment (data not shown), suggesting that ATRA effects the dissociation of C/EBPß from the Runx2 promoter via an indirect mechanism.


Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 7. ATRA Reduces C/EBPß Occupancy of the Runx2 Promoter

A, Chromatin immunoprecipitation of C/EBPß from C3H10T1/2 cells treated with vehicle or 10–6 M ATRA for 48 h. The antibodies used were anti-C/EBPß (ß) or a type matched nonspecific antibody (NS). Inputs represent approximately 25% of the material used for immunoprecipitation. B, Quantification of C/EBPß occupancy of the Runx2 promoter in C3H10T1/2 cells after retinoic treatment as in panel A. C, Western analysis of C/EBPß protein expression after a 24- or 48-h ATRA treatment or treatment with vehicle (0). Actin is used as a loading control.

 
The Effects of ATRA in C3H10T1/2 Cells Are Mediated through Retinoic Acid Receptor (RAR) {alpha} and {gamma}
Both RAR{alpha} and RAR{gamma} are detectable in C3H10T1/2 cells by Western blotting and their levels were unaffected by treatment with ATRA (Fig. 8AGo); RARß was undetectable in C3H10T1/2 cells even after ATRA treatment (data not shown). RAR{alpha} and RAR{gamma} functionally overlap in vivo, with concomitant null mutation of both transcription factors required to elicit a severe phenotype (33). To investigate the role of RAR{alpha} and RAR{gamma} in the displacement of C/EBPß from the Runx2 promoter, we first determined whether endogenous C/EBPß interacted with these receptors in C3H10T1/2 cells treated with vehicle or with ATRA for 4, 24, or 48 h, and found that both RAR{alpha} and RAR{gamma} coprecipitated with C/EBPß in a ligand-dependent fashion (Fig. 8BGo). Interestingly, this interaction is likely to be indirect as GST-C/EBPß did not interact with in vitro-translated RARs (data not shown). Moreover, although a physical interaction occurs between RAR{alpha}/{gamma} and C/EBPß in C3H10T1/2 cells, these receptors are not recruited to the Runx2 P1 promoter, as assessed by ChIP, though they did efficiently coprecipitate with the c-jun promoter as previously demonstrated (Fig. 8CGo) (34). The C/EBPß-displacing effects of ATRA are receptor specific as cotreatment with the RAR{alpha} antagonist Ro 41-5253 efficiently blocked C/EBPß displacement from the Runx2 promoter, indicating that the effects of retinoid treatment are mediated at least in part by RAR{alpha} (Fig. 8Go, D and E).


Figure 8
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 8. The Effects of ATRA Are Mediated by RAR{alpha}

A, Analysis of RAR expression in C3H10T1/2 cells treated for the indicated times with ATRA or vehicle and harvested for Western blotting. Actin is used as a loading control. B, Coimmunoprecipitation of RAR{alpha} and RAR{gamma} with endogenous C/EBPß in C3H10T1/2 cells hormone treated as in panel A. C, Chromatin immunoprecipitation of RAR{alpha}/{gamma} with the Runx2 promoter and the c-jun promoter in C3H10T1/2 cells after a 48-h treatment with ATRA or vehicle as indicated. Inputs represent approximately 25% of the material used for immunoprecipitation. D, Occupancy of the Runx2 promoter by C/EBPß as measured by ChIP after a 48-h treatment with vehicle, ATRA or ATRA and 10–6 M Ro 41–5253 (RAR{alpha} antagonist). Inputs represent approximately 25% of the material used for immunoprecipitation. E, Quantification of C/EBPß occupancy ChIPs performed in panel D by PhosphorImager analysis.

