Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M002031200 on June 19, 2000

J. Biol. Chem., Vol. 275, Issue 37, 29066-29075, September 15, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/37/29066    most recent
M002031200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, Y. L.
Right arrow Articles by Snead, M. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, Y. L.
Right arrow Articles by Snead, M. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Functional Antagonism between Msx2 and CCAAT/Enhancer-binding Protein alpha  in Regulating the Mouse Amelogenin Gene Expression Is Mediated by Protein-Protein Interaction*

Yan Larry Zhou, Yaping Lei, and Malcolm L. SneadDagger

From the The Center for Craniofacial Molecular Biology, The University of Southern California, Los Angeles, California 90033

Received for publication, March 10, 2000, and in revised form, June 14, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha  (C/EBPalpha ) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBPalpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBPalpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBPalpha , -beta , and -gamma ), Msx2 preferentially interacts with C/EBPalpha . Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Enamel is the only epithelially derived calcified tissue in vertebrates. Amelogenin, the major organic component of enamel matrix, is essential to the proper regulation of enamel mineralization. Amelogenin proteins comprise ~90% of the enamel matrix proteins. Several mutations in the human X-chromosomal amelogenin gene have been identified from patients with the inherited enamel defect X-linked Amelogenesis imperfecta (1-3). Disruption of amelogenin synthesis during tooth development with either antisense oligonucleotides or ribozymes results in disorganized enamel (4, 5). Amelogenin expression is ameloblast specific and developmentally regulated at the temporal and spatial level (6-13). A 2263-nucleotide proximal promoter element from the mouse X-chromosomal amelogenin gene has been demonstrated by transgenic mouse analysis to recapitulate the spatiotemporal expression pattern of the endogenous amelogenin gene (10). Extensive homologies (70% identity) in the 300-nucleotide region upstream of the transcription initiation site exist between the murine, bovine, and human X-chromosomal amelogenin gene, suggesting that this region is likely involved in the transcriptional regulation of tissue-specific amelogenin gene expression. In a previous study, the minimal promoter of the mouse amelogenin gene (-70/+52) was identified, which contains a CCAAT/enhancer-binding protein (C/EBP)1 consensus binding site, and C/EBPalpha activated amelogenin transcription in a cell-type specific manner through binding to its cognate site (14).

The C/EBPs consist of a family of related basic region leucine zipper transcription factors that are critical regulators of cellular differentiation and function in multiple tissues. Six different members of the family (C/EBPalpha , -beta , -gamma , -delta , -epsilon , and -zeta ) have been isolated and characterized. The expression of C/EBPs is tissue- and stage-specific during development. C/EBPs have been shown to play a key role in regulating cellular differentiation, terminal function, and response to inflammatory insults (15-19).

The Msx2 gene family (20) is the mammalian counterpart of the Drosophila msh (muscle segment homeobox) gene. Three unlinked members, Msx1 (21, 22), Msx2 (23), and Msx3 (24), have homeobox sequences very similar to each other and to the Drosophila msh gene. The murine Msx3 is expressed only in the dorsal neural tube (25-27), which appears to exclude the possibility of functional redundancy of Msx3 on the role of Msx1 and Msx2 in tooth development. During odontogenesis, Msx1 is expressed at all stages in dental mesenchymal cells but not in epithelial cells (28, 29). The expression pattern of Msx2 changes with the differentiation of different germ layers. Msx2 is strongly expressed in undifferentiated inner enamel epithelia in which amelogenin expression is barely detectable; but Msx2 is absent in differentiated ameloblasts in which robust expression of amelogenin is detected. On the other hand, Msx2 is weakly expressed/absent in undifferentiated dental papilla mesenchyme, whereas it is strongly expressed in odontoblasts and differentiated dental papilla cells (30). Msx2 has been shown to function as a transcriptional repressor independent of its intrinsic DNA binding activity through the homeodomain. Instead, the repression is mediated by protein-protein interaction with either components of basal transcription machinery or other transcription factors (31-35).

To investigate the role of Msx2 in the regulation of amelogenin gene expression, various amelogenin-promoter reporter constructs were transiently transfected into ameloblast-like LS8 cells with a Msx2 expression plasmid. The functional relationship between Msx2 and C/EBPalpha , a transcriptional activator of amelogenin, was examined with cotransfection assays. The potential of Msx2 to interfere with the binding of C/EBPalpha to its cognate site on the amelogenin minimal promoter as well as the ability of Msx2 itself to bind to the promoter was assessed with electrophoresis mobility shift assays (EMSA). Whether Msx2 is able to interact with C/EBPalpha in LS8 cells was further determined with co-immunoprecipitation analyses. The functional antagonism between Msx2 and C/EBPalpha was tested on a stably transfected amelogenin-promoter reporter construct. Finally, the ability of Msx2 to interact with two other C/EBP family members, C/EBPbeta and C/EBPgamma , was examined with cotransfection assays.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Plasmids-- A mouse ameloblast cell line (LS8) established by immortalizing primary cultures of enamel organ epithelium with SV40 large T antigen was maintained in Dulbecco's Modified Eagle's Medium (Sigma) supplemented with fetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 µg/ml) (36). The mouse amelogenin-promoter reporter constructs were described in a previous study (14). The expression plasmids of Msx2FL, MsxDelta N, and Msx2Delta C (31) were generously provided by Dr. Dwight Towler (Washington University).

Transfection and Luciferase Assay-- Transient transfection and luciferase assays were performed as described previously (14). Stable cell line LS8/p2207 was established by transfecting LS8 cells with amelogenin-promoter reporter construct p2207. After selection with hygromycin (750 ng/ml), the resistant colonies were isolated, expanded, and screened with luciferase assays for reporter gene activity. Seven clones with luciferase activity 1,000-10,000-fold of that in parental LS8 cells were selected for further studies. One of the selected clones with the highest basal luciferase activity was designated LS8/p2207 and maintained in the presence of hygromycin (750 ng/ml).

Fluorescence-activated Cell Sorting (FACS)-- The plasmid pCMV-lacZ (0.2 µg) was cotransfected into LS8/p2207 cells with an empty vector pcDNA3 (4 µg), a Msx2 expression plasmid pcMsxFL (2 µg) plus pcDNA3 (2 µg), a C/EBPalpha expression plasmid pcC/EBPalpha (2 µg) plus pcDNA3 (2 µg), and pcMsx2FL (2 µg) plus pcC/EBPalpha (2 µg) in 60-mm plates. Twenty four hours after transfection, cells were harvested, washed in phosphate-buffered saline (pH 7.4), resuspended in 50 µl of staining medium (phosphate-buffered saline, 4% fetal bovine serum, and 10 mM HEPES, pH 7.4), and warmed at 37 °C for 5 min. An equal volume (50 µl) of prewarmed (37 °C) 2 mM di-beta -D-galactopyranoside (Molecular Probes) was added, mixed, and incubated at 37 °C for 1 min. The mixture was then placed on ice, and 1 ml of ice-cold isotonic Dulbecco's modified Eagle's medium was added. The beta -galactosidase positive cells were sorted by flow cytometry (Moflo, Cytomation, Inc.).

Protein Purification-- An expression plasmid pQE-Msx2 was used to produce recombinant 6xHis-Msx2 protein. Bacterial culture, isopropyl-1-thio-beta -D-galactopyranoside induction, cell lysis, and nickel-nitrilotriacetic acid resin (QIAGEN) loading were performed according to the manual (77). The recombinant 6xHis-Msx2 protein was eluted with 20 ml of buffer D (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 5.9) followed by 20 ml of buffer E (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 4.5). Fractions (3 ml) from each elution were collected and analyzed by SDS-polyacrylamide gel electrophoresis. Fractions containing 6xHis-Msx2 protein were pooled and dialyzed sequentially against 8, 6, 4, 2, and 1 M urea and distilled H2O at 4 °C for 3 h each. After lyophilization, the sample was stored at 4 °C.

EMSA-- Double-stranded oligonucleotide probes were generated by annealing antisense strand to a 10-fold excess of sense strand and filling in with [alpha -32P]dATP (NEN Life Science Products) and Klenow (exo-). The GelShift buffer kit (Stratagene) was used for the binding reaction, which was performed as recommended by the manufacturer. The reaction mixtures were resolved in a 6% nondenaturing polyacrylamide gel provided in the kit. The gel was dried, and the bands were visualized by autoradiography. The sequences of the oligonucleotides were: amel antisense strand 5'-GAACAGCCAATCAGGTTTCTGAATGAA-3', amel sense strand 5'-TTTTTCATTCAGAAACCTGA TTGGCTGTTC-3' (nucleotide 1510-1539, GenBankTM accession number AF083091); msx2 antisense strand 5'-ACTTTGAACAGCCAATTAGT TTCTGAATGAA-3', msx2 sense strand 5'-TTCATTCAGAAACTAATTGGCTGTTCA-3'. Nuclear extracts were prepared from LS8 cells transfected with a C/EBPalpha expression plasmid, as described previously (14).

Immunoprecipitation and Western Blot Analysis-- LS8 cells, 80-90% confluent in a 100-mm cell culture plate, were collected in ice-cold radioimmune precipitation buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin (Sigma), and 1 mM sodium orthovanadate) and lysed by passing through a 22-gauge needle six times at 4 °C. After centrifugation at 3,000 rpm for 15 min at 4 °C, the protein concentration of the supernatant was determined using a Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin standards. A primary antibody (2 µg) was added to 1 ml of cell lysate (500 µg of total cellular protein) and incubated overnight at 4 °C with rotation. Twenty-five microliters of protein G PLUS-Agarose (Santa Cruz Biotechnology) was added and incubated for 6 h at 4 °C with rotation. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4 °C. Pellets were washed three times with 1.0 ml of radioimmune precipitation buffer and once with 1.0 ml of phosphate-buffered saline (pH 7.4) at 4 °C. After final wash, pellets were resuspended in an equal volume of 2× SDS loading buffer, boiled for 3 min, and stored at -80 °C. Samples (10 µl) were analyzed by Western blot as described previously (14).

In Vitro Expression of Protein with TNT-coupled Wheat Germ Extract System-- The in vitro transcription-coupled translation reactions were performed using a TNT T7-coupled wheat germ extract system, according to the manufacturer's recommendation (Promega). Briefly, 25 µl of TNT wheat germ extract, 2 µl of TNT reaction buffer, 1 µl of TNT RNA polymerase, 0.5 µl of amino acid mixture (minus leucine, 1 mM), 0.5 µl of amino acid mixture (minus methionine, 1 mM), 1 µl of RNasin ribonuclease inhibitor (40u/µl), 2 µl of template DNA (0.5 mg/ml), and 18 µl of nuclease-free H2O were added into a 0.6-ml centrifuge tube. The translation mixture was incubated at 30 °C for 90 min and then stored at -80 °C. The [35S]methionine was included in the reactions in a parallel experiment to determine the translation efficiency of individual protein.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Msx2 Was a Transcriptional Repressor of Mouse Amelogenin Promoter-- To determine whether Msx2 could regulate the promoter activity of the mouse amelogenin gene, a Msx2 expression plasmid was cotransfected into ameloblast-like LS8 cells with a series of 5'-deletion amelogenin-promoter reporter constructs. In a previous study, we demonstrated that the -70/+51 region of the mouse amelogenin promoter functions as a minimal promoter in LS8 cells (14). Exogenous Msx2 was able to down-regulate the promoter activity of each reporter construct so long as that construct contained the mouse amelogenin minimal promoter (p2207, p454, p349, p194, and p70 all contained the region -70/+51), whereas no inhibitory effect was observed on either the reporter construct p51 or the promoter-less construct pGL3-Basic (Fig. 1A). The reporter construct p51 by itself exhibited very little promoter activity in LS8 cells (Fig. 1A (14)). The effect of Msx2 on the full-length promoter (p2207) and the minimal promoter (p70) was further tested in LS8 cells with transient cotransfection assays. Transfection of different amounts of a Msx2 expression plasmid resulted in a roughly linear increase in the expression level of the exogenous Msx2 protein (Fig. 1B, middle panel, Msx2FL). Compared with that of the exogenous Msx2 protein (Msx2FL), the expression level of endogenous Msx2 protein (Msx2) in LS8 cells was very low (Fig. 1B, lower panel). In response to an increasing amount of exogenous Msx2 protein, the reporter gene activity was decreased up to 15-fold for p2207 and up to 7-fold for p70 (Fig. 1B), respectively. Furthermore, overexpression of Msx2 in LS8 cells had no effect on both the simian virus 40 (SV40) and the human cytomegalovirus immediate-early gene (CMV) promoter (data not shown). Taken together, these data indicated that Msx2 repressed the mouse amelogenin promoter in a dose-dependent manner in ameloblast-like LS8 cells.


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1.   Dose-dependent repression of the mouse amelogenin promoter by Msx2. A, the left panel summarizes the reporter constructs used. The numeral represents the number of nucleotides upstream of the transcription initiation site in the 5'-proximal region of the mouse amelogenin gene promoter. The transcription initiation site is indicated by an arrow. The amelogenin promoter region was subcloned into pGL3-Basic (Promega) to drive the reporter gene, luciferase. The right panel shows the results of transfection experiments. Equal amounts of various reporter constructs were transiently transfected into LS8 cells with or without a Msx2 expression plasmid. pCMV-lacZ was used as an internal control for transfection efficiency. The relative luciferase activity is the normalization of luciferase activity with beta -galactosidase activity. The mean ± S.D. from at least three independent experiments is represented, and the level of pGL3-Basic in the absence of Msx2 was set as 1. B, various amounts (31.25, 62.5, 125, 250, and 500 ng) of Msx2 expression plasmid were transiently cotransfected into LS8 cells with 250 ng of p2207 or p70 reporter construct. pCMV-lacZ was used as an internal control for transfection efficiency. The upper panel shows the results of transfection experiments. The relative luciferase activity is the normalization of luciferase activity with beta -galactosidase activity. The mean ± S.D. from at least three independent experiments is represented, and the level of p2207 in the absence of exogenous Msx2 was set as 1. The middle and lower panel show the results of Western blot analysis. Whole cell lysates were prepared 24 h after transfection. Comparable amounts of cell lysate were electrophoresesed, transferred to Immobilon-P membrane, and immunoblotted with a goat anti-actin antibody (Santa Cruz Biotechnology) and a rabbit anti-Msx2 antibody. The lower panel had a 30-fold longer exposure time than the middle panel to detect the endogenous Msx2 protein in addition to the exogenous FLAG-tagged Msx2FL protein.

C/EBPalpha and Msx2 Antagonized Each Other in Regulating Mouse Amelogenin Promoter Activity-- C/EBPalpha has been demonstrated to function as a transcriptional activator of the mouse amelogenin gene through its cognate binding site in the -70/-52 region of the mouse amelogenin promoter (14). To investigate the functional relationship between C/EBPalpha and Msx2 in regulating the mouse amelogenin promoter activity, C/EBPalpha and Msx2 expression plasmids were cotransfected into LS8 cells with either amelogenin-promoter reporter construct p2207 or p70. Three different Msx2 expression plasmids were used in the study to generate amino-terminal FLAG epitope-tagged Msx2 proteins. Msx2FL was a full-length protein, including murine Msx2 residues 2-267. Msx2Delta N was an amino-terminal deletion containing residues 98-267, whereas Msx2Delta C was a carboxyl-terminal deletion containing residues 2-183.

Increasing amounts of Msx2FL were able to attenuate the C/EBPalpha -mediated transactivation of amelogenin-promoter reporter construct p2207 (Fig. 2A, lanes 1-5) or p70 (Fig. 2B, lanes 1-5), whereas Msx2FL by itself was a potent transcriptional repressor. On the other hand, C/EBPalpha was capable of overcoming the repressive effect of Msx2FL on the reporter construct p2207 (Fig. 3A, lanes 1-5) or p70 (Fig. 3B, lanes 1-5) in a dose-dependent manner. As a first step to identify the functional domain within Msx2, two deleted forms of Msx2 were tested. A less potent repression by Msx2Delta N and little repression by Msx2Delta C on the basal promoter activity of p2207 (Fig. 2A, lanes 10 and 15) or p70 (Fig. 2B, lanes 10 and 15) were observed. Msx2Delta C had little effect on the C/EBPalpha -mediated transactivation of the mouse amelogenin-promoter reporter construct p2207 (Fig. 2A, lanes 11-14) and p70 (Fig. 2B, lanes 11-14), respectively. The antagonistic capacity of Msx2Delta N fell in between Msx2FL and Msx2Delta C on both reporter construct p2207 (Fig. 2A, lanes 6-9) and p70 (Fig. 2B, lanes 6-9). Similar amounts of C/EBPalpha transactivated p2207 (Fig. 3A, lanes 6-10 and 11-15) or p70 (Fig. 3B, lanes 6-10 and 11-15) to a greater extent in the presence of Msx2Delta N or Msx2Delta C than in the presence of Msx2FL. These transfection analyses indicated that there was a functional antagonism between Msx2 and C/EBPalpha in the transcriptional regulation of the mouse amelogenin gene. Furthermore, these data suggested that there was a difference in the activities of the two Msx2 deletion mutants.


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 2.   Functional antagonism of C/EBPalpha -mediated transactivation of the mouse amelogenin promoter by Msx2. In the presence of 250 ng of p2207 (A) or p70 (B) reporter construct, 500 ng of empty vector (pcDNA3), 250 ng of C/EBPalpha expression plasmid (pcC/EBPalpha ) with 250 ng of pcDNA3, 250 ng of pcC/EBPalpha with 125 ng of pcDNA3 plus 125 ng of Msx2FL expression plasmid (pcMsx2FL), 250 ng of pcC/EBPalpha with 250 ng of pcMsx2FL, and 250 ng of pcMsx2FL with 250 ng of pcDNA3 were cotransfected into LS8 cells, respectively. Similar parameters for transfection experiments were used for Msx2Delta N and Msx2Delta C, respectively. pCMV-lacZ was used as an internal control for transfection efficiency. The relative luciferase activity is the normalization of luciferase activity with beta -galactosidase activity. The mean ± S.D. from at least three independent experiments is represented, and the basal level of p2207 (A) and p70 (B) was set as 1, respectively.


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 3.   Functional antagonism of Msx2-mediated repression of the mouse amelogenin promoter by C/EBPalpha . In the presence of 250 ng of p2207 (A) or p70 (B) reporter construct, 500 ng of empty vector (pcDNA3), 250 ng of Msx2FL expression plasmid (pcMsx2FL) with 250 ng of pcDNA3, 250 ng of pcMsx2FL with 125 ng of pcDNA3 plus 125 ng of C/EBPalpha expression plasmid (pcC/EBPalpha ), 250 ng of pcMsx2FL with 250 ng of pcC/EBPalpha , and 250 ng of pcC/EBPalpha with 250 ng of pcDNA3 were cotransfected into LS8 cells, respectively. Similar parameters for transfection experiments were used for Msx2Delta N and Msx2Delta C, respectively. pCMV-lacZ was used as an internal control for transfection efficiency. The relative luciferase activity is the normalization of luciferase activity with beta -galactosidase activity. The mean ± S.D. from at least three independent experiments is represented, and the basal level of p2207 (A) and p70 (B) was set as 1, respectively.

Msx2 Interfered with the Binding of C/EBPalpha to Its Cognate Site on Mouse Amelogenin Promoter-- As a first step to understand the mechanism underlying the functional antagonism between Msx2 and C/EBPalpha , the effect of Msx2 on the binding of C/EBPalpha to the mouse amelogenin promoter was assessed with an EMSA. Various forms of Msx2 protein were generated using an in vitro transcription-coupled translation system (TNT-coupled wheat germ extract system, Promega). A functional C/EBP cognate site has been identified in the -70/-52 region of the mouse amelogenin promoter (14). A 32P-labeled double-stranded oligonucleotide containing the C/EBP cognate site (Fig. 4C, amel probe) was able to form a C/EBPalpha -containing protein-DNA complex using nuclear extracts prepared from LS8 cells overexpressing C/EBPalpha protein (Fig. 4A, lane 12). Only modest changes in the intensity of the C/EBPalpha -probe complex were observed when different amounts of either sham-treated TNT extracts (Fig. 4A, lanes 2 and 3) or TNT-expressed luciferase protein (Fig. 4A, lanes 10 and 11) were included in the EMSA reactions. On the contrary, TNT-expressed Msx2FL diminished the intensity of the EMSA complex to 20% (Fig. 4A, lanes 4 and 5). Reduction in the intensity of the EMSA complex was also observed for the two deleted forms of Msx2, down to 25% for Msx2Delta N (Fig. 4A, lanes 6 and 7) and 37% for Msx2Delta C (Fig. 4A, lanes 8 and 9). Furthermore, increasing amounts of purified full-length Msx2 protein was capable of reducing the intensity of the C/EBPalpha -probe complex to 27% (Fig. 4B, lanes 4-8), whereas the highest concentration of bovine serum albumin had little effect (Fig. 4B, lane 9). Neither purified Msx2 protein nor bovine serum albumin alone bound to the probe (Fig. 4B, lanes 2 and 3).


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 4.   Msx2-mediated interruption of the binding of C/EBPalpha to the mouse amelogenin promoter. A, nuclear extracts were prepared from near confluent LS8 cells transfected with a C/EBPalpha expression plasmid (NE/alpha ). The extracts were incubated with 32P-labeled double-stranded oligonucleotides, 5'-TTTTTCATTCAGAAACCTGATTGGCTGTTC-3', containing the murine amelogenin C/EBP binding site. Based on the incorporation of [35S]methionine, a comparable amount of Msx2FL, Msx2Delta N, Msx2Delta C, or luciferase protein was produced in TNT extract (Promega). Sham-treated TNT extract (TNT) and TNT extract expressing Msx2FL (TNT/FL), Msx2Delta N (TNT/Delta N), Msx2Delta C (TNT/Delta C), or luciferase protein (TNT/luc) was included in the reactions of EMSA, respectively. For each TNT extract, two doses with a 2-fold difference were used. Complexes were separated by electrophoresis and visualized by autoradiography. The intensity of the EMSA complex was quantified by densitometry and plotted. The density level of the EMSA complex in the absence of TNT extract was set as "100." B, the same nuclear extracts (NE/alpha ) and probe as described above were utilized. His-tagged Msx2 proteins (His-Msx2) purified by nickel-nitrilotriacetic acid resin (Promega) were included in the EMSA reactions. Four different doses of His-Msx2 (50, 100, 150, and 200 ng) or 200 ng of bovine serum albumin were used. Complexes were separated by electrophoresis and visualized by autoradiography. The intensity of the EMSA complex was quantified by densitometry and plotted. The density level of the EMSA complex in the absence of His-Msx2 or bovine serum albumin was set as 100. C, a 32P-labeled double-stranded oligonucleotide containing a consensus Msx2 binding site (msx2 probe) was used in addition to the probe described above (amel probe). Purified Msx2 protein (His-Msx2) and TNT extract expressing Msx2FL (TNT/FL), Msx2Delta N (TNT/Delta N), Msx2Delta C (TNT/Delta C), or luciferase protein (TNT/luc) was incubated with the two probes, respectively. Complexes were separated by electrophoresis and visualized by autoradiography. The sequences of the two probes were shown, with C/EBP- and Msx2 binding site in bold, respectively. The amelogenin probe (amel probe) was derived from the mouse amelogenin promoter (GenBankTM accession number AF083091).

To ascertain that both TNT-expressed and -purified Msx2 proteins were capable of binding to DNA, a 32P-labeled double-stranded oligonucleotide containing a Msx2 cognate site (Fig. 4C, msx2 probe) was used in gel shift analyses. The TNT-expressed full-length (Msx2FL) and amino-terminal deleted (Msx2Delta N) Msx2 protein efficiently bound to the msx2 probe (Fig. 4C, lanes 7, 8, and 12), whereas the TNT-expressed carboxyl-terminal deleted Msx2 protein (Msx2Delta C) and luciferase protein failed to form an EMSA complex, respectively (Fig. 4C, lanes 9 and 10). An EMSA complex with the msx2 probe was also observed for the purified Msx2 protein (Fig. 4C, lane 12). Neither of the various Msx2 proteins nor the luciferase control protein could bind to the amel probe that contained a C/EBP binding site instead of a Msx2 cognate site (Fig. 4C, lanes 2-5 and 11). The EMSA analyses indicated that Msx2 protein was able to interfere with the binding of C/EBPalpha to the mouse amelogenin promoter in a dose-dependent manner. Given the fact that Msx2 protein itself did not bind to the mouse amelogenin promoter, the observed interference appeared to result from protein-protein interaction instead of competition for binding to overlapping cognate sites on the promoter. Deletion of either amino- or carboxyl-terminal domain of Msx2 only had a modest effect on the ability of Msx2 to interfere with the binding of C/EBPalpha to the amelogenin promoter in the EMSA analyses (Fig. 4A). However, the same deletions resulted in a dramatic decrease in the antagonistic potency of Msx2 on C/EBPalpha in the transfection studies (Fig. 2).

Msx2 Interacted with C/EBPalpha in LS8 Cells-- To investigate whether Msx2 could interact with C/EBPalpha , a co-immunoprecipitation analysis was performed in LS8 cells. A C/EBPalpha expression plasmid was cotransfected into LS8 cells with an empty vector, Msx2FL, Msx2Delta N, and Msx2Delta C expression plasmid, respectively. Comparable amounts of C/EBPalpha protein were expressed in all four transfected cell populations (Fig. 5, lanes 1-4, panels III and IV). The C/EBPalpha protein was co-immunoprecipitated efficiently with Msx2FL (Fig. 5, lane 2, panel I) but to a lesser extent with Msx2Delta N (Fig. 5, lane 3, panel I). However, C/EBPalpha protein was barely detected in the Msx2Delta C-containing immunocomplex (Fig. 5, lane 4, panel I). A similar amount of FLAG-tagged Msx2 protein was present in each immunoprecipitated complex (Fig. 5, lanes 2-4, panel II), whereas no protein was detected in empty vector-transfected cells (Fig. 5, lane 1, panel II) using the same monoclonal anti-FLAG antibody (M2Ab) as that in the immunoprecipitation process. The reciprocal experiment was also performed, in which immunoprecipitation with a C/EBPalpha -specific antibody was followed by Western blot analysis using the anti-FLAG M2Ab (data not shown). The Msx2FL protein was readily detected in the C/EBPalpha -containing immunocomplex and so was Msx2Delta N to a less extent. However, the migration rate of the C/EBPalpha -specific antibody light chain was very close to that of Msx2Delta C in SDS-polyacrylamide gel electrophoresis, thereby compromising the detection of Msx2Delta C band in Western blot analyses. These data indicated that Msx2 interacted with C/EBPalpha at the protein level in LS8 cells. Moreover, the carboxyl-terminal domain (residues 183-267) of Msx2 was required, whereas the amino-terminal domain (residues 2-97) of Msx2 contributed somehow to the interaction between Msx2 and C/EBPalpha protein. Taken together with the transfection data in Figs. 2 and 3, the carboxyl-terminal domain of Msx2 was indispensable for the repressive effect as well as the antagonism between Msx2 and C/EBPalpha on the promoter activity of the mouse amelogenin gene, whereas the amino-terminal domain of Msx2 played a less critical role.


View larger version (57K):
[in this window]
[in a new window]
  		Fig. 5.   Co-immunoprecipitation of Msx2 and C/EBPalpha in LS8 cells. In a 100-mm cell culture plate, 2 µg of C/EBPalpha expression plasmid was cotransfected into LS8 cell with 2 µg of empty vector (lane 1, vector) or with the expression plasmid for Msx2FL (lane 2, Msx2FL), Msx2Delta N (lane 3, Msx2Delta N), Msx2Delta C (lane 4, Msx2Delta C). Whole cell lysates were prepared 24 h after transfection. Comparable amounts of cell lysate from four cell populations were electrophoresesed, transferred to Immobilon-P membrane, and immunoblotted with a C/EBPalpha -specific antibody (Santa Cruz Biotechnology; panel III). Equal loading was demonstrated using an anonymous invariant band (panel IV). Equal amounts of protein from the four cell lysates were subject to immunoprecipitation with a monoclonal anti-FLAG antibody (M2Ab, Sigma). The immunoprecipitates were then electrophoresesed, transferred to Immobilon-P membrane, and immunoblotted with a C/EBPalpha -specific antibody (panel I). After stripping, the same membrane was reprobed with the M2Ab (panel II). IP, immunoprecipitate; WB, Western blot.

Functional Antagonism between Msx2 and C/EBPalpha Was Observed on the 2.2-Kilobase Mouse Amelogenin Promoter in the Context of the Chromosome-- Several stable cell lines were established by stably transfecting the amelogenin-promoter reporter construct p2207 into LS8 cells. The basal level of luciferase activity in these stable transfected cells was 1,000- to 10,000-fold of that in the parental LS8 cells (data not shown), indicating that the 2.2-kilobase mouse amelogenin promoter in the context of chromosome was very efficient in directing the expression of the reporter gene luciferase. LS8/p2207, one of the cell lines with the highest basal luciferase activity, was selected for further studies on the Msx2- and C/EBPalpha -mediated regulation of the mouse amelogenin promoter. Transient transfection of a C/EBPalpha expression plasmid into LS8/p2207 cells resulted in a 3-4-fold increase in the reporter gene activity (Fig. 6A, lane 2), whereas little effect on the basal promoter activity was observed for each of the Msx2 constructs alone (Fig. 6A, lanes 3-5). Furthermore, cotransfection of Msx2FL with C/EBPalpha was able to decrease the C/EBPalpha -mediated transactivation from 3.3-fold to 2-fold of the basal activity, whereas either Msx2Delta N or Msx2Delta C interfered little with C/EBPalpha function (Fig. 6A, lanes 6-8).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6.   Functional antagonism between Msx2 and C/EBPalpha on the mouse amelogenin promoter in a stable cell line LS8/p2207. The reporter construct p2207 was transfected into LS8 cells to establish a stably transfected cell line LS8/p2207, in which the reporter gene luciferase is constitutively expressed at high level under the control of the 2.2-kilobase mouse amelogenin promoter. A, LS8/p2207 cells in 12-well plates were transfected, respectively, with equal amounts (500 ng) of empty vector (vector), C/EBPalpha expression plasmid (C/EBPalpha ) and Msx2 expression plasmids (Msx2FL, Msx2Delta N, and Msx2Delta C), or cotransfected with C/EBPalpha and Msx2 expression plasmid together (C/EBPalpha +Msx2FL, C/EBPalpha +Msx2Delta N, and C/EBPalpha +Msx2Delta C). pCMV-lacZ was used as an internal control for transfection efficiency. The relative luciferase activity is the normalization of luciferase activity with beta -galactosidase activity. B, LS8/p2207 cells in 60-mm dishes were cotransfected with pCMV-lacZ (0.2 µg) and expression plasmids for C/EBPalpha , Msx2FL, or C/EBPalpha plus Msx2FL as described under "Materials and Methods." The beta -galactosidase positive cells were sorted by FACS analysis and subjected to luciferase assay. The relative luciferase activity is the normalization of luciferase activity with protein concentration of the cell lysate. The mean ± S.D. from three independent experiments is represented, and the level of luciferase activity in the presence of empty vector was set as 1.

In our transient transfection experiments, 20-30% transfection efficiency was consistently achieved. In other words, 70-80% of the LS8/p2207 cells assayed for luciferase activity were not transfected with an expression plasmid, but these nontransfected cells still contributed to the background signal. To circumvent this problem, FACS analyses were performed. A beta -galactosidase expression plasmid pCMV-lacZ was included in each transfection. After incubation with a fluorescent beta -galactosidase substrate, di-beta -D-galactopyranoside, cells containing the plasmid pCMV-lacZ were sorted with flow cytometry and assayed for luciferase activity. Given the fact that pCMV-lacZ only comprised 5% of the total amount of the plasmid DNA in each transfection, the beta -galactosidase positive cells should also contain the plasmid of interest (Msx2 or C/EBPalpha ). A 6-7-fold increase and 75% decrease in reporter gene activity were observed for C/EBPalpha and Msx2FL, respectively. Furthermore, cotransfection of equal amounts of C/EBPalpha and Msx2FL expression plasmids gave rise to a 3-fold increase in reporter gene activity (Fig. 6B). Therefore, the 2.2-kilobase mouse amelogenin promoter in the chromosome context was responsive not only to the C/EBPalpha -mediated activation and Msx2-mediated repression but also to the functional antagonism between C/EBPalpha and Msx2.

Little Functional Interaction with Msx2 Was Observed for Two Other C/EBP Family Members-- To determine whether Msx2 is also able to interact with other C/EBP family members in regulating the mouse amelogenin promoter activity, C/EBPbeta and C/EBPgamma expression plasmid was transfected into LS8 cells together with a Msx2 expression plasmid, respectively. The amelogenin-promoter construct p2207 or p70 was used as a reporter in this study. C/EBPalpha not only potently activated the basal promoter activity but also efficiently overcame the Msx2-mediated transcriptional repression of p2207 (Fig. 7A, lanes 1-6) or p70 (Fig. 7B, lanes 1-6). C/EBPbeta by itself weakly activated the basal promoter activity of the reporter construct, and an increasing amount of C/EBPbeta modestly antagonized Msx2 repression of p2207 (Fig. 7A, lanes 7-12) or p70 (Fig. 7B, lanes 7-12). C/EBPgamma had little effect on either the basal promoter activity or the Msx2-mediated transcriptional repression of p2207 (Fig. 7A, lanes 13-18) or p70 (Fig. 7B, lanes 13-18). These cotransfection data indicated that C/EBP family members functioned differentially in the regulation of the mouse amelogenin promoter. Furthermore, only C/EBPalpha , but not C/EBPbeta or C/EBPgamma , was able to efficiently antagonize the repressive effect of Msx2 on the amelogenin gene in the functional analyses.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 7.   Differential interaction between Msx2 and C/EBP family members in the regulation of the mouse amelogenin promoter. In the presence of 250 ng of p2207 (A) or p70 (B) reporter construct, 500 ng of empty vector (pcDNA3), 250 ng of Msx2 expression plasmid (pcMsx2) with 250 ng of pcDNA3, 250 ng of pcMsx2 with 125 ng of pcDNA3 plus 125 ng of C/EBPalpha expression plasmid (pcC/EBPalpha ), 250 ng of pcMsx2 with 250 ng of pcC/EBPalpha , 125 ng of pcC/EBPalpha with 375 ng of pcDNA3, and 250 ng of pcC/EBPalpha with 250 ng of pcDNA3 were cotransfected into LS8 cells, respectively. Similar parameters for transfection experiments were used for C/EBPbeta and C/EBPgamma , respectively. pCMV-lacZ was used as an internal control for transfection efficiency. The relative luciferase activity is the normalization of luciferase activity with beta -galactosidase activity. The mean ± S.D. from at least three independent experiments is represented, and the basal level of p2207 (A) and p70 (B) was set as 1, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Msx2-mediated Transcriptional Repression of the Mouse Amelogenin Gene Is DNA Binding Independent-- We tested the hypothesis that Msx2 is involved in the regulation of mouse amelogenin gene expression. Amelogenin expression is ameloblast specific and spatiotemporally regulated during tooth development. The transcription of amelogenin is restricted to inner enamel epithelial cells that undergo terminal differentiation to the ameloblast phenotype (6-8). In transgenic mouse analyses, the same 2.2-kilobase mouse amelogenin promoter as that used in the p2207 construct is able to recapitulate the spatiotemporal expression pattern of the endogenous amelogenin gene (10). Furthermore, the minimal promoter of the mouse amelogenin gene has been identified, in which a 19-nucleotide stretch (-71/-52 region) is required for the basal promoter activity (14).

The expression patterns of Msx2 during initiation and development of the murine teeth have been identified with in situ hybridization (30). In molar teeth, the expression pattern of Msx2 changes with the differentiation of each germ layer derivative, being strongly expressed in undifferentiated inner enamel epithelia but absent in differentiated ameloblasts and weakly expressed/absent in undifferentiated dental papilla mesenchyme but strongly expressed in odontoblasts and differentiated dental papilla cells. At the late bell stage (E18), moderate levels of Msx2 are expressed in the stratum intermedium and stellate reticulum (30). The reciprocal expression pattern of Msx2 and amelogenin in ameloblast cell lineage suggests that Msx2 may repress the expression of the mouse amelogenin gene.

Here, we demonstrate that Msx2 functions as a transcriptional repressor of the mouse amelogenin gene in a dose-dependent manner (Fig. 1). Two lines of evidence indicate that the primary repressive effect of Msx2 on amelogenin promoter is not DNA binding mediated. First, there is no Msx2 consensus binding site in the mouse amelogenin minimal promoter that is effectively repressed by Msx2. Second, Msx2 itself does not bind to the amelogenin minimal promoter as shown in Fig. 4C. The repression is most pronounced on the full-length promoter (p2207) and decreases gradually with the shortening of the promoter. This is consistent with the observation that the longer promoter constructs have lower basal activity than the minimal promoter does, which may be because of the endogenous Msx2 protein expressed in LS8 cells. Msx2 transcripts are detected in LS8 cells with reverse transcriptase-polymerase chain reaction (data not shown), and very low levels of endogenous Msx2 protein are expressed in LS8 cells as detected by an antipeptide antibody specific for Msx2 (Fig. 1B). During odontogenesis, Msx1 is expressed in all stages in dental mesenchymal cells but not in epithelial cells (28, 29). In cotransfection assays, Msx1 can repress amelogenin-promoter reporter constructs only modestly in ameloblast-like LS8 cells (data not shown).

Functional Antagonism between Msx2 and C/EBPalpha Results from Protein-Protein Interaction-- Msx2Delta C, the carboxyl-terminal deletion form of Msx2, interacts very poorly with C/EBPalpha in vivo, evidenced by the cotransfection and immunoprecipitation analyses in LS8 cells. However, Msx2Delta C is able to interfere with the binding of C/EBPalpha to its cognate site on the mouse amelogenin minimal promoter in vitro, although less potently than Msx2FL, the full-length protein. Residues 182-193 in murine Msx2 consist of homeodomain helix 3 that is the DNA recognition helix. The nuclear localization signal of Msx2 is overlapped with homeodomain helix 3. Deletion of residues 183-267, as in Msx2Delta C, not only results in the loss of DNA binding activity but also affects nuclear localization (31). Therefore, different subcellular localization most likely accounts for the inability of Msx2Delta C to antagonize C/EBPalpha in cotransfection assays, with Msx2Delta C in cytoplasm and C/EBPalpha in nucleus. Little C/EBPalpha protein is co-immunoprecipitated with Msx2Delta C from a whole cell lysate of LS8 cells in which both Msx2Delta C and C/EBPalpha are overexpressed. The weak interaction between Msx2Delta C and C/EBPalpha cannot withstand the stringent washing condition in co-immunoprecipitation assays; however, the high local concentration of Msx2Delta C in gel shift analyses likely enables Msx2Delta C proteins to interfere with the binding of C/EBPalpha to its cognate site. Therefore, in vivo, the nuclear localization signal in Msx2 is necessary for the functional antagonism between Msx2 and C/EBPalpha . However, in co-immunoprecipitation assays, being accessible to each other alone is not sufficient for effective interaction between C/EBPalpha and Msx2 in the absence of Msx2 amino acid residues 183-267. Deletion of the amino-terminal domain (residues 2-97) of Msx2 attenuates, but does not abolish the interaction between Msx2 and C/EBPalpha , suggesting that Msx2 amino acid residues 2-97 play a less critical role. In the future, it will be of interest to further delineate the domains(s) responsible for the interaction between Msx2 and C/EBPalpha .

Msx2-mediated transcriptional repression has been extensively studied in osteoblasts. Gene repression by Msx2 is independent of the intrinsic DNA binding activity of the Msx homeodomain. Protein-protein interactions are essential to the repressive function of Msx2 (31-33). Two repressive mechanisms have been proposed, general repression and promoter-specific repression. Msx2 can bind to transcription factor F for RNA polymerase II (TFIIF), a component of the preinitiation complex, thereby repressing basal transcription (31). The DNA binding activity of other interacting partners is required to achieve promoter specificity for Msx2-dependent transcriptional repression (32-35). Dlx5, another homeodomain transcription factor, up-regulates transcription of the osteoblast-specific osteocalcin gene through binding to its cognate site. Msx2 antagonizes the function of Dlx-5 by forming a Msx2-Dlx5 heterodimer that cannot bind DNA (33). Msx2 also abrogates the induction of the osteocalcin promoter by fibroblast growth factor 2 through inhibiting a DNA binding activity to the fibroblast growth factor 2-response element without Msx2 itself binding to this element (32). However, the identity of this DNA binding activity remains unclear.

The mechanism underlying the Msx2-mediated transcriptional repression on the mouse amelogenin promoter appears to fall into the second category, in which Msx2 inhibits the DNA binding activity of C/EBPalpha , a transcriptional activator of amelogenin promoter. By antagonizing C/EBPalpha , Msx2 fulfills its role as a promoter-specific transcriptional repressor of the mouse amelogenin gene in ameloblasts.

Various transcription factors in concert with C/EBPalpha have been shown to synergistically activate the responsive promoters (37-42). Negative regulation of C/EBP-mediated transactivation through protein-protein interaction has also been reported (43-46). To our knowledge, Msx2, a homeodomain protein, is the first nonbasic region leucine zipper protein identified to date that functions as a transcriptional repressor through its interaction with C/EBPalpha protein. However, the nature of the interaction remains to be delineated. Our data demonstrate that the interaction between C/EBPalpha and Msx2 requires the carboxyl-terminal domain of Msx2, although the interaction is not mediated by the binding of Msx2 to the mouse amelogenin minimal promoter. Notably, this domain contains the third helix of the homeodomain, which is responsible for the binding of Msx2 to its cognate sites.

Transcription factors of the nuclear factor-kappa B families have been reported to have interacted directly with C/EBPbeta via the Rel homology domain of nuclear factor-kappa B and the basic leucine-zipper domain of C/EBPbeta (47, 48). The glucocorticoid receptor, transcription factor v-myb or Sp1, interacts directly with C/EBPbeta , resulting in synergistic activation of the target genes (49-51). Among these factors, Sp1 synergizes with C/EBPbeta but not with C/EBPalpha , suggesting that the C/EBP family members may interact differentially with Msx2. In the present study, Msx2 preferentially interacts with C/EBPalpha but not with either C/EBPbeta or C/EBPgamma in the regulation of the mouse amelogenin promoter activity.

A previous study has demonstrated an important role for C/EBPalpha in regulating the mouse amelogenin promoter (14). A C/EBP binding site (-70 to -61) is located at the minimal promoter of the mouse amelogenin gene and cotransfection of a C/EBPalpha expression plasmid transactivates amelogenin-promoter reporter constructs in a cell type-specific manner. Mutation or deletion of the C/EBP site within the amelogenin promoter not only results in the loss of C/EBPalpha -mediated transactivation but also abolishes the basal promoter activity (Fig. 1A (14)). Furthermore, both the endogenous amelogenin gene and the stably transfected amelogenin-promoter reporter construct (p2207) in ameloblast-like LS8 cells are responsive to the transactivation mediated by C/EBPalpha (Fig. 6 (14)). These data indicate that C/EBPalpha is likely to play a critical role in regulating ameloblast-specific expression of the amelogenin gene.

C/EBPalpha has been demonstrated to mediate cell cycle arrest, cellular differentiation, and transcriptional regulation of tissue-specific genes in adipocytes, hepatocytes, keratinocytes, pneumocytes, and ovarian follicles (52-66). In liver and adipose, peak levels of C/EBPalpha mRNA are detected only in differentiated tissues (67, 68). C/EBPalpha functions as a transcriptional activator in adipocytes, and the accumulation of C/EBPalpha late in preadipocyte differentiation is correlated to the expression of differentiation markers (69-71).

Msx2 has been demonstrated to regulate cellular proliferation and differentiation during development (72-75). Msx2 prevents differentiation and stimulates proliferation in primary cultured chick calvarial osteoblast (72). In transgenic mouse analyses, enhanced expression of Msx2 transiently inhibits osteoblast differentiation. As a consequence, the increase in osteoblast precursors in growth centers of the developing skull results in augmented bone growth and ultimately craniosynostosis (73). Tissue-specific gene expression during development has been shown to be regulated by Msx2. For example, Msx2 has been suggested to repress the expression of osteocalcin gene in the craniofacial skeleton at stages immediately preceding odontoblast and osteoblast terminal differentiation (76). It is conceivable that Msx2 may function in an analogous way to regulate the amelogenin gene during ameloblast differentiation.

In summary, we demonstrate that the functional antagonism between Msx2 and C/EBPalpha results from the Msx2-mediated interference with the binding of C/EBPalpha to its cognate site on the mouse amelogenin minimal promoter. Protein-protein interaction rather than competition for overlapping binding sites are responsible for the observed antagonism. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for the interaction, whereas the amino-terminal residues 2-97 play a less critical role. These data, together with the identification of C/EBPalpha as a transactivator of amelogenin gene in a previous study (14), support our interpretation that Msx2-mediated repression and C/EBPalpha -mediated activation operate in concert to regulate the spatiotemporal expression of amelogenin gene during tooth development.

    ACKNOWLEDGEMENTS

We thank Drs. Laurence Kedes, Charles Shuler, David Ann, David Crowe, and Yi-Hsin Liu for their critical reading of the manuscript; Dr. Paul Denny for providing the anti-Msx2 antibody; and Dr. Dwight Towler for providing the FLAG-tagged Msx2 expression plasmids.

    FOOTNOTES

* This work was supported by Grant DE06988 from NIDCR, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: CSA142, CCMB, The University of Southern California, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-3178; Fax: 323-442-2981; E-mail: mlsnead@ hsc.usc.edu.

Published, JBC Papers in Press, June 19, 2000, DOI 10.1074/jbc.M002031200

    ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; EMSA, electrophoresis mobility shift assay; FACS, fluorescence-activated cell sorting; CMV, cytomegalovirus; TNT, transcription and translation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Aldred, M. J., Crawford, P. J., Roberts, E., and Thomas, N. S. (1992) Hum. Genet. 90, 413-416
2. Lagerstrom, M., Dahl, N., Nakahori, Y., Nakagome, Y., Backman, B., Landegren, U., and Pettersson, U. (1991) Genomics 10, 971-975
3. Lagerstrom-Fermer, M., Nilsson, M., Backman, B., Salido, E., Shapiro, L., Pettersson, U., and Landegren, U. (1995) Genomics 26, 159-162
4. Diekwisch, T., David, S., Bringas, P., Jr., Santos, V., and Slavkin, H. C. (1993) Development 117, 471-482
5. Lyngstadaas, S. P., Risnes, S., Sproat, B. S., Thrane, P. S., and Prydz, H. P. (1995) EMBO J. 14, 5224-5229
6. Snead, M. L., Bringas, P., Jr., Bessem, C., and Slavkin, H. C. (1984) Dev. Biol. 104, 255-258
7. Snead, M. L., Lau, E., Zeichner-David, M., Nanci, T., Bendayan, N., Bringas, P., Bessem, C., and Slavkin, H. C. (1987) in Molecular Approaches to Developmental Biology (Firtel, A. , and Davidson, E., eds) , pp. 641-652, Alan Liss, New York
8. Snead, M. L., Luo, W., Lau, E. C., and Slavkin, H. C. (1988) Development 104, 77-85
9. Couwenhoven, R. I., Schwartz, S. A., and Snead, M. L. (1993) J. Craniofac. Genet. Dev. Biol. 13, 259-269
10. Snead, M. L., Paine, M. L., Chen, L. S., Luo, B. Y., Zhou, D. H., Lei, Y. P., Liu, Y. H., and Maxson, R. E., Jr. (1996) Connect. Tissue Res. 35, 41-47
11. Diekwisch, T. G., Ware, J., Fincham, A. G., and Zeichner-David, M. (1997) J. Histochem. Cytochem. 45, 859-866
12. Zeichner-David, M., Vo, H., Tan, H., Diekwisch, T., Berman, B., Thiemann, F., Alcocer, M. D., Hsu, P., Wang, T., Eyna, J., Caton, J., Slavkin, H. C., and MacDougall, M. (1997) Int. J. Dev. Biol. 41, 27-38
13. Chen, E., Piddington, R., Decker, S., Park, J., Yuan, Z. A., Abrams, W. R., Rosenbloom, J., Feldman, G., and Gibson, C. W. (1994) Dev. Dyn. 199, 189-198
14. Zhou, Y. L., and Snead, M. L. (2000) J. Biol. Chem. 275, 12273-12280
15. Lekstrom-Himes, J., and Xanthopoulos, K. G. (1998) J. Biol. Chem. 273, 28545-28548
16. Poli, V. (1998) J. Biol. Chem. 273, 29279-29282
17. Darlington, G. J., Ross, S. E., and MacDougald, O. A. (1998) J. Biol. Chem. 273, 30057-30060
18. Diehl, A. M. (1998) J. Biol. Chem. 273, 30843-30846
19. Croniger, C., Leahy, P., Reshef, L., and Hanson, R. W. (1998) J. Biol. Chem. 273, 31629-31632
20. Davidson, D. (1995) Trends Genet. 11, 405-411
21. Hill, R. E., Jones, P. F., Rees, A. R., Sime, C. M., Justice, M. J., Copeland, N. G., Jenkins, N. A., Graham, E., and Davidson, D. R. (1989) Genes Dev. 3, 26-37
22. Robert, B., Sassoon, D., Jacq, B., Gehring, W., and Buckingham, M. (1989) EMBO J. 8, 91-100
23. Monaghan, A. P., Davidson, D. R., Sime, C., Graham, E., Baldock, R., Bhattacharya, S. S., and Hill, R. E. (1991) Development 112, 1053-1061
24. Holland, P. W. (1991) Gene (Amst.) 98, 253-257
25. D'Alessio, M., and Frasch, M. (1996) Mech. Dev. 58, 217-231
26. Shimeld, S. M., McKay, I. J., and Sharpe, P. T. (1996) Mech. Dev. 55, 201-210
27. Wang, W., Chen, X., Xu, H., and Lufkin, T. (1996) Mech. Dev. 58, 203-215
28. Mackenzie, A., Leeming, G. L., Jowett, A. K., Ferguson, M. W., and Sharpe, P. T. (1991) Development 111, 269-285
29. MacKenzie, A., Ferguson, M. W., and Sharpe, P. T. (1991) Development 113, 601-611
30. MacKenzie, A., Ferguson, M. W., and Sharpe, P. T. (1992) Development 115, 403-420
31. Newberry, E. P., Latifi, T., Battaile, J. T., and Towler, D. A. (1997) Biochemistry 36, 10451-10462
32. Newberry, E. P., Boudreaux, J. M., and Towler, D. A. (1997) J. Biol. Chem. 272, 29607-29613
33. Newberry, E. P., Latifi, T., and Towler, D. A. (1998) Biochemistry 37, 16360-16368
34. Zhang, H., Hu, G., Wang, H., Sciavolino, P., Iler, N., Shen, M. M., and Abate-Shen, C. (1997) Mol. Cell. Biol. 17, 2920-2932
35. Mann, R. S., and Affolter, M. (1998) Curr. Opin. Genet. Dev. 8, 423-429
36. Dhamija, S., Liu, Y., Yamada, Y., Snead, M. L., and Krebsbach, P. H. (1999) J. Biol. Chem. 274, 20738-20743
37. Zhang, D. E., Hetherington, C. J., Meyers, S., Rhoades, K. L., Larson, C. J., Chen, H. M., Hiebert, S. W., and Tenen, D. G. (1996) Mol. Cell. Biol. 16, 1231-1240
38. Puigserver, P., Ribot, J., Serra, F., Gianotti, M., Bonet, M. L., Nadal-Ginard, B., and Palou, A. (1998) Eur. J. Cell Biol. 77, 117-123
39. Choi, B. H., Park, G. T., and Rho, H. M. (1999) J. Biol. Chem. 274, 2858-2865
40. Metzger, S., Halaas, J. L., Breslow, J. L., and Sladek, F. M. (1993) J. Biol. Chem. 268, 16831-16838
41. Wu, K. J., Wilson, D. R., Shih, C., and Darlington, G. J. (1994) J. Biol. Chem. 269, 1177-1182
42. Vietor, I., Oliveira, I. C., and Vilcek, J. (1996) J. Biol. Chem. 271, 5595-5602
43. Cooper, C., Henderson, A., Artandi, S., Avitahl, N., and Calame, K. (1995) Nucleic Acids Res. 23, 4371-4377
44. Ron, D., and Habener, J. F. (1992) Genes Dev. 6, 439-453
45. Hsu, W., Kerppola, T. K., Chen, P. L., Curran, T., and Chen-Kiang, S. (1994) Mol. Cell. Biol. 14, 268-276
46. Hollenberg, A. N., Susulic, V. S., Madura, J. P., Zhang, B., Moller, D. E., Tontonoz, P., Sarraf, P., Spiegelman, B. M., and Lowell, B. B. (1997) J. Biol. Chem. 272, 5283-5290
47. Matsusaka, T., Fujikawa, K., Nishio, Y., Mukaida, N., Matsushima, K., Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10193-10197
48. Stein, B., Cogswell, P. C., and Baldwin, A. S., Jr. (1993) Mol. Cell. Biol. 13, 3964-3974
49. Nishio, Y., Isshiki, H., Kishimoto, T., and Akira, S. (1993) Mol. Cell. Biol. 13, 1854-1862
50. Mink, S., Kerber, U., and Klempnauer, K. H. (1996) Mol. Cell. Biol. 16, 1316-1325
51. Lee, Y. H., Williams, S. C., Baer, M., Sterneck, E., Gonzalez, F. J., and Johnson, P. F. (1997) Mol. Cell. Biol. 17, 2038-2047
52. Timchenko, N. A., Wilde, M., Nakanishi, M., Smith, J. R., and Darlington, G. J. (1996) Genes Dev. 10, 804-815
53. Timchenko, N. A., Harris, T. E., Wilde, M., Bilyeu, T. A., Burgess-Beusse, B. L., Finegold, M. J., and Darlington, G. J. (1997) Mol. Cell. Biol. 17, 7353-7361
54. Freytag, S. O., and Geddes, T. J. (1992) Science 256, 379-382
55. Constance, C. M., Morgan, J. I. t., and Umek, R. M. (1996) Mol. Cell. Biol. 16, 3878-3883
56. Wang, N. D., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112
57. Flodby, P., Barlow, C., Kylefjord, H., Ahrlund-Richter, L., and Xanthopoulos, K. G. (1996) J. Biol. Chem. 271, 24753-24760
58. Li, F., Rosenberg, E., Smith, C. I., Notarfrancesco, K., Reisher, S. R., Shuman, H., and Feinstein, S. I. (1995) Am. J. Physiol. 269, L241-L247
59. Brockes, J. P. (1997) Science 276, 81-87
60. Michalopoulos, G. K., and DeFrances, M. C. (1997) Science 276, 60-66
61. Stocum, D. L. (1997) Science 276, 15
62. Piontkewitz, Y., Enerback, S., and Hedin, L. (1993) Endocrinology 133, 2327-2333
63. Maytin, E. V., and Habener, J. F. (1998) J. Invest. Dermatol. 110, 238-246
64. Umek, R. M., Friedman, A. D., and McKnight, S. L. (1991) Science 251, 288-292
65. Swart, G. W., van Groningen, J. J., van Ruissen, F., Bergers, M., and Schalkwijk, J. (1997) Biol. Chem. Hoppe-Seyler 378, 373-379
66. Freytag, S. O., Paielli, D. L., and Gilbert, J. D. (1994) Genes Dev. 8, 1654-1663
67. Birkenmeier, E. H., Gwynn, B., Howard, S., Jerry, J., Gordon, J. I., Landschulz, W. H., and McKnight, S. L. (1989) Genes Dev. 3, 1146-1156
68. Antonson, P., and Xanthopoulos, K. G. (1995) Biochem. Biophys. Res. Commun. 215, 106-113
69. MacDougald, O. A., and Lane, M. D. (1995) Annu. Rev. Biochem. 64, 345-373
70. Yeh, W. C., Cao, Z., Classon, M., and McKnight, S. L. (1995) Genes Dev. 9, 168-81
71. Lane, M. D., Lin, F. T., MacDougald, O. A., and Vasseur-Cognet, M. (1996) Int. J. Obes. Relat. Metab. Disord. 20 Suppl. 3, 91-99
72. Dodig, M., Tadic, T., Kronenberg, M. S., Dacic, S., Liu, Y. H., Maxson, R., Rowe, D. W., and Lichtler, A. C. (1999) Dev. Biol. 209, 298-307
73. Liu, Y. H., Tang, Z., Kundu, R. K., Wu, L., Luo, W., Zhu, D., Sangiorgi, F., Snead, M. L., and Maxson, R. E. (1999) Dev. Biol. 205, 260-274
74. Mina, M., Gluhak, J., Upholt, W. B., Kollar, E. J., and Rogers, B. (1995) Dev. Dyn. 202, 195-214
75. Mina, M., Gluhak, J., and Rodgers, B. (1996) Connect. Tissue Res. 35, 79-84
76. Bidder, M., Latifi, T., and Towler, D. A. (1998) J. Bone Miner. Res. 13, 609-619
77. Qiagen Inc.. (1997) The QIAexpressionist , pp. 38-73, Valencia, CA

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J BiochemHome page
L. Xu, H. Harada, and A. Taniguchi
The Effects of LAMP1 and LAMP3 on M180 Amelogenin Uptake, Localization and Amelogenin mRNA Induction by Amelogenin Protein
J. Biochem., October 1, 2008; 144(4): 531 - 537.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S.-L. Cheng, J.-S. Shao, J. Cai, O. L. Sierra, and D. A. Towler
Msx2 Exerts Bone Anabolism via Canonical Wnt Signaling
J. Biol. Chem., July 18, 2008; 283(29): 20505 - 20522.
[Abstract] [Full Text] [PDF]

Home page
 JDRHome page
M.L. Paine, M.L. Snead, H.J. Wang, N. Abuladze, A. Pushkin, W. Liu, L.Y. Kao, S.M. Wall, Y.-H. Kim, and I. Kurtz
Role of NBCe1 and AE2 in Secretory Ameloblasts
Journal of Dental Research, April 1, 2008; 87(4): 391 - 395.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Xu, Y. L. Zhou, F. J. Gonzalez, and M. L. Snead
CCAAT/Enhancer-binding Protein {delta} (C/EBP{delta}) Maintains Amelogenin Expression in the Absence of C/EBP{alpha} in Vivo
J. Biol. Chem., October 12, 2007; 282(41): 29882 - 29889.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Xu, H. Harada, and A. Taniguchi
The Exon 6ABC Region of Amelogenin mRNA Contribute to Increased Levels of Amelogenin mRNA through Amelogenin Protein-enhanced mRNA Stabilization
J. Biol. Chem., October 27, 2006; 281(43): 32439 - 32444.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Xu, Y. L. Zhou, W. Luo, Q.-S. Zhu, D. Levy, O. A. MacDougald, and M. L. Snead
NF-Y and CCAAT/Enhancer-binding Protein {alpha} Synergistically Activate the Mouse Amelogenin Gene
J. Biol. Chem., June 9, 2006; 281(23): 16090 - 16098.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Xu, H. Harada, T. Yokohama-Tamaki, S. Matsumoto, J. Tanaka, and A. Taniguchi
Reuptake of Extracellular Amelogenin by Dental Epithelial Cells Results in Increased Levels of Amelogenin mRNA through Enhanced mRNA Stabilization
J. Biol. Chem., January 27, 2006; 281(4): 2257 - 2262.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
S. Fukumoto, T. Kiba, B. Hall, N. Iehara, T. Nakamura, G. Longenecker, P. H. Krebsbach, A. Nanci, A. B. Kulkarni, and Y. Yamada
Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts
J. Cell Biol., December 6, 2004; 167(5): 973 - 983.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Q. Hassan, A. Javed, M. I. Morasso, J. Karlin, M. Montecino, A. J. van Wijnen, G. S. Stein, J. L. Stein, and J. B. Lian
Dlx3 Transcriptional Regulation of Osteoblast Differentiation: Temporal Recruitment of Msx2, Dlx3, and Dlx5 Homeodomain Proteins to Chromatin of the Osteocalcin Gene
Mol. Cell. Biol., October 15, 2004; 24(20): 9248 - 9261.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Ichida, R. Nishimura, K. Hata, T. Matsubara, F. Ikeda, K. Hisada, H. Yatani, X. Cao, T. Komori, A. Yamaguchi, et al.
Reciprocal Roles of Msx2 in Regulation of Osteoblast and Adipocyte Differentiation
J. Biol. Chem., August 6, 2004; 279(32): 34015 - 34022.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. Brunelli, E. Tagliafico, F. G. De Angelis, R. Tonlorenzi, S. Baesso, S. Ferrari, M. Niinobe, K. Yoshikawa, R. J. Schwartz, I. Bozzoni, et al.
Msx2 and Necdin Combined Activities Are Required for Smooth Muscle Differentiation in Mesoangioblast Stem Cells
Circ. Res., June 25, 2004; 94(12): 1571 - 1578.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. Yoshizawa, F. Takizawa, F. Iizawa, O. Ishibashi, H. Kawashima, A. Matsuda, N. Endo, and H. Kawashima
Homeobox Protein Msx2 Acts as a Molecular Defense Mechanism for Preventing Ossification in Ligament Fibroblasts
Mol. Cell. Biol., April 15, 2004; 24(8): 3460 - 3472.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S.-L. Cheng, J.-S. Shao, N. Charlton-Kachigian, A. P. Loewy, and D. A. Towler
Msx2 Promotes Osteogenesis and Suppresses Adipogenic Differentiation of Multipotent Mesenchymal Progenitors
J. Biol. Chem., November 14, 2003; 278(46): 45969 - 45977.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/37/29066    most recent
M002031200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, Y. L.
Right arrow Articles by Snead, M. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, Y. L.
Right arrow Articles by Snead, M. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement