The 1,25(OH)2D3-Regulated Transcription Factor MN1 Stimulates Vitamin D Receptor-Mediated Transcription and Inhibits Osteoblastic Cell Proliferation

Amelia L. M. Sutton, Xiaoxue Zhang, Tara I. Ellison and Paul N. MacDonald

Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Paul N. MacDonald, Ph.D., Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106. E-mail: pnm2{at}po.cwru.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
The vitamin D endocrine system is essential for maintaining mineral ion homeostasis and preserving bone density. The most bioactive form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] elicits its effects by binding to the vitamin D receptor (VDR) and regulating the transcription of target genes. In osteoblasts, the bone-forming cells of the skeleton, 1,25-(OH)2D3 regulates cell proliferation, differentiation, and mineralization of the extracellular matrix. Despite these well-characterized biological functions, relatively few 1,25-(OH)2D3 target genes have been described in osteoblasts. In this study, we characterize the regulation and function of MN1, a novel 1,25-(OH)2D3-induced gene in osteoblastic cells. MN1 is a nuclear protein first identified as a gene disrupted in some meningiomas and leukemias. Our studies demonstrate that MN1 preferentially stimulates VDR-mediated transcription through its ligand-binding domain and synergizes with the steroid receptor coactivator family of coactivators. Furthermore, forced expression of MN1 in osteoblastic cells results in a profound decrease in cell proliferation by slowing S-phase entry, suggesting that MN1 is an antiproliferative factor that may mediate 1,25-(OH)2D3-dependent inhibition of cell growth. Collectively, these data indicate that MN1 is a 1,25-(OH)2D3-induced VDR coactivator that also may have critical roles in modulating osteoblast proliferation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
THE VITAMIN D endocrine system is crucial for maintaining calcium and phosphate homeostasis and protecting skeletal integrity (1, 2). 1,25-(OH)2D3 is the bioactive hormonal form of vitamin D. This hormone functions through the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, to regulate transcription of target genes. 1,25-(OH)2D3 induces expression of various calcium binding and transport proteins in the intestine to stimulate active calcium uptake, thus preserving normocalcemia and, indirectly, maintaining bone mineralization. However, 1,25-(OH)2D3 also acts directly on osteoblasts, the resident bone-forming cells of the skeleton, to inhibit proliferation, modulate differentiation, and regulate mineralization of the extracellular matrix (3).

Upon binding 1,25-(OH)2D3, VDR heterodimerizes with retinoid X receptor (RXR) and associates with vitamin D response elements (VDREs) in the promoter regions of target genes (4). The active VDR/RXR heterodimer forms multiple protein complexes with general transcription factors and coactivator proteins, factors that interact with nuclear receptors and enhance their transcriptional activity. A variety of coactivators for VDR have been described, including the steroid receptor coactivator (SRC) proteins, the DRIP (VDR-interacting proteins) complex, and nuclear coactivator 62 kDa (NCoA-62)/ski-interacting protein (SKIP) (5). The SRC family represents the first nuclear receptor coactivators identified and includes SRC-1 (6), SRC-2 [glucocorticoid receptor (GR)-interacting protein (GRIP1)/transcriptional intermediary factor 2 (7, 8)], and SRC-3 [RAC3/ACTR/AIB1 (9, 10, 11)]. SRCs are thought to stimulate nuclear receptor activity by recruiting histone acetyl transferases, such as cAMP response element binding protein (CREB)-binding protein (CBP)/p300, to the promoter regions of target genes (11, 12, 13). SRCs also possess intrinsic histone acetyl transferase activity (14) and, in concert with CBP/p300, modify histones, thereby allowing the transcriptional machinery better access to regulatory regions to initiate transcription. DRIP is a multimeric complex that includes more than 10 proteins and is nearly identical with the transcriptional coactivators thyroid receptor-activating proteins and the mammalian mediator complex (15, 16). Because DRIP interacts with the RNA polymerase II holoenzyme (17), it may serve as a bridge between liganded VDR/RXR and the basal transcriptional machinery. Finally, NCoA-62 or SKIP represents a distinct VDR coactivator (18) that may couple transcriptional activation to mRNA splicing (19). SRCs and DRIP205, the anchoring subunit of the DRIP complex, interact with liganded VDR through LXXLL motifs, hydrophobic domains that interact with a complementary hydrophobic cleft in the nuclear receptor ligand binding domain (LBD) (20, 21). Although these LXXLL motifs are essential for SRCs and DRIP205 to interact with VDR, other coactivators interact with the receptor through different mechanisms. For example, NCoA-62/SKIP associates with VDR through a central domain that does not contain LXXLL sequences (22). All three classes of coactivators, acting through distinct mechanisms, are necessary for 1,25-(OH)2D3-mediated transcription (15, 22). The complexity of VDR-activated transcriptional processes is highlighted by the ever-expanding number of protein factors that are involved in this mechanism.

The cellular consequences of 1,25-(OH)2D3-mediated transcriptional activation include inhibition of proliferation and stimulation of differentiation in many tissues (23, 24). In fact, a number of synthetic 1,25-(OH)2D3 analogs have been developed to treat cancer and other hyperproliferative diseases. How 1,25-(OH)2D3 accomplishes this growth inhibition is not fully understood, but it likely involves alterations in the expression of numerous growth factors, cell cycle-related proteins, transcription factors, and other genes (23). Two well-established target genes of 1,25-(OH)2D3 are the cell cycle-dependent kinase inhibitors p21WAF1/cip1 (25) and p27kip1 (26). 1,25-(OH)2D3 stimulates expression of these genes resulting in cell cycle accumulation at the G1/S checkpoint (27). GADD45{alpha}, another cell cycle regulator and DNA-damage repair protein, is also induced by 1,25-(OH)2D3 (28, 29) and causes growth arrest at G2/M (29). Further identification of potential 1,25-(OH)2D3 target genes involved in cell proliferation and differentiation has accelerated recently with the advent of microarray technology (30), but the functional implications of these genes remain largely unexplored.

Here, we identify and characterize MN1 (meningioma-1) as a novel target of 1,25-(OH)2D3 in osteoblasts. MN1 was first identified as a gene disrupted in some meningiomas as part of a balanced translocation t(4;22) (31) and as part of a fusion with the ETS (E26 transformation specific) domain transcription factor TEL (translocation ets leukemia) in some myeloid leukemias (32). Sequence analysis reveals that MN1 exhibits several attributes indicative of a transcription factor, including an N-terminal nuclear localization signal, proline-rich sequences, and two polyglutamine stretches (31). MN1 localizes to the nucleus and activates transcription of a Moloney sarcoma virus long terminal repeat (MSV-LTR) reporter gene (33). Subsequent studies showed that MN1 associates with a retinoic acid-responsive element (RARE) in the MSV-LTR and that MN1 stimulates RAR-dependent transcription of the MSV-LTR, a previously unrecognized RAR-responsive promoter (34). These studies support a role for MN1 in transcriptional regulation. However, beyond these initial reports, there are currently no studies addressing the role of MN1 in other nuclear receptor pathways, the factors that control expression of MN1, or the potential function of MN1 in cellular events such as proliferation or differentiation.

In the current study, we demonstrate that 1,25-(OH)2D3 strongly increased MN1 mRNA levels in osteoblastic cells. We provide data supporting a coactivator role for MN1 in VDR/1,25-(OH)2D3-mediated transcription through a mechanism that involves the VDR LBD and SRC coactivators. Finally, expression of MN1 in osteoblastic cells potently inhibited cellular proliferation by reducing the proportion of cells entering S-phase of the cell cycle, uncovering an important role for MN1 in regulating osteoblastic cell growth. This study is the first demonstration that MN1 stimulates VDR transcriptional activity and inhibits osteoblastic cell proliferation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
Characterization of MN1 as a 1,25-(OH)2D3 Target Gene in Osteoblastic Cells
Microarray analysis was used as an initial screen to identify 1,25-(OH)2D3-regulated genes in MG-63 cells, a human osteoblastic cell line. In this analysis, MN1 was induced approximately 8-fold in response to a 6-h treatment with 10 nM 1,25-(OH)2D3 (data not shown). To validate the microarray results in MG-63 cells, Northern blot analysis was performed. As shown in Fig. 1Go, 1,25-(OH)2D3 increased MN1 mRNA levels in a time- and dose-dependent manner. This increase is apparent as early as 3 h and is maximal at 12 h (Fig. 1AGo). As little as 0.1 nM 1,25-(OH)2D3 stimulates MN1 expression, and mRNA levels continue to rise up to 100 nM 1,25-(OH)2D3 (Fig. 1BGo). This effect was specific for 1,25-(OH)2D3 because cholicalciferol (vitamin D3), an inactive precursor molecule of 1,25-(OH)2D3, and 24,25(OH)2D3, a metabolite of vitamin D3, had little effect on MN1 mRNA levels (Fig. 1AGo and data not shown). RNA synthesis was required for this response since inhibition of transcription by actinomycin D abolished the 1,25-(OH)2D3-mediated induction of MN1 (Fig. 2AGo). Furthermore, when de novo protein synthesis was inhibited with cycloheximide, 1,25-(OH)2D3-mediated induction of MN1 mRNA was preserved (Fig. 2BGo). However, the basal expression of MN1 was enhanced upon cycloheximide treatment, diminishing the extent of MN1 induction by 1,25-(OH)2D3. These data also suggest that synthesis of other proteins is required for maximal induction of MN1 mRNA. Taken together, these data imply that 1,25-(OH)2D3 increases steady-state levels of MN1 mRNA by process that, in part, involves active transcription and does not require on-going protein synthesis.



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Fig. 1. 1,25-(OH)2D3 Induces MN1 Expression in a Time- and Dose-Dependent Manner

A, MG-63 cells were treated for the indicated times with 10 nM 1,25-(OH)2D3 or 10 nM cholecalciferol (chol). mRNA was analyzed by Northern blots for MN1 and ß-actin. B, MG-63 cells were treated with ethanol vehicle control (–) or 0.1–100 nM 1,25-(OH)2D3 for 6 h. mRNA was analyzed by Northern blots for MN1 and ß-actin. Fold increases in MN1 mRNA levels are expressed relative to ß-actin mRNA levels.

 


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Fig. 2. 1,25-(OH)2D3 Induction of MN1 Requires de Novo RNA Synthesis But Not Protein Synthesis

A, MG-63 cells were pretreated with methanol vehicle control (– ACTD) or 1 µg/ml actinomycin D (+ ACTD) for 1 h. Cells were then treated with ethanol control (Et) or 10 nM 1,25-(OH)2D3 (1 25 ) for 6 h. mRNA was analyzed by Northern blots for MN1 and ß-actin. B, MG-63 cells were pretreated with 10 µg/ml cycloheximide (+ CHX) or ethanol control (– CHX) for 1 h. Cells were then treated with ethanol control (Et) or 10 nM 1,25-(OH)2D3 (1 25 ) for 6 h. mRNA was analyzed by Northern blots for MN1 and ß-actin. Fold increases in MN1 mRNA levels are expressed relative to ß-actin mRNA levels. –, MN1 mRNA levels are undetectable in actinomycin D-treated samples.

 
MN1 Augments VDR-Mediated Transcription
Given recent evidence indicating that MN1 stimulates RAR-mediated transcription of MSV-LTR (34), we tested whether MN1 modulates VDR-mediated transcription. COS-7 cells were transfected with a VDR expression vector and a 1,25-(OH)2D3-responsive reporter gene composed of four copies of a VDRE and a minimal TATA promoter fused upstream of firefly luciferase. Transfection of an expression plasmid encoding MN1 augmented 1,25-(OH)2D3-mediated expression of the reporter gene approximately 6- to 7-fold (Fig. 3AGo). MN1 also stimulated 1,25-(OH)2D3-mediated induction of a reporter gene driven by the 24-hydroxylase promoter, a native 1,25-(OH)2D3-responsive regulatory sequence (data not shown). The effect of MN1 was both VDR- and 1,25-(OH)2D3-dependent because MN1 expression had minimal effects on basal reporter gene activity in the absence of VDR (Fig. 3BGo) and in the absence of 1,25-(OH)2D3 (Fig. 3AGo). MN1 also enhanced the ligand-dependent transcriptional activity of a fusion protein composed of the GAL4 DNA binding domain (DBD) and the ligand binding domain (LBD) of VDR (Fig. 3CGo). Because the LBD of VDR was sufficient for the increase in transcriptional activation, it is likely that MN1 acts through the VDR LBD to stimulate VDR-mediated transcription.



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Fig. 3. MN1 Augments 1,25-(OH)2D3-Dependent Transcription through the VDR Ligand-Binding Domain

A, COS-7 cells were transiently transfected with (VDRE)4-TATA-luc, SG5-VDR, CMV-renilla luciferase, and either CMVTAG2B (vector) or CMVTAG2B-MN1. Cells were treated with either ethanol vehicle control or 10 nM 1,25-(OH)2D3 for 24 h. B, COS-7 cells with transiently transfected with (VDRE)4-TATA-luc, CMV-renilla, SG5 (vector) or SG5-VDR, and CMVTAG2B (vector) or CMVTAG2B-MN1. Cells were treated with either ethanol vehicle control or 100 nm 1,25-(OH)2D3 for 24 h. C, COS-7 cells were transiently transfected with (GAL4)5-TK-luc, CMV-renilla, GAL4 or GAL4-VDR LBD, and CMVTAG2B (vector) or CMVTAG2B-MN1. Cells were treated with either ethanol vehicle control or indicated doses of 1,25-(OH)2D3 for 24 h.

 
MN1 Selectively Stimulates Other Nuclear Receptors
To test the putative role of MN1 in other nuclear receptor pathways, MN1 was cotransfected along with plasmids encoding the GAL4 DBD fused to the LBDs of two other nuclear receptors that function as RXR heterodimers. The activity of these fusion proteins was measured by activation of a GAL4-responsive reporter gene. As shown in Fig. 4AGo, MN1 selectively stimulated the VDR LBD. In addition to ligand-activated nuclear receptors, we also tested whether MN1 impacts the transcriptional activity of the ROR orphan nuclear receptors. MN1 modestly stimulated ROR{gamma} activity but did not enhance ROR{alpha} or RORß activity (Fig. 4BGo). Although MN1 failed to activate the LBD of RAR{alpha} (Fig. 4AGo), it did augment the transcriptional activity of the full-length RAR{alpha} on a synthetic RARE-driven reporter gene (Fig. 4CGo). In contrast, MN1 inhibited the transcriptional activity of the GR. Collectively, these data indicate that MN1 selectively stimulates the transcriptional activity of VDR, RAR, and potentially ROR{gamma}. Furthermore, it is likely that the mechanisms underlying MN1 transactivation are distinct among these nuclear receptors because only the VDR LBD is required for activation by MN1, whereas the full-length RAR{alpha} is necessary for MN1-mediated stimulation.



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Fig. 4. Effect of MN1 on Other Nuclear Receptors

A, COS-7 cells were transiently transfected with (GAL4)5-TK-luc, CMV-renilla, GAL4 fusions of the ligand binding domain of the indicated nuclear receptor, and either CMVTAG2B (vector) or CMVTAG2B-MN1. Cells were treated with either ethanol vehicle control or 10 nM 1,25-(OH)2D3 (VDR), 10 nM thyroid hormone (TRß), or 100 nM all-trans-retinoic acid (RAR{alpha}). B, COS-7 cells were transiently transfected with (GAL4)5-TK-luc, CMV-renilla, GAL4 fusions of the ligand-binding domain of the indicated receptor, and either CMVTAG2B (vector) or CMVTAG2B-MN1. C, COS-7 cells were transiently transfected with (VDRE)4-TK-luc, (GRE)2-TK-luc, or (RARE)4-TK-luc, CMV-renilla, SG5-VDR, -GR, or -RAR{alpha}, and either CMVTAG2B (vector) or CMVTAG2B-MN1. Cells were treated with either ethanol vehicle control or 10 nM 1,25-(OH)2D3 (VDR), 1 µM dexamethasone (GR), or 100 nM all-trans-retinoic acid (RAR{alpha}) for 24 h.

 
MN1 Synergizes with SRCs
The SRC proteins are well-characterized coactivators of VDR and other nuclear receptors (35). To test whether MN1 synergizes with SRCs to stimulate VDR-mediated transcription, COS-7 cells were transfected with VDR, a VDRE-driven reporter gene, and MN1, either alone or in combination with SRC-1 or SRC-2 (GRIP1/transcriptional intermediary factor 2). SRC-1 and SRC-2 both stimulated the VDR-mediated transcription by approximately 3- and 4-fold, respectively (Fig. 5Go, A and B). MN1 alone similarly stimulated VDR activity by about 2- to 3-fold (Fig. 5Go, A and B). However, MN1 and SRC-1 together augmented VDR-mediated transcription by 20-fold, whereas MN1 and SRC-2 stimulated VDR transactivation by 16-fold. These data suggest that MN1 cooperates with the SRCs to synergistically stimulate VDR-mediated transcription.



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Fig. 5. MN1 Synergizes with SRC Coactivators

A and B, COS-7 cells were transiently transfected with (VDRE)4-TATA-luc, CMV-renilla, SG5-VDR, and either vector control or the indicated coactivators. Cells were treated with either ethanol vehicle control or 100 nM 1,25-(OH)2D3 for 24 h.

 
MN1 Inhibits Osteoblastic Cell Proliferation
In agreement with previous studies (36, 37), 1,25-(OH)2D3 inhibited the proliferation of MG-63 osteoblastic cells in a time-dependent manner (Fig. 6AGo). MN1 was first described as a gene disrupted in some meningiomas and leukemias (31, 32, 38), suggesting that it functions as a tumor suppressor. Given these data, we hypothesized that MN1 may be one of the 1,25-(OH)2D3-regulated genes responsible for its growth inhibition. Because we were unable to isolate stable clones that maintain elevated MN1 expression, we chose to use a short-term stable pool approach to study the effect of MN1 expression on cell growth (39). MG-63 cells were transfected with empty vector, MN1, or RAR-related orphan receptor-ß (RORß) as an irrelevant control plasmid. Stable transfectants were selected and grown in G418-containing media for 1 wk, and resulting colonies were stained with crystal violet. Cells transfected with MN1 formed much smaller colonies than those transfected with empty vector (Fig. 6BGo). Spectrophotometric quantification of the crystal violet staining as a measure of total cell number revealed that forced expression of MN1 resulted in a greater than 90% decrease in cell growth (Fig. 6CGo). In contrast, transfection of RORß had no effect on colony size or total cell number (Fig. 6Go, B and C), indicating that the effect observed with MN1 expression was unlikely to be caused by nonspecific toxicity due to overexpression of a nuclear protein. Transfection efficiency, as measured by luciferase activity of a cotransfected reporter gene plasmid, was similar among all three groups (data not shown). These data indicate that MN1 dramatically inhibits the growth of osteoblastic cells.



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Fig. 6. MN1 Inhibits Cell Proliferation in Osteoblastic Cells

A, MG-63 cells were cultured in the presence of either ethanol vehicle control or 10 nM 1,25-(OH)2D3 and counted at the indicated times. B, MG-63 cells were transfected with either CMVTAG2B (vector) or CMVTAG2B-MN1. Stable transfectants were selected by culture in the presence of G418 for 7 d. Cells were visualized by staining with crystal violet. C, Total cell number was quantified by solubilizing the crystal violet dye in 0.1 M sodium citrate in 50% ethanol, and absorbance was measured at 540 nm.

 
MN1 Decreases S-Phase Entry in Osteoblastic Cells
To determine whether the MN1-mediated decrease in cell proliferation is due to alterations in cell cycle progression, MG-63 cells transiently expressing MN1 were analyzed for 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of S-phase entry (Fig. 7AGo and B). To easily identify transfected cells, MN1 was expressed using a dual expression vector that drives transcription of enhanced green fluorescent protein (EGFP) and MN1 from two separate promoters on a single plasmid. Whereas 38% of MG-63 cells transfected with the EGFP vector alone were BrdU positive, only 13% of cells transfected with MN1 were BrdU positive (Fig. 7BGo). The growth inhibitory effect was specific for MN1 because expression of another VDR coactivator, SRC-1, did not affect cell proliferation (Fig. 7Go, A and B). These data indicate that MN1 expression inhibits osteoblastic cell proliferation by slowing entry into the S-phase of the cell cycle.



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Fig. 7. MN1 Decreases S-Phase Entry in Osteoblastic Cells

A, MG-63 cells were transiently transfected with either pCMS-EGFP, pCMS-EGFP-MN1, or pCMS-EGFP-SRC-1 and allowed to express for 24 h. Cells were pulse labeled with BrdU for 1 h, fixed, and immunostained to visualize BrdU incorporation. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Representative fields are shown. EGFP-positive BrdU-positive cells are indicated by open arrows, and EGFP-positive BrdU-negative cells are indicated by closed arrows. B, Quantification of percentage of EGFP-expressing cells that were BrdU-positive.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
The VDR and 1,25-(OH)2D3 control a variety of biological process in a diverse array of tissues. One of the most important effects of 1,25-(OH)2D3 is to stimulate active calcium transport across the intestinal epithelium to maintain normocalcemia and preserve bone mineralization (1, 2, 4). In addition to these systemic effects on mineral ion homeostasis, 1,25-(OH)2D3 also acts directly on osteoblasts to control proliferation, differentiation, and mineralization. Beyond a handful of target genes, such as p21WAF1/cip1, p27kip1, and GADD45, little is known about the mechanisms through which 1,25-(OH)2D3 inhibits proliferation of target cells. Microarray analysis has led to an expansion of our knowledge of 1,25-(OH)2D3-regulated genes in a number of cell types (30, 40, 41, 42, 43, 44, 45, 46). The precise molecular and cellular functions of these novel target genes are just now being uncovered. Functional characterization of 1,25-(OH)2D3-regulated genes is essential to understand the mechanisms underlying the pleiotropic effects, including growth regulation, of 1,25-(OH)2D3. In this report, we show that MN1 is a novel 1,25-(OH)2D3-induced gene in osteoblastic cells and that MN1 augments VDR-mediated transcription and potently inhibits osteoblastic cell growth.

MN1 was revealed as a 1,25-(OH)2D3-induced gene in MG-63 osteoblastic cells in our microarray studies. Another recent microarray study also showed that MN1 mRNA expression was increased by the synthetic 1,25-(OH)2D3 analog EB1089 in squamous cell carcinoma cells (30). Although the regulation of MN1 expression and its functional roles were not addressed in this global expression profiling study, together our two studies clearly point to MN1 as a 1,25-(OH)2D3-induced gene in multiple 1,25-(OH)2D3 target cells. In agreement with our microarray results, Northern blot analysis confirmed that 1,25-(OH)2D3 increased MN1 mRNA levels in a time- and dose-dependent manner in osteoblastic cells (Fig. 1Go). 1,25-(OH)2D3 likely induces MN1 expression through a transcriptional mechanism because inhibition of transcription with actinomycin D completely abolished the increase in MN1 mRNA observed with 1,25-(OH)2D3 (Fig. 2AGo). Furthermore, it appears that protein synthesis is not necessary, although may enhance, 1,25-(OH)2D3-mediated induction of MN1 (Fig. 2BGo). These data suggest that VDR directly regulates MN1 expression by binding to and activating the promoter region of MN1. Although the putative promoter of MN1 does not contain any canonical VDREs, the sequence contains many nuclear receptor response element half-sites that may support binding of the VDR/RXR heterodimer. Further studies, including promoter analysis, are required to dissect the molecular details of the 1,25-(OH)2D3-mediated induction of MN1 expression.

MN1 is a nuclear protein with transcriptional activity (33) and its transcriptional targets and mechanisms of actions are currently being uncovered (34). The present study shows that MN1 stimulates VDR-dependent transcriptional activation. This stimulation occurs through the LBD of VDR because MN1 increased the transcriptional activity of a GAL4 DBD-VDR LBD chimeric protein (Fig. 3CGo). We did not detect a similar activation of GAL4 DBD-RAR LBD (Fig. 4AGo) but did observe an increase in the transcriptional activation of full-length RAR{alpha} by MN1 using a canonical RARE-driven reporter gene (Fig. 4CGo). This corroborates and extends a previous study that demonstrated that MN1, cooperating with RAR, activates the MSV-LTR, a previously unrecognized RAR-responsive promoter (34). It is not surprising that MN1 augments the activity of both VDR and RAR because these two nuclear receptors are structurally related and both utilize RXR as their heterodimeric partner (47). However, MN1 likely enhances the transcriptional activity of these two nuclear receptors through distinct mechanisms. Whereas MN1 stimulates VDR through its LBD, RAR{alpha} activation does not occur exclusively through the LBD and potentially requires an intact amino-terminal region (the activation function-1) and/or DBD of the receptor. Furthermore, these data demonstrate that MN1 is not a general nuclear receptor coactivator; rather, it selectively stimulates VDR, RAR, and, to a lesser extent, ROR{gamma} activity, inhibits GR activity, and does not affect the transactivation potential of the other nuclear receptors analyzed in this study. Although the exact mechanism underlying the transcriptional stimulation of nuclear receptors is not known, the current study suggests that MN1 cooperates with both SRC-1 and SRC-2 in a synergistic manner (Fig. 5Go). Coupled with the previous report that showed that MN1 synergizes with SRC-3 in RAR-dependent transcription (34), these data provide further evidence of a functional link between MN1 and the SRC family of coactivators.

Although it is clear from our studies and others (33, 34) that MN1 is a transcription factor that stimulates nuclear receptor-dependent transactivation, the cellular consequences of this activity previously had not been explored. Gene expression profiling has revealed that TGF-ß, which is growth inhibitory in epithelial cells, induces expression of MN1 in breast cancer cells (48), untransformed breast epithelial cells, keratinocytes, and lung epithelial cells (49). Likewise, the current study and a recent microarray analysis (30) demonstrate that MN1 expression is induced by 1,25-(OH)2D3 and its synthetic analog, which inhibit proliferation in a wide array of tissues. These data, coupled with the clinical findings that MN1 is disrupted in some meningiomas and leukemias (31, 32, 38), point to a role for MN1 in modulating cell proliferation. However, until now, this concept has not been directly tested. In the current study, we present the first experimental evidence that MN1 negatively regulates cell growth. Stable expression of MN1 in MG-63 osteoblasts results in a greater than 90% reduction in cell growth (Fig. 6Go, B and C). This antiproliferative effect is likely due to decreased entry into S-phase as MN1-transfected cells display a reduction in BrdU incorporation (Fig. 7Go). Together, these data indicate that MN1 inhibits osteoblastic cell proliferation by slowing cell cycle progression. It is likely that MN1 regulates cell growth in a variety of other tissues because it is mutated in tumors, such as meningiomas and leukemia, and is induced by the growth inhibitory factors TGF-ß and EB1089 in numerous cell types. Evaluation of the potential antiproliferative activity of MN1 in these other tissues will be important in understanding whether MN1 acts in a cell type-selective manner or as a more global regulator of cell growth.

The mechanism of this growth suppression by MN1 is not clear, but given the transcriptional activity of MN1, likely involves regulating expression of downstream targets involved in cell cycle progression and proliferation. Although most studies suggest that 1,25-(OH)2D3/VDR directly regulates the expression of cell cycle regulatory genes such as p21WAF1/cip1 (25) and p27kip1 (26), 1,25-(OH)2D3-mediated induction of GADD45{alpha} expression is partially blocked by cycloheximide (28), suggesting that synthesis of other transcription factors are involved in this response. It is possible that 1,25-(OH)2D3 increases MN1 levels and that VDR and MN1 cooperate to regulate the expression of genes such as GADD45{alpha} that inhibit cell proliferation. However, because MN1 expression inhibits cell growth in the absence of 1,25-(OH)2D3 (Figs. 6Go and 7Go), it is unlikely that the growth inhibitory effects of MN1 are completely dependent on liganded VDR. Furthermore, given that SRC-1 expression did not affect cell proliferation (Fig. 7Go), it is unlikely that MN1 suppresses cell growth by acting as a general VDR coactivator. MN1 may modulate cell proliferation through a number of transcriptional mechanisms given that it is induced by more than one growth regulatory signal. Further studies aimed at understanding whether MN1 modulates the expression or activity of cell cycle regulators are required to delineate the molecular details underlying the growth inhibitory effects of MN1.

In summary, we have demonstrated that MN1 is a novel transcriptional target of 1,25-(OH)2D3 in osteoblastic cells. 1,25-(OH)2D3 appears to directly regulate the expression of MN1 mRNA through a transcriptional mechanism. Our studies uncover both a molecular and cellular function of MN1 in regulating VDR- and RAR-mediated transcription by synergizing with SRC proteins and in potently inhibiting osteoblastic cell proliferation by decreasing S-phase entry. Thus, VDR and MN1 may be involved in a positive feedback circuit in which 1,25-(OH)2D3 stimulates expression of MN1, and MN1, in turn, augments VDR-mediated transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
Cell Culture
MG-63 human osteosarcoma cells were maintained in MEM supplemented with 10% fetal bovine serum. COS-7 green monkey kidney cells were maintained in DMEM supplemented with 10% bovine calf serum. For experiments using 1,25-(OH)2D3 compounds, cells were grown in media supplemented with charcoal-stripped bovine calf serum for 3 d before the experiment and during the experiment.

Northern Blot Analysis
mRNA was isolated from MG-63 cells with the FastTrack system (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. mRNA was separated on a formaldehyde/agarose gel and transferred to a Duralon membrane (Stratagene, La Jolla, CA) by capillary action. {alpha}32P-labeled probes were synthesized using the Prime-A-Gene kit (Promega, Madison, WI) according to the manufacturer’s instructions and hybridized to the blots using standard methods. After exposure to x-ray film, blots were quantitated by densitometry.

Plasmids
The reporter plasmids (VDRE)4-TATA-luciferase (luc), (VDRE)4-TK-luc, (GRE)2-TK-luc, (RARE)4-TK-luc, and (GAL4)5-TK-luc were generated by subcloning the promoter regions from (VDRE)4-TATA-GH (22), (VDRE)4-TK-GH (50), (GRE)2-TK-GH (51), (RARE)4-TK-GH (18), and (GAL4)5-TK-GH (52), respectively, into pGL3 (Promega). The rat 24-hydroxylase promoter-luciferase reporter plasmid (r24OHase-luc) was constructed by cloning the PCR-amplified promoter sequence (–369 to +113) from ROS 17/2.8 cells into pGL3. pSG5-VDR (50), pSG5-GAL4-VDR (53), pSG5-RAR{alpha} (18), pSG5-GR (54), pCR-SRC-1 (55), and pSG5-SRC-2/GRIP1 (56) have been previously described. Plasmids encoding GAL4 DBD fusions with all other nuclear receptor LBDs were constructed by PCR amplification of the LBDs from either a human fetal brain or human placental cDNA library (BD Biosciences Clontech, Palo Alto, CA) and ligating them in frame with the GAL4 DBD. CMVTAG2B-MN1 was constructed by subcloning the MN1 cDNA from pSCTOP-MN1 (33) in frame with the FLAG epitope tag in the CMVTAG2B vector (Stratagene). CMVTAG2A-RORß was created by PCR amplification of the full-length RORß coding sequence from a human fetal brain library (BD Biosciences Clontech) and ligating it in frame with FLAG. The dual expression vector pCMS-EGFP-MN1 drives expression of EGFP and MN1 from two separate promoters on a single plasmid. This plasmid was constructed by subcloning FLAG-tagged MN1 from CMVTAG2B-MN1 into pCMS-EGFP (BD Biosciences Clontech). pCMS-EGFP-SRC1 was created by subcloning full-length human SRC1 from pCR-SRC1 (55) into pCMS-EGFP.

Transient Transfection Assays
COS-7 cells were seeded at a density of 70,000 per well of six-well plates and transfected by the standard calcium phosphate precipitation method as previously described (50). CMV-renilla (Promega) was cotransfected with other plasmids to normalize for transfection efficiency. Plasmids encoding MN1 or other coactivators and their respective vector controls were transfected in molar balance in all experiments. Cells were dosed with the indicated amounts of ligand for 24 h. Cell lysates were isolated after 24 h of ligand stimulation, and the luciferase and renilla reporter gene activities were determined using a commercial dual luciferase assay kit (Promega) according to the manufacturer’s recommendations. An LMax microplate luminometer (Molecular Devices, Sunnyvale, CA) was used to measure luciferase activity. Data presented the mean of duplicate or triplicate wells ± SD of a representative experiment that has been repeated at least three times.

MG-63 Cell Growth Assay
MG-63 cells were plated at a density of 8 x 104 cells/well of a six-well dish in media containing charcoal-stripped serum. Cells were treated with either ethanol vehicle control or 10 nM 1,25-(OH)2D3 for up to 5 d. Viable cells were quantified by counting trypan blue-excluding cells with a hemocytometer. Data presented represent the mean of triplicate wells ± SD of a representative experiment performed twice.

MG-63 Stable Pool Growth Assay
MG-63 cells were plated the day before transfection at a density of 1.4 x 106 cells/150-mm plate. Cells were transfected with CMVTAG2B, CMVTAG2B-MN1, or CMVTAG2A-RORß using Fugene (Roche Applied Science, Indianapolis, IN). After 48 h, cells were trypsinized and 250,000 cells/well were plated in G418-containing media to select for transfected cells. After 7 d, resulting colonies were stained with 0.25% crystal violet in 50% methanol. After extensive washing in PBS, colonies were air-dried for at least 1 h and photographed. Dye was extracted in 100 µl of 0.1 M sodium citrate in 50% ethanol and quantified spectrophotometrically at 540 nm using a Versimax microplate reader (Molecular Devices). Data presented represent the mean of triplicate wells ± SD of a representative experiment performed three times.

BrdU Incorporation
A total of 22,000 MG-63 cells were seeded per chamber of a four-well Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY) coated with poly-L-lysine and bovine calf serum to promote cell attachment. Cells were transfected with pCMS-EGFP, pCMS-EGFP-MN1, or pCMS-EGFP-SRC1 with Fugene (Roche Applied Science) and allowed to express for 24 h. Cells were pulse-labeled with 10 µM BrdU for 1 h, fixed in 4% paraformaldehyde, and permeabilized in PBS containing 3% BSA and 0.3% Triton X-100. BrdU incorporation sites were exposed by treating cells with 100 U/ml of bovine pancreatic deoxyribonuclease I (Amersham Biosciences, Piscataway, NJ). Cells were immunostained with an anti-BrdU mouse monoclonal antibody (BD Biosciences) followed by a goat antimouse secondary antibody conjugated to Alexa Fluorophore 594 (Molecular Probes, Eugene, OR). Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (Roche Applied Science). Between 100 and 150 EGFP-positive cells were counted and scored for BrdU positivity in each sample. Data represent the mean percentage of BrdU-positive cells of three independent experiments ± SD.


    Note Added in Proof
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 Note Added in Proof
 REFERENCES
 
While this manuscript was in press, a mouse model with a targeted deletion in MN1 was described (57). MN1-deficient mice have defects in numerous cranial bones formed by intramembraneous ossification. These findings provide in vivo evidence for an important role of MN1 in skeletal biology. Coupled with the data in the current study that indicate that MN1 regulates osteoblastic cell proliferation, the phenotype of the MN1-knockout mice suggests that this protein controls osteoblast differentiation or mineralization and provide the groundwork for further investigation into the molecular details of MN1 functions in osteoblasts in vivo and in vitro.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Gerald Grosveld (St. Jude Children’s Research Hospital, Memphis, TN) for providing pSCTOP-MN1, Dr. Bert O’Malley (Baylor College of Medicine, Houston, TX) for providing pCR-SRC-1, and Dr. Michael Stallcup (University of Southern California, Los Angeles, CA) for providing pSG5-SRC-2/GRIP1. We would also like to thank Dr. Milan Uskokovic (Bioxell, Inc., Nutley, NJ) for supplying 1,25-(OH)2D3.


    FOOTNOTES
 
This work was supported in part by National Institutes of Health (NIH) Grants R01 DK50348 and R01 DK 53980 (to P.N.M.), by an Institutional National Research Service Award from the National Institutes of General Medical Sciences GM 08803 (to T.I.E), by an award from the Medical Scientist Training Program NIH Grant T32 GM007250 (to A.L.M.S.), and by a Pharmaceutical Manufacturers’ Association (PhRMA) Foundation Pre-Doctoral Fellowship (to A.L.M.S.).

First Published Online May 12, 2005

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CBP, cAMP response element binding protein (CREB)-binding protein; DBD, DNA binding domain; DRIP, VDR-interacting protein; EGFP, enhanced green fluorescent protein; GR, glucocorticoid receptor; GRIP, GR-interacting protein; LBD, ligand-binding domain; MN1, gene disrupted in meningioma-1; MSV-LTR, Moloney sarcoma virus long terminal repeat; NCoA-62, nuclear coactivator 62 kDa; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; RAR, retinoic acid receptor; RARE, retinoic acid response element; RORß, RAR-related orphan receptor; RXR, retinoid X receptor; SKIP, ski-interacting protein; SRC-1, steroid receptor coactivator-1; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication February 14, 2005. Accepted for publication May 4, 2005.


    REFERENCES
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 ABSTRACT
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 DISCUSSION
 MATERIALS AND METHODS
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