 
Overexpressed C/EBPß Is Retained at the Runx2 P1 Promoter after ATRA Treatment
If ATRA triggers the dissociation of C/EBPß from the Runx2 P1 promoter, overexpression of C/EBPß in C3H10T1/2 cells may compromise osteoblastic differentiation by remaining associated with the P1 promoter. Using ChIP analyses, we evaluated the interaction of ectopically expressed C/EBPß with the Runx2 promoter in C3H10T1/2 cells that had been treated with vehicle or ATRA for 48 h. As in control cultures, in C/EBPß-overexpressing cells we observe occupancy of the Runx2 promoter in cells treated with vehicle (Fig. 9AGo). After ATRA treatment we observe only modest reduction in C/EBPß occupancy, which was not statistically significant (Fig. 9Go, A and B). Thus the level of C/EBPß overexpression in these cells (2-fold) may be sufficient to saturate the ATRA mechanism that normally acts to dislodge this factor from the Runx2 promoter. If so, overexpression of RAR in these cells would be anticipated to restore the sensitivity to ATRA and result in the displacement of exogenous C/EBPß. To assess this, we used ChIP to directly measure C/EBPß occupancy on the Runx2 promoter in cells overexpressing C/EBPß in the presence or absence of transfected RAR{alpha}. In RAR{alpha}-transfected cells, we observed a 50% decrease in C/EBPß occupancy upon ATRA treatment compared with mock transfected cells (Fig. 9Go, C and D). These results are consistent with a role for RAR in controlling transcription of Runx2 through titration of promoter-bound C/EBPß.


Figure 9
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 9. Ectopically Expressed C/EBPß Is Retained at the Runx2 Promoter after ATRA Treatment

A, ChIP of the Runx2 promoter in C3H10T1/2 cells retrovirally transduced to express C/EBPß and treated for 48 h with vehicle or 10–6 M ATRA. Inputs represent approximately 25% of the material used for immunoprecipitation. B, Quantification of C/EBPß occupancy of the Runx2 promoter by ChIP after retinoic treatment as in panel A. C, ChIP of C/EBPß occupancy of the Runx2 promoter after transient RAR{alpha} overexpression. D, Quantification of C/EBPß occupancy of the Runx2 promoter as in panel B.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
CCAAT/enhancer binding proteins, and in particular C/EBPß, have been implicated in the transcriptional control of various differentiation processes including adipogenesis and liver regeneration (19, 21). Here, we present evidence suggesting that C/EBPß regulates osteoblastogenesis through direct repression of the Runx2 P1 promoter. In vivo, ectopic expression of C/EBPß reduced Runx2 expression and a concomitant decrease in early osteoblast differentiation as measured by alkaline phosphatase activity and osteopontin expression. When cultures were allowed to differentiate for 4 wk, C/ EBPß overexpression resulted in reduced matrix mineralization, although osteoblast-specific gene expression was no longer perturbed. These observations suggest that overexpression of C/EBPß reduced, but did not completely abolish, Runx2 expression resulting in attenuated osteoblastogenesis during early differentiation stages. In the longer term, C/EBPß overexpressing cultures appeared to catch up to the empty vector controls in terms of osteoblast gene expression, although prolonged culture times may be needed to achieve maximal calcium deposition. Because the Runx2 P1 promoter is autoregulated, the low levels of Runx2 expression in C/EBPß overexpressing cultures may accumulate and eventually assume dominant regulation of the promoter. Given these observations, it is possible that higher levels of C/EBPß may produce stronger repression of late osteoblastic differentiation in these cells.

The mechanism by which ATRA triggers the displacement of C/EBPß remains unknown. Classical ATRA response elements are absent from the Runx2 promoter and prolonged exposure to ATRA is required to dislodge C/EBPß from the promoter. Together, these observations suggest that ATRA acts indirectly to stimulate Runx2 expression. In this regard, we have observed that C/EBPß and RAR{alpha} and {gamma} interact in vivo in a ligand-dependent fashion, but do not form a complex on the Runx2 promoter. This DNA-independent interaction may serve to sequester C/EBPß away from promoter response elements (Fig. 10AGo). Upon hormone treatment, C/EBPß that is not associated with DNA can interact with RAR{alpha}/{gamma}. With prolonged treatment, more and more C/EBPß may be complexed, resulting in a titration of this factor from the Runx2 promoter. Alternatively, ATRA may act via RAR{alpha}/{gamma} to up-regulate the expression of a secondary displacement factor that may interact with C/EBPß and trigger its dissociation from the Runx2 promoter (Fig. 10BGo). One such factor is C/EBP homologous protein (CHOP), which has been shown to be induced by ATRA treatment in myeloid cells and can bind C/EBP factors preventing their interaction with DNA (35). Our data also does not exclude the possibility that activating factors are required to stimulate transcription from the Runx2 P1 promoter and these factors may themselves be directly induced by ATRA treatment, and such regulators may also serve to displace C/EBPß from the Runx2 promoter (Fig. 10CGo).


Figure 10
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 10. Proposed Model for the Regulation of Runx2 Transcription by C/EBPß and ATRA

In the absence of ATRA and presence of C/EBPß expression, C/EBPß occupies the Runx2 P1 promoter at site II (–591/–576) and transcription from the promoter is inhibited. Upon ATRA treatment, C/EBPß is displaced from the promoter, an event that corresponds with an increase in Runx2 expression. We propose three possible mechanisms for ATRA action. A, Ligand-dependent interaction of RAR{alpha} and/or {gamma} with C/EBPß may sequester C/EBPß away from DNA thereby preventing C/EBPß from interacting with the Runx2 promoter. B, The stimulation of transcription of ATRA-responsive genes by RAR{alpha}/{gamma} upon ligand treatment induces the expression of a secondary factor which could interact with C/EBPß and prevent/destabilize C/EBPß:DNA interactions. C, The stimulation of transcription of an ATRA-responsive gene may produce a factor that stimulates Runx2 gene expression. Its interaction with the promoter may dislodge C/EBPß and prevent its reassociation.

 
Studies into the role of C/EBP factors during osteoblast differentiation have produced varying results. Our observation that C/EBPß attenuates RunX2 promoter activity is in accordance with previously reported studies that indirectly interfere with C/EBP factor DNA binding activity through the overexpression of Fra1 or {Delta}FosB (36, 37). Similarly, CHOP null mutant mice have decreased bone formation rates and reduced collagen and osteocalcin expression (38). These mouse models provide strong biological evidence that C/EBP activity attenuates osteoblast differentiation and function. In addition, in uncommitted precursor cells, CHOP overexpression represses adipogenesis and stimulates osteoblast differentiation (39, 40). In accordance with these observations, we identified an early defect in osteoblastogenesis in C3H10T1/2 cells overexpressing C/EBPß stimulated to differentiate with either ATRA or 5-azacytidine.

Several lines of evidence point to a dual role for C/EBPß during osteoblastogenesis. In committed preosteoblastic MC3T3-E1 cells, C/EBPß overexpression has no effect on the efficiency of osteoblast differentiation, whereas ectopic CHOP expressed in osteoblastic cells reduces osteoblast gene expression, in contrast to studies in uncommitted cells (40, 41). In preosteoblasts, C/EBPß and Runx2 have been shown to act together to stimulate the expression of osteocalcin in osteoblasts and a cooperative interaction between the negative acting C/EBPß isoform liver-enriched inhibitory protein and Runx2 has also been described (42, 43).

To consolidate the above observations, we propose the following model. In uncommitted cells, C/EBPß binds to its negative response element within the Runx2 P1 promoter and represses transcription. The consequence of this interaction would be to prevent expression of Runx2, which would trigger inappropriate osteoblastic differentiation of the precursor cell. C/EBPß remains at the promoter until inducing factors, such as ATRA, trigger its displacement from the response element. Loss of C/EBPß occupancy, possibly coupled with the arrival of positive-acting transcription factors at the promoter, results in an increase in Runx2 expression, which in turn stimulates osteoblastogenesis. Promoter occupancy of such positive acting transcription factors and/or posttranslational modification of C/EBPß may prevent its reassociation with the negative DNA element. Once transcription from the P1 promoter is initiated and Runx2 is expressed, it can autoregulate its own production, presumably in a manner that excludes C/EBPß (3, 9, 15). Runx2 and C/EBPß can then act together to stimulate the transcription of osteoblastic genes such as osetocalcin (35). Because C/EBPß protein levels are stable during the induction of osteoblast differentiation in C3H10T1/2 cells, the switch from a transcriptional repressor to a transcriptional activator is modulated by the ability of C/EBPß to both associate with relevant DNA response elements and its interactions with Runx2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Expression plasmids for C/EBPß and RAR{alpha} and the wild-type C/EBP{alpha}-luciferase reporter gene (–350/+7) and the wild type –976/+111 Runx2 promoter have been described previously (16, 27, 44). Site-directed mutations in the Runx2 promoter were accomplished using the QuikChange Mutagenesis kit (Stratagene, La Jolla, CA) using the following primers: 5'- CCTCTCCAGTAATAGTCGTCTCGAGAAAAA-3' (site I), 5'-CCACACACTCAGTTGAGATCTTTTTG-3' (site II).

Retroviral Infection and Cellular Differentiation
C3H10T1/2 cells were maintained in DMEM containing 4.5 g liter–1 glucose supplemented with 10% fetal bovine serum in 5% CO2. MC3T3-E1 cells were maintained in {alpha}MEM lacking ascorbic acid and supplemented with 10% fetal bovine serum. Replication incompetent pLXSN-based (CLONTECH, Palo Alto, CA) retroviruses were generated in Phoenix Ampho (ATCC, Manassas, VA) packaging cells. 10 cm dishes of 50% confluent cells were infected using 1 ml of viral supernatant in the presence of 4 µg µl–1 polybrene (Sigma, Oakville, Ontario, Canada). Cells were selected in media containing 400 µg µl–1 G418 (Invitrogen, Burlington, Ontario, Canada) for 10 d before differentiation to ensure retroviral expression in all cells.

To stimulate osteoblastogenesis, confluent C3H10T1/2 cells were treated with 10–6 M ATRA, whereas MC3T3-E1 cells were treated with 10–6 M dexamethasone, 50 µg/ml ascorbic acid, and 10 mM ß-glycerophosphate. In both cases, media were refreshed every 3 d.

Analysis of Osteoblastic and Adipogenic Phenotypes
Alkaline phosphatase staining was performed according to manufacturer’s instructions (Sigma kit 85) and was assessed 6 d after induction to differentiate. Alkaline phosphatase activity was determined in extracts by hydrolysis of p-nitrophenyl phosphate to p-nitrophenol according to manufacturer’s instructions. Data were corrected for protein content using a standard Bradford assay and are expressed as a percent of activity observed in C3H10T1/2 cells transduced with empty virus. Alizarin Red staining and Oil red O staining were performed as described (27, 45). To assess expression of osteoblast and adipocyte markers, the following antibodies were used in Western blot analyses: anti-AML3 (Calbiochem, La Jolla, CA), C/EBPß C-19, osteopontin Akm2A1, RAR{gamma} (all Santa Cruz Biotechnology, Santa Cruz, CA), RAR{alpha} (Affinity Bioreagents, Golden, CO) and actin (Sigma). For RT-PCR, RNA was extracted using the RNeasy kit (QIAGEN, Mississauga, Ontario, Canada) and was reverse transcribed using oligo-deoxythymidine and Superscript III (Invitrogen) according to manufacturer’s instructions. PCRs were optimized to determine the linear phase of amplification and results were compared with glyceraldehyde-3-phosphate message. Primer sequences used for amplification are available upon request.

Analysis of Reporter Gene Expression
NIH 3T3 and C3H10T1/2 cells maintained under standard culture conditions were transfected using ExGen 500 (MBI Fermentas, Burlington, Ontario, Canada) or Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Quantities of 200 ng of reporter DNA and 400 ng of C/EBPß expression vector were used for transfection. Luciferase assays were performed according to standard protocol. Error bars represent the SEM of a minimum of three experiments performed in duplicate. Transfection efficiency was monitored by cotransfection of Rous sarcoma virus-ß-galactosidase expression plasmid and measured by ß-gal enzyme activity.

ABCD Assay
Five hundred nanograms of whole cell lysates from C3H10T1/2 cells were incubated with 2 µg of 5' biotin-tagged double-stranded oligo in binding buffer [20 mM HEPES (pH 7.7), 50 mM KCl, 20% glycerol, 0.1% Nonidet P-40] in the presence of 2 µg of sheared salmon sperm. DNA fragments used were: site I, site II, site II mutant, and region of the Runx2 promoter not predicted to bind C/EBP factors (nonspecific). Oligos were then immunoprecipitated with strepavidin-agarose beads (Sigma) and precipitates were washed extensively in binding buffer and resolved by SDS-PAGE. Binding was evaluated by Western blot analysis using anti-C/EBPß antibody (C-19; Santa Cruz Biotechnology).

ChIP Assay
C3H10T1/2 cells were treated as indicated in the figure legends for 24 h and cells were washed 2x in serum-free media and treated with 1% formaldehyde at room temperature for 10 min. ChIP was performed essentially as described (27) using C/EBPß C-19 or RAR M-454 (Santa Cruz Biotechnology) for precipitation at 4 C overnight. A type-matched nonspecific antibody was used as a negative control. DNA fragments were purified using the Qiaquick PCR purification kit (QIAGEN) and amplified by PCR using the following primers to amplify –126 to +83 within the murine Runx2 P1 promoter. Results shown are representative of a minimum of three independent experiments. Occupancy of the C/EBPß promoter was quantified using PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis and corrected for input levels. Error bars are representative of the SD across a minimum of three independent experiments.

Coimmunoprecipitation Assays
C3H10T1/2 cells treated with vehicle or ATRA for the indicated times were incubated overnight with anti-C/EBPß or nonspecific antibody in binding buffer. Complexes were precipitated with protein G-Sepharose beads for 1 h, followed by extensive washing with 30 mM HEPES (pH 7.5), 300 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 0.2 mM dithiothreitol. Complexes were analyzed by Western blot using Protein A-HRP to avoid detection of the heavy chain of the immunoprecipitating antibody. Data are representative of three independent experiments. Results were subject to analysis of variance and statistically significant results are indicated on the appropriate figures with their P values.


   ACKNOWLEDGMENTS
 
The authors thank Drs. P. Ducy, S. K. McKnight, I. S. Skerjanc, and G. Nolan for reagents and Dr. David Lohnes and S. Jeganathan for critical review of this manuscript.


   FOOTNOTES
 
This work was supported by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society (to J.M.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2007

Abbreviations: ABCD, Avidin-biotin conjugated DNA; ALP, alkaline phosphatase; ATRA, all-trans RA; C/EBP, CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; CHOP, C/EBP homologous protein; DEX, dexamethasone; GST, glutathione S-transferase; HDAC1, histone deacetylase 1; RA, retinoic acid; RAR, RA receptor; TSA, trichostatin A.

Received for publication October 31, 2006. Accepted for publication June 11, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T 1997 Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764[CrossRef][Medline]
  2. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ 1997 Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765–771[CrossRef][Medline]
  3. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G 1997 Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754[CrossRef][Medline]
  4. Harada H, Tagashira S, Fujiwara M, Ogawa S, Katsumata T, Yamaguchi A, Komori T, Nakatsuka M 1999 Cbfa1 isoforms exert functional differences in osteoblast differentiation. J Biol Chem 274:6972–6978[Abstract/Free Full Text]
  5. Kern B, Shen J, Starbuck M, Karsenty G 2001 Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J Biol Chem 276:7101–7107[Abstract/Free Full Text]
  6. Alliston T, Choy L, Ducy P, Karsenty G, Derynck R 2001 TGF-ß-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation. EMBO J 20:2254–2272[CrossRef][Medline]
  7. Gersbach CA, Byers BA, Pavlath GK, Garcia AJ 2004 Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Exp Cell Res 300:406–417[CrossRef][Medline]
  8. Xiao ZS, Hinson TK, Quarles LD 1999 Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells. J Cell Biochem 74:596–605[CrossRef][Medline]
  9. Ducy P, Starbuck M, Priemel M, Shen J, Pinero G, Geoffroy V, Amling M, Karsenty G 1999 A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 13:1025–1036[Abstract/Free Full Text]
  10. Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, Albright S, Lindhout D, Cole WG, Henn W, Knoll JH, Owen MJ, Mertelsmann R, Zabel BU, Olsen BR 1997 Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 89:773–779[CrossRef][Medline]
  11. Geoffroy V, Corral DA, Zhou L, Lee B, Karsenty G 1998 Genomic organization, expression of the human CBFA1 gene, and evidence for an alternative splicing event affecting protein function. Mamm Genome 9:54–57[CrossRef][Medline]
  12. Xiao ZS, Thomas R, Hinson TK, Quarles LD 1998 Genomic structure and isoform expression of the mouse, rat and human Cbfa1/Osf2 transcription factor. Gene 214:187–197[CrossRef][Medline]
  13. Fujiwara M, Tagashira S, Harada H, Ogawa S, Katsumata T, Nakatsuka M, Komori T, Takada H 1999 Isolation and characterization of the distal promoter region of mouse Cbfa1. Biochim Biophys Acta 1446:265–272[Medline]
  14. Xiao ZS, Liu SG, Hinson TK, Quarles LD 2001 Characterization of the upstream mouse Cbfa1/Runx2 promoter. J Cell Biochem 82:647–659[CrossRef][Medline]
  15. Drissi H, Luc Q, Shakoori R, Chuva De Sousa Lopes S, Choi JY, Terry A, Hu M, Jones S, Neil JC, Lian JB, Stein JL, Van Wijnen AJ, Stein GS 2000 Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene. J Cell Physiol 184:341–350[CrossRef][Medline]
  16. Zambotti A, Makhluf H, Shen J, Ducy P 2002 Characterization of an osteoblast-specific enhancer element in the CBFA1 gene. J Biol Chem 277:41497–41506[Abstract/Free Full Text]
  17. Greenbaum LE, Cressman DE, Haber BA, Taub R 1995 Coexistence of C/EBP {alpha}, ß, growth-induced proteins and DNA synthesis in hepatocytes during liver regeneration. Implications for maintenance of the differentiated state during liver growth. J Clin Invest 96:1351–1365[Medline]
  18. Grimm SL, Contreras A, Barcellos-Hoff MH, Rosen JM 2005 Cell cycle defects contribute to a block in hormone-induced mammary gland proliferation in CCAAT/enhancer-binding protein (C/EBPß)-null mice. J Biol Chem 280:36301–36309[Abstract/Free Full Text]
  19. Darlington GJ, Ross SE, MacDougald OA 1998 The role of C/EBP genes in adipocyte differentiation. J Biol Chem 273:30057–30060[Free Full Text]
  20. Cao Z, Umek RM, McKnight SL 1991 Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev 5:1538–1552[Abstract/Free Full Text]
  21. Schrem H, Klempnauer J, Borlak J 2004 Liver-enriched transcription factors in liver function and development. Part II: the C/EBPs and D site-binding protein in cell cycle control, carcinogenesis, circadian gene regulation, liver regeneration, apoptosis, and liver-specific gene regulation. Pharmacol Rev 56:291–330[Abstract/Free Full Text]
  22. Buck M, Poli V, Hunter T, Chojkier M 2001 C/EBPß phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival. Mol Cell 8:807–816[CrossRef][Medline]
  23. Park BH, Qiang L, Farmer SR 2004 Phosphorylation of C/EBPß at a consensus extracellular signal-regulated kinase/glycogen synthase kinase 3 site is required for the induction of adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes. Mol Cell Biol 24:8671–8680[Abstract/Free Full Text]
  24. Ron D, Habener JF 1992 CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev 6:439–453[Abstract/Free Full Text]
  25. Buck M, Zhang L, Halasz NA, Hunter T, Chojkier M 2001 Nuclear export of phosphorylated C/EBPß mediates the inhibition of albumin expression by TNF-{alpha}. EMBO J 20:6712–6723[CrossRef][Medline]
  26. Wiper-Bergeron N, Salem HA, Tomlinson JJ, Wu D, Hache RJ 2007 Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPß by GCN5. Proc Natl Acad Sci USA 104:2703–2708[Abstract/Free Full Text]
  27. Wiper-Bergeron N, Wu D, Pope L, Schild-Poulter C, Hache RJ 2003 Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex. EMBO J 22:2135–2145[CrossRef][Medline]
  28. Lamb J, Ramaswamy S, Ford HL, Contreras B, Martinez RV, Kittrell FS, Zahnow CA, Patterson N, Golub TR, Ewen ME 2003 A mechanism of cyclin D1 action encoded in the patterns of gene expression in human cancer. Cell 114:323–334[CrossRef][Medline]
  29. Mink S, Haenig B, Klempnauer KH 1997 Interaction and functional collaboration of p300 and C/EBPß. Mol Cell Biol 17:6609–6617[Abstract]
  30. Kowenz-Leutz E, Leutz A 1999 A C/EBPß isoform recruits the SWI/SNF complex to activate myeloid genes. Mol Cell 4:735–743[CrossRef][Medline]
  31. Gazit D, Ebner R, Kahn AJ, Derynck R 1993 Modulation of expression and cell surface binding of members of the transforming growth factor-ß superfamily during retinoic acid-induced osteoblastic differentiation of multipotential mesenchymal cells. Mol Endocrinol 7:189–198[Abstract/Free Full Text]
  32. Jiang MS, Lane MD 2000 Sequential repression and activation of the CCAAT enhancer-binding protein-{alpha} (C/EBP{alpha}) gene during adipogenesis. Proc Natl Acad Sci USA 97:12519–12523[Abstract/Free Full Text]
  33. Lohnes D, Mark M, Mendelsohn C, Dolle P, Dierich A, Gorry P, Gansmuller A, Chambon P 1994 Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120:2723–2748[Abstract]
  34. Borras E, Zaragoza R, Morante M, Garcia C, Gimeno A, Lopez-Rodas G, Barber T, Miralles VJ, Vina JR, Torres L 2003 In vivo studies of altered expression patterns of p53 and proliferative control genes in chronic vitamin A deficiency and hypervitaminosis. Eur J Biochem 270:1493–1501[Medline]
  35. Gery S, Park DJ, Vuong PT, Chih DY, Lemp N, Koeffler HP 2004 Retinoic acid regulates C/EBP homologous protein expression (CHOP), which negatively regulates myeloid target genes. Blood 104:3911–3917[Abstract/Free Full Text]
  36. Jochum W, David JP, Elliott C, Wutz A, Plenk Jr H, Matsuo K, Wagner EF 2000 Increased bone formation and osteosclerosis in mice overexpressing the transcription factor Fra-1. Nat Med 6:980–984[CrossRef][Medline]
  37. Kveiborg M, Sabatakos G, Chiusaroli R, Wu M, Philbrick WM, Horne WC, Baron R 2004 {Delta}FosB induces osteosclerosis and decreases adipogenesis by two independent cell-autonomous mechanisms. Mol Cell Biol 24:2820–2830[Abstract/Free Full Text]
  38. Pereira RC, Stadmeyer L, Marciniak SJ, Ron D, Canalis E 2006 C/EBP homologous protein is necessary for normal osteoblastic function. J Cell Biochem 97:633–640[CrossRef][Medline]
  39. Pereira RC, Delany AM, Canalis E 2004 CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation. Endocrinology 145:1952–1960[Abstract/Free Full Text]
  40. Tang QQ, Lane MD 2000 Role of C/EBP homologous protein (CHOP-10) in the programmed activation of CCAAT/enhancer-binding protein-ß during adipogenesis. Proc Natl Acad Sci USA 97:12446–12450[Abstract/Free Full Text]
  41. Shirakawa K, Maeda S, Gotoh T, Hayashi M, Shinomiya K, Ehata S, Nishimura R, Mori M, Onozaki K, Hayashi H, Uematsu S, Akira S, Ogata E, Miyazono K, Imamura T 2006 CCAAT/enhancer-binding protein homologous protein (CHOP) regulates osteoblast differentiation. Mol Cell Biol 26:6105–6116[Abstract/Free Full Text]
  42. Gutierrez S, Javed A, Tennant DK, van Rees M, Montecino M, Stein GS, Stein JL, Lian JB 2002 CCAAT/enhancer-binding proteins (C/EBP) ß and {delta} activate osteocalcin gene transcription and synergize with Runx2 at the C/EBP element to regulate bone-specific expression. J Biol Chem 277:1316–1323[Abstract/Free Full Text]
  43. Hata K, Nishimura R, Ueda M, Ikeda F, Matsubara T, Ichida F, Hisada K, Nokubi T, Yamaguchi A, Yoneda T 2005 A CCAAT/enhancer binding protein ß isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes. Mol Cell Biol 25:1971–1979[Abstract/Free Full Text]
  44. Legraverend C, Antonson P, Flodby P, Xanthopoulos KG 1993 High level activity of the mouse CCAAT/enhancer binding protein (C/EBP{alpha}) gene promoter involves autoregulation and several ubiquitous transcription factors. Nucleic Acids Res 21:1735–1742[Abstract/Free Full Text]
  45. Wan DC, Shi YY, Nacamuli RP, Quarto N, Lyons KM, Longaker MT 2006 Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling. Proc Natl Acad Sci USA 103:12335–12340[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
S. Zanotti, L. Stadmeyer, A. Smerdel-Ramoya, D. Durant, and E. Canalis
Misexpression of CCAAT/enhancer binding protein beta causes osteopenia
J. Endocrinol., May 1, 2009; 201(2): 263 - 274.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Zhong, H. J. Armbrecht, and S. Christakos
Calcitonin, a Regulator of the 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene
J. Biol. Chem., April 24, 2009; 284(17): 11059 - 11069.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wiper-Bergeron, N.
Right arrow Articles by Lee, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wiper-Bergeron, N.
Right arrow Articles by Lee, J. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals