Nuclear Factor of Activated T-cells (NFAT)c2 Inhibits Notch Receptor Signaling in Osteoblasts*

Background: Notch and nuclear factor of activated T-cells (NFAT)c signaling regulate cell function and interact in osteoblasts. Results: Notch stabilizes NFATc2 transcripts, NFATc2 inhibits Notch by competing for DNA binding with the Notch transcriptional complex, and Notch and NFATc2 suppress osteoblast gene markers. Conclusion: Notch and NFATc2 interact to inhibit osteoblast function. Significance: Notch and NFAT signaling suppress osteoblastogenesis. Notch receptors regulate osteoblastogenesis, and Notch activation induces cleavage and nuclear translocation of the Notch intracellular domain (NICD), which associates with Epstein-Barr virus latency C-promoter binding factor-1/suppressor of hairless/lag-1 (CSL) and induces transcription of Notch target genes, such as hairy enhancer of split-related with YRPW motif (Hey)1 and Hey2. Nuclear factors of activated T-cells (NFAT) are transcription factors that regulate osteoclastogenesis, but their function in osteoblasts is not clear. Notch inhibits NFATc1 transcription, but interactions between Notch and NFAT are understood poorly. To determine the regulation of NFAT expression by Notch, osteoblasts from RosaNotch mice, where NICD is transcribed following excision of a loxP flanked STOP cassette, were used. Alternatively, wild-type C57BL/6 osteoblasts were exposed to the Notch ligand Delta-like (Dll)1 to induce Notch signaling or to bovine serum albumin as control. In RosaNotch osteoblasts, Notch suppressed NFATc1 expression, increased Nfatc2 mRNA by post-transcriptional mechanisms, and had no effect on NFATc3 and NFATc4 transcripts. Induction of Nfatc2 transcripts by Notch was confirmed in C57BL/6 osteoblasts exposed to Dll1. To investigate NFATc2 function in osteoblasts, constitutively active NFATc2 was overexpressed in RosaNotch osteoblasts. NFATc2 suppressed Notch transactivation and expression of Hey genes. Electrophoretic mobility shift assays revealed that NFATc2 and CSL bind to similar DNA sequences, and chromatin immunoprecipitation indicated that NFATc2 displaced CSL from the Hey2 promoter. The effects of NICD and NFATc2 in RosaNotch osteoblasts were assessed, and both proteins inhibited osteoblast function. In conclusion, Notch stabilizes Nfatc2 transcripts, NFATc2 suppresses Notch signaling, and both proteins inhibit osteoblast function.

The Notch signaling pathway regulates developmental processes, cell renewal, and cell fate (1). Notch1 to Notch4 are a family of transmembrane receptors that interact with transmembrane ligands expressed by neighboring cells (2). Ligandreceptor interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD) 2 (3). Epstein-Barr virus latency C promoter binding factor 1, suppressor of hairless and lag-1 (CSL), also known as Rbp-j in mice, is a nuclear protein constitutively bound to DNA, able to suppress gene expression by recruiting transcriptional co-repressors. In the canonical signaling pathway, NICD translocates to the nucleus and forms a multimeric protein complex with CSL, displacing the transcriptional co-repressors and recruiting coactivators of transcription (4). These events result in the expression of Notch target genes, such as hairy enhancer of split (Hes) and Hes related with YRPW motif (Hey)1 and Hey2 (5).
Nuclear factors of activated T-cells (NFAT)c1 to NFATc4 are transcription factors that regulate growth and differentiation of multiple cell types. NFAT transactivation is induced by calcineurin, a phosphatase that dephosphorylates specific serine residues in the SRR and SPXX repeat motifs of the regulatory domain of NFAT (6). Dephosphorylated NFAT translocates to the nucleus, binds to specific DNA consensus sequences, such as those present upstream of exon 4 of regulator of calcineurin (Rcan1.4), and as a consequence induces expression of NFAT target genes (6,7). NFAT phosphorylation by protein kinases, such as casein kinase 1 and glycogen synthase kinase 3␤, induces NFAT nuclear export, preventing its binding to DNA, and as a consequence inhibiting NFAT transactivation (6).
Osteoblast cell fate and function are regulated by signaling networks that include the Notch and calcineurin/NFAT signal-ing pathways (8,9). Notch promotes osteoblast proliferation, possibly by inducing expression of cyclin D1 and D3, and suppresses osteoblast differentiation by inhibiting Wnt/␤-catenin signaling and by opposing runt-related transcription factor (Runx)2 transactivation (10). The role played by NFAT proteins in the regulation of osteoblast differentiation and function is not clear, and suppression of NFAT signaling by inactivation of calcineurin in mice has generated controversial results (11)(12)(13). We demonstrated that overexpression of a constitutively active form of NFATc1 suppresses the expression of osteoblast gene markers in vitro (14). Accordingly, NFATc1 inhibits osteoblastic differentiation and recruits histone deacetylases to the osteocalcin promoter to suppress osteocalcin expression (15). Unexpectedly, global Nfatc1 null mice are osteopenic and display defective bone formation, although indirect nonspecific effects are possible (16,17). Similarly, global Nfatc2 null mice display osteopenia and suppressed osteoblastic function, but hyperproliferation of B and T-cells may be responsible for this skeletal phenotype (18). In osteoblasts, Notch suppresses NFATc1 transcription and NFAT transactivation (14), but the effects of Notch on the expression of alternate NFAT paralogs have not been reported, and interactions between these two signaling pathways are poorly understood.
In the present study, we investigated the effects of Notch on the expression of the four NFATc paralogs in primary calvarial osteoblasts. In addition, we explored the regulation of Notch canonical signaling by NFATc2 and assessed the effects of Notch and NFATc2 on osteoblast function.

EXPERIMENTAL PROCEDURES
Cell Cultures-Osteoblast-enriched cells were isolated by sequential collagenase digestion from parietal bones of 3-5day-old Rosa Notch mice, generated in a 129SvJ/C57BL/6 mixed genetic background (D. A. Melton, Harvard University, Cambridge, MA, obtained from The Jackson Laboratory, Bar Harbor, ME), or wild-type C57BL/6 mice, as described (19,20). In Rosa Notch mice, the Rosa26 locus is targeted by homologous recombination with a DNA construct encoding NICD, preceded by a STOP cassette flanked by loxP sequences, cloned downstream of the Rosa26 promoter. Expression of NICD from the targeted Rosa26 locus occurs following the excision of the STOP cassette by CRE recombination of loxP sequences. Notch receptors can be activated by Notch ligands adherent to the cell culture substrate (21). Tissue culture plates were exposed to 250 ng/ml of the Notch ligand Delta-like (Dll)1 (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS, Amresco, Solon, OH) for 1 h at room temperature to obtain immobilized Dll1 at a density of 62.5 ng/cm 2 . As control, an equimolar amount of bovine serum albumin (BSA, Sigma-Aldrich) in PBS was immobilized per cm 2 of cell culture substrate. Wild-type C57BL/6 osteoblasts were seeded on immobilized Dll1 to induce Notch signaling or seeded on BSA as control. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with nonessential amino acids (Life Technologies), 20 mM HEPES, 100 g/ml ascorbic acid (both from Sigma-Aldrich), and 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), at 37°C in a humidified 5% CO 2 incubator. Adenoviral Infection-At 70% confluence, osteoblasts were transferred to medium containing 2% FBS for 1 h and exposed overnight to 100 multiplicity of infection of replication defective recombinant adenoviruses. An adenoviral vector expressing CRE recombinase under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-CRE, Vector Biolabs, Philadelphia, PA) was delivered to Rosa Notch cells to induce recombination of the loxP sequences and NICD expression. An adenoviral vector expressing green fluorescent protein (GFP) under the control of the CMV promoter (Ad-CMV-GFP, Vector Biolabs) was used as control.
A 2.8-kb DNA fragment containing the sequence coding for amino acids 98 -106 of the human influenza hemagglutinin (HA) followed by the coding sequence of murine Nfatc2, where serine to alanine mutations in the SRR and SPXX repeat motifs of the regulatory domain render NFATc2 constitutively active, was created by A. Rao (Harvard Medical School, Boston, MA) (22). This construct was obtained from Addgene (Cambridge, MA, Addgene plasmid 11792) and used to create an adenoviral vector where the expression of constitutively active NFATc2 (caNFATc2) is directed by the CMV promoter (Ad-CMV-caNFATc2; Vector Biolabs). Wild-type C57BL/6 osteoblasts were transduced with Ad-CMV-caNFATc2 or with control Ad-CMV-GFP, and in selected experiments, Rosa Notch osteoblasts transduced with Ad-CMV-CRE or with control Ad-CMV-GFP were co-transduced with Ad-CMV-caNFATc2 or with control Ad-CMV-GFP. After transduction, cells were cultured in the presence of DMEM containing 10% FBS.
To assess Nfatc2 transcription, heterogeneous nuclear RNA (hnRNA) levels were determined. For this purpose, 0.5 g of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Life Technologies), in the presence of a specific antisense primer targeted to intron 6 of Nfatc2 (supplemental Table 1). Reverse-transcribed cDNA was amplified in the presence of primers flanking the exon 6 intron 6 junction of Nfatc2 hnRNA (supplemental Table 1) and Platinum quantitative PCR SuperMix-UDG (Life Technologies) at 60°C for 45 cycles. -Fold changes in Nfatc2 hnRNA, normalized to Rpl38 expression, were determined by performing amplification reactions in a CFX96 real time system and by analyzing the results with the 2 Ϫ⌬⌬CT method using as a reference the corrected expression levels of Nfatc2 hnRNA in control cells. Amplification efficiency was estimated by comparison with a standard curve generated by parallel amplification of a dilution series of genomic murine 129SvJ/C57BL/6 DNA (28). Fluorescence was monitored during every PCR cycle at the annealing step, and specificity of the reaction was confirmed by the presence of a single peak in the melt curve analysis of PCR products.
Western Blot Analysis-To detect changes in endogenous NFATc2 protein levels or to assess overexpression of caNFATc2, Rosa Notch osteoblasts were washed with PBS and extracted in 50 mM Tris, 15 mM EGTA, 100 mM NaCl, and 0.5% Triton X-100 (all from Sigma-Aldrich) or cell lysis buffer (Cell Signaling Technology, Beverly, MA), respectively. Extraction was performed in the presence of protease and phosphatase inhibitors and 1 mM dithiothreitol (DTT) (all from Sigma-Aldrich) at 4°C for 30 min, and cell debris was removed by centrifugation at 4°C. Protein concentrations were determined using a DC protein assay kit (Bio-Rad), and 20 -50 g of total protein were fractionated by gel electrophoresis in 10% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were blocked with 3% BSA in PBS and exposed to a 1:1000 dilution of primary antibody. A rabbit polyclonal antibody raised against the N terminus of NFATc2 (antibody 4389, Cell Signaling Technologies) was used for the detection of endogenous NFATc2, whereas a mouse monoclonal antibody (clone 25A10.D6.D2, Thermo Scientific) was used for the detection of caNFATc2. Blots were exposed to either anti-rabbit IgG or anti-mouse IgG conjugated to horseradish peroxidase (Sigma-Aldrich) and incubated with a chemiluminescence detection reagent (Bio-Rad), and digital images were acquired with a cooled charged coupled device camera, mounted on the Chemidoc XSR molecular imager (Bio-Rad). To assess even loading of the samples, membranes were stripped and reprobed with a 1:1000 dilution of a goat polyclonal antibody against actin (I-19, Santa Cruz Biotechnology, Santa Cruz, CA) and exposed to anti-goat IgG conjugated to horseradish peroxidase, and digital images were acquired with a Chemidoc XSR molecular imager. Intensity of the chemiluminescent signal was determined by densitometry with Image Lab v2.0.1 (Bio-Rad).
RNA Decay Experiments-The effects of NICD on the stability of Nfatc2 mRNA were assessed in Rosa Notch osteoblasts grown to confluence and exposed to 75 M 5,6-dichloro-1-␤-Dribofuranosylbenzimidazole (DRB, BioMol, Plymouth Meeting, PA) to arrest transcription. Total RNA was extracted and subjected to qRT-PCR analysis to determine Nfatc2 mRNA levels following different times of exposure to DRB. To establish the slopes of Nfatc2 mRNA decay, Nfatc2 copy numbers corrected for Rpl38 transcript levels, expressed as a percentage of the corrected Nfatc2 mRNA levels measured before exposure to DRB, were transformed by a base 10 logarithmic function and fitted against time by linear regression.
Transient Transfections-To study the effects of NFATc2 on Notch transactivation, Rosa Notch osteoblasts were transduced with Ad-CMV-caNFATc2 or control Ad-CMV-GFP, either under basal conditions or in the context of Notch overexpression. Cells were transiently transfected with a construct containing six multimerized dimeric CSL binding sites, linked to the ␤-globin basal promoter (12ϫCSL-Luc; L. J. Strobl, Munich, Germany) or with 2.9-and 2.0-kb fragments of the Hey1 (Hey1-Luc; M. M. Maier, Wuerzburg, Germany) and Hey2 (Hey2-Luc; T. Iso) promoters, cloned upstream of luciferase (29 -31). Transfections were conducted in cells cultured to 70% confluence using FuGENE 6 (3 l of FuGENE 6/2 g of DNA), according to the manufacturer's instructions (Roche Applied Science). A construct where the CMV promoter directs the expression ␤-galactosidase (CMV/␤-galactosidase; Clontech) was used to correct for transfection efficiency. Cells were exposed to the FuGENE 6-DNA mix for 16 h, transferred to fresh medium for 24 h, and harvested. Luciferase activity was corrected for ␤-galactosidase activity; both were measured using an Optocomp luminometer (MGM Instruments, Hamden, CT).
Electrophoretic Mobility Shift Assay (EMSA)-To analyze DNA binding by CSL and NFATc2, nuclear extracts were obtained from Rosa Notch osteoblasts transduced with Ad-CMV-caNFATc2 or control Ad-CMV-GFP, in the context or not of NICD overexpression (32,33). Synthetic doublestranded oligonucleotides containing consensus sequences for Csl or Nfatc, found in the Epstein-Barr virus nuclear antigen 2 (EBNA2) or in the natriuretic peptide type B (Bnp) promoters (Integrated DNA Technology, Coralville, IA), were labeled with [␥-32 P]ATP, using T 4 polynucleotide kinase (Promega Corp., Madison, WI) (supplemental Table 2A) (34,35). Nuclear extracts and radiolabeled oligonucleotides were incubated for 20 min at room temperature in 10 mM Tris buffer (pH 7.5) containing 3 g of poly(dI-dC) (Sigma-Aldrich). To assess whether CSL and NFATc2 recognize reciprocal consensus sequences, binding to the radiolabeled Csl oligonucleotide was performed in the presence of a 200-fold excess of unlabeled oligonucleotide containing the Nfatc consensus sequence from the Bnp promoter (supplemental Table 2A) (35). In the converse experiment, binding to the radiolabeled Nfatc oligonucleotide was performed in the presence of a 200-fold excess of unlabeled oligonucleotide containing the Csl sequence from the EBNA2 promoter (supplemental Table 2A) (34). To determine specificity of the binding of the nuclear extracts to the consensus sequences, unlabeled homologous or mutated oligonucleotides were added in 200-fold excess (supplemental Table  2A). DNA-protein complexes were resolved on nondenaturing, nonreducing 6% polyacrylamide gels, and the complexes were visualized by autoradiography.
Chromatin Immunoprecipitation (ChIP) Assay-The association of NFATc2 to the Rcan1.4 promoter was assessed in wildtype C57BL/6 osteoblasts transduced with Ad-CMV-caNFATc2, or with Ad-CMV-GFP as control. The interactions of NFATc2 and CSL with the Hey2 promoter were investigated in Rosa Notch osteoblasts transduced with Ad-CMV-caNFATc2, or control Ad-CMV-GFP, in the context or not, of NICD overexpression. ChIP analysis was carried out with the ChIP-IT express magnetic chromatin immunoprecipitation and sonication shearing kit following the manufacturer's instructions with slight modifications (Active Motifs, Carlsbad, CA). To crosslink DNA-interacting proteins and DNA, confluent osteoblasts were exposed to DMEM and 1% formalin (Sigma-Aldrich) at room temperature for 5 min. The cross-linking reaction was arrested by exposure to 125 mM glycine in PBS, and the cell layer was collected in PBS in the presence of protease and phosphatase inhibitors. Nuclei were released by Dounce homogenization, and DNA fragments about 400 -800 bp long were obtained by sonication. DNA was incubated with constant agitation overnight at 4°C with a ChIP grade rabbit polyclonal HA antibody (Abcam, Cambridge, MA), a rabbit CSL (H-50, Santa Cruz Biotechnology) antibody, or control rabbit immunoglobulin G (IgG; Santa Cruz Biotechnology), conjugated to protein G magnetic beads (Cell Signaling Technologies). Beads were washed, and DNA-nuclear protein complexes were eluted. The cross-linking reaction was reversed at 65°C for 2.5 h, and following incubation with proteinase K, DNA was purified with DNeasy columns according to the manufacturer's instructions (Qiagen). Purified DNA was amplified by quantitative PCR (qPCR) performed in the presence of specific primers flanking Nfatc consensus sequences required for the activity of the Rcan1.4 promoter, or Csl consensus sequences that regulate the activity of the Hey2 promoter (supplemental Table 2B), and iQ SYBR Green Supermix at 60°C for 40 cycles (31,36). Amplification reactions were conducted in a CFX96 real time system, fluorescence was monitored during every PCR cycle at the annealing step, and specificity of the reaction was confirmed by the presence of a single peak in the melt curve analysis of PCR products.
Cytochemical Assays and Alkaline Phosphatase Activity-To determine mineralization of the culture, cells were rinsed in PBS, fixed with 3.7% formaldehyde, and stained with a 2% Alizarin Red solution in H 2 O (37). Color images were acquired with a Coolpix 995 digital camera (Nikon Inc., Melville, NY) imported to ImageJ software (38) and converted to grayscale, and threshold was defined as the intensity of gray above the background. Identical threshold settings were used for each image analyzed, and the mineralized area was defined as the area occupied by pixels surpassing the preset threshold, quantified by using the measure function of ImageJ.
Alkaline phosphatase activity was determined in 0.5% Triton X-100 cell extracts by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol and measured by spectroscopy at 405 nm after 10 min of incubation at room temperature, according to the manufacturer's instructions (Sigma-Aldrich). Data are expressed as nanomoles of p-nitrophenol released per minute per g of protein. Total protein content was determined in cell extracts by the DC protein assay.
Statistical Analysis-Data are expressed as means Ϯ S.E. Statistical differences were determined by Student's t test or analysis of variance with Scheffé post hoc analysis for pairwise or multiple comparisons. Statistical differences for the slopes of mRNA decay were analyzed by analysis of covariance (39).

Notch Induces the Expression of NFATc2 by Post-transcriptional Mechanisms-
The effects of NICD on the expression of the four Nfatc paralogs were tested in primary calvarial osteoblast cultures. Osteoblasts from Rosa Notch mice were transduced with an adenoviral vector expressing CRE under the control of the CMV promoter (Ad-CMV-CRE) to excise a STOP cassette flanked by lox P sequences and allow NICD expression under the control of the Rosa26 promoter. Control cultures were infected with an adenoviral vector where the CMV promoter directs the expression of GFP (Ad-CMV-GFP). In accordance with our previous observations demonstrating that NICD inhibits the transcription of NFATc1, NICD decreased Nfatc1 mRNA levels (14). NICD induced Nfatc2 transcripts and protein levels and did not modify the expression of Nfatc3 and Nfatc4 in osteoblasts (Fig. 1, A and B). To confirm whether Notch activation induces Nfatc2 mRNA levels, wild-type C57BL/6 osteoblasts were exposed to immobilized Dll1 to induce Notch signaling or to BSA as control. Following 3 days of Wild-type C57BL/6 osteoblasts were exposed to immobilized Dll1 (Dll1, black bars), or control BSA (BSA, white bars). In A and C, Rosa Notch osteoblasts were cultured for 3 days (A) or 1 day (C) after confluence, and total RNA was extracted, reverse-transcribed, and amplified by qRT-PCR. Data are expressed as ratio of Nfatc1, Nfatc2, Nfatc3, Nfatc4 (A), and NFATc2 hnRNA (C) copy number corrected for Rpl38 expression, relative to corrected expression in GFP. Values are means Ϯ S.E., n ϭ 4. *, significantly different between CRE and GFP, p Ͻ 0.05. Data are from representative experiments performed in duplicate. In B, total cellular extracts from Rosa Notch osteoblasts were fractioned by gel electrophoresis and transferred to an Immobilon-P membrane, which was incubated with an antibody against NFATc2, stripped, and reprobed with an antibody against actin. Digital images were acquired, and signal from NFATc2 and actin antibodies was quantified by densitometry to estimate protein expression. Data are expressed as ratio of NFATc2 levels corrected for actin expression relative to corrected NFATc2 levels in GFP. Values are means of four independent experiments Ϯ S.E. *, significantly different between CRE and GFP, p Ͻ 0.05. In D, osteoblasts from wild-type C57BL/6 were cultured for 3 days after confluence, and total RNA was extracted, reverse-transcribed, and amplified by qRT-PCR. Data are expressed as ratio of Hey2 or Nfatc2 copy number corrected for Rpl38 expression, relative to corrected expression in BSA. Values are means Ϯ S.E., n ϭ 4. *, significantly different between Dll1 and BSA, p Ͻ 0.05. In E, Rosa Notch osteoblasts were transcriptionally arrested with DRB (time 0) and harvested at the indicated times. Total RNA was extracted and amplified by qRT-PCR, and data are expressed as the percentage of Nfatc2 mRNA copy number corrected for Rpl38 expression, relative to the corrected Nfatc2 mRNA expression before DRB treatment, plotted versus time. Data are means Ϯ S.E., n ϭ 4. Nfatc2 decay slopes for CRE and GFP are statistically different, p Ͻ 0.05. Data are from representative experiments performed in triplicate. culture, osteoblasts exposed to Dll1 exhibited increased Hey2 expression in comparison with cells exposed to BSA, indicating activation of Notch signaling by Dll1. In agreement with the stimulatory effects of NICD overexpression on Nfatc2 transcripts in Rosa Notch osteoblasts, Dll1 increased Nfatc2 mRNA levels, confirming that Notch induces Nfatc2 in osteoblasts (Fig. 1D). The mechanisms mediating the increase of Nfatc2 mRNA levels by Notch were investigated in Rosa Notch osteoblasts. Notch did not change Nfatc2 hnRNA levels, indicating that Notch does not regulate Nfatc2 transcription (Fig. 1C). To determine whether Notch increased Nfatc2 expression by posttranscriptional mechanisms, the decay of Nfatc2 transcripts was assessed in Rosa Notch osteoblasts transcriptionally arrested with DRB. NICD prolonged the half-life of Nfatc2 mRNA from 4 to 13 h, demonstrating that Notch stabilizes Nfatc2 transcripts (Fig. 1E).
NFATc2 Suppresses Notch Canonical Signaling-To investigate the effects of NFATc2 on Notch signaling, Rosa Notch osteoblasts transduced with Ad-CMV-CRE or Ad-CMV-GFP, and wild-type C57BL/6 osteoblasts, were transduced with an adenoviral vector where the CMV promoter controls the expression of constitutively active NFATc2 (Ad-CMV-caNFATc2) or control Ad-CMV-GFP. Expression of caNFATc2 in Rosa Notch osteoblasts was documented by Western blot and qRT-PCR analysis (Fig. 2, A and B). We confirmed that Notch induced Nfatc2 mRNA in control Rosa Notch osteoblasts, and Notch caused an additional increase in Nfatc2 transcripts in cells transduced with Ad-CMV-caNFATc2 (Fig. 2B). Because transactivation of the CMV promoter by Notch is not likely, this result suggests that the NFATc2 coding sequence contains regulatory motifs in part responsible for the post-transcriptional effects of Notch on Nfatc2 mRNA. In C57BL/6 osteoblasts transduced with Ad-CMV-caNFATc2 tagged with HA, ChIP analysis revealed that immunoprecipitation with an HA antibody enriched DNA fragments containing Nfatc consensus sequences required for the activity of the Rcan1.4 promoter, demonstrating direct interaction of NFATc2 with this promoter ( Fig. 2C) (36). Accordingly, caNFATc2 increased Rcan1.4 mRNA levels in Rosa Notch osteoblasts, although the induction was similar in control cells and in the context of Notch induction, suggesting that the increase in Nfatc2 mRNA levels caused by Notch does not translate in enhanced NFATc2 activity (Fig. 2D).
To test the effects of NFATc2 on Notch transactivation, Rosa Notch osteoblasts were transfected either with the 12ϫCSL-Luc reporter, which is induced by activation of Notch canonical signaling, or with fragments of the Hey1 (Hey1-Luc) and Hey2 (Hey2-Luc) promoters, which are targets of Notch signaling. NICD transactivated the 12ϫCSL-Luc reporter and the Hey1-Luc and Hey2-Luc promoter constructs, whereas caNFATc2 opposed this effect (Fig. 3A). In accordance with these results, NICD induced Hey1 and Hey2 transcripts, and this effect was opposed by caNFATc2, demonstrating that NFATc2 suppresses Notch canonical signaling in osteoblasts (Fig. 3B).
NFATc2 Competes with CSL for Binding to DNA-The mechanism of the inhibitory effect of NFATc2 on Notch canonical signaling was analyzed by EMSA in Rosa Notch osteoblasts transduced with Ad-CMV-CRE or Ad-CMV-GFP and co-trans-duced with Ad-CMV-caNFATc2 or control Ad-CMV-GFP. A radiolabeled Csl consensus oligonucleotide was bound by nuclear protein extracts from cells overexpressing NICD and controls, and an excess of unlabeled Csl oligonucleotides prevented this effect, demonstrating specificity of the binding. An excess of unlabeled oligonucleotides containing an Nfatc consensus sequence decreased the binding of the nuclear extracts to the Csl consensus oligonucleotide, indicating that CSL can bind to Nfatc consensus sequences. In the converse experiment, nuclear extracts from osteoblasts expressing caNFATc2 bound to a radiolabeled Nfatc consensus oligonucleotide to a greater extent than control extracts, confirming increased DNA binding by NFATc2. Formation of nuclear protein complexes with the radiolabeled Nfatc oligonucleotide was opposed by unlabeled Nfatc and Csl oligonucleotides, demonstrating specificity of the binding reaction and suggesting that NFATc2 can bind to Csl consensus sequences (Fig. 4B). Unlabeled oligonucleotides containing mutated Csl or Nfatc consensus sequences did not preclude formation of nuclear protein complexes with the radiolabeled oligonucleotides, demonstrating that nuclear protein binding to DNA requires intact Csl or Nfatc consensus sequences (Fig. 4, A and B). These findings indicate that CSL and NFATc2 recognize similar consensus FIGURE 2. NFATc2 is expressed and activated in osteoblasts. Rosa Notch or wild-type C57BL/6 calvarial osteoblasts were transduced with Ad-CMV-caN-FATc2 tagged with HA (NFATc2, black bars) or control Ad-CMV-GFP (Control, white bars). Rosa Notch osteoblasts were co-transduced with Ad-CMV-CRE (CRE) to induce Notch, or control Ad-CMV-GFP (GFP), and cultured for 72 h. In A, total cellular extracts from Rosa Notch osteoblasts were fractioned by gel electrophoresis and transferred to an Immobilon-P membrane, which was incubated with an antibody against NFATc2. Membranes were stripped and reprobed with an antibody against actin. In B and D, total RNA was extracted from Rosa Notch osteoblasts and amplified by qRT-PCR. Data are expressed as ratio of Nfatc2 and Rcan1.4 copy number corrected for Rpl38 expression, relative to corrected expression in control cells co-transduced with Ad-CMV-GFP. Values are means Ϯ S.E., n ϭ 4. *, significantly different between NFATc2 and control, p Ͻ 0.05. ϩ, significantly different between CRE and GFP, p Ͻ 0.05. In C, wild-type C57BL/6 osteoblasts were cultured to confluence, and DNA was cross-linked to associated proteins and fragmented by sonication. DNA-protein complexes were obtained by immunoprecipitation with an HA antibody or control IgG, and DNA was purified following reversal of the cross-linking reaction. Immunoprecipitated DNA and fragmented input DNA were amplified by qPCR with primers flanking Nfatc consensus sequences required for the activity of the Rcan1.4 promoter. Data are expressed as enrichment of DNA by immunoprecipitation with the HA antibody relative to control IgG, corrected for input DNA levels. Values are means of three qPCR reactions. Data are from representative experiments performed in duplicate.
sequences and that NFATc2 competes with CSL for binding to Csl consensus sequences.
The association of NFATc2 and CSL to the Hey2 promoter was investigated by ChIP analysis in Rosa Notch osteoblasts transduced with Ad-CMV-caNFATc2 or control Ad-CMV-GFP, in the context of Notch induction, or under basal conditions. In agreement with the results obtained by EMSA, qPCR analysis revealed that in control cells co-transduced with Ad-CMV-caNFATc2, immunoprecipitation with an HA antibody, in comparison with a control IgG, enriched DNA fragments from the Hey2 promoter containing Csl consensus sequences (Fig. 5A). However, under conditions of Notch induction, association of NFATc2 to the Hey2 promoter was not detected (Fig.  5A). Under basal conditions, DNA fragments from the Hey2 promoter were immunoprecipitated with a CSL antibody, and a modest increase in this effect was observed in the presence of NICD (Fig. 5B), confirming that the Csl consensus sequences analyzed regulate the activity of the Hey2 promoter (31). This effect was opposed by caNFATc2 (Fig. 5B), confirming the competition between CSL and NFATc2 for DNA binding observed by EMSA and indicating that NFATc2 displaces CSL from promoter sequences that regulate Hey2 expression.
Notch and NFATc2 Regulate Osteoblast Expression of Gene Markers-The effects of NFATc2 expression on osteoblast function were determined in Rosa Notch osteoblasts transduced with Ad-CMV-caNFATc2 or control Ad-CMV-GFP and cotransduced with Ad-CMV-CRE to induce Notch or Ad-CMV-GFP as control. In agreement with the inhibition of osteoblast function by Notch, NICD suppressed Alpl, Bsp, Dlx5, Runx2, and osteocalcin transcripts, alkaline phosphatase activity, and mineralization of the culture, although no effect on Col1a1 expression was observed (Fig. 6, A-C) (14). NFATc2 suppressed the expression of all the osteoblastic gene markers analyzed, either in the context of Notch induction or under basal conditions (Fig. 6, A and B). Accordingly, NFATc2 inhibited alkaline phosphatase activity and reduced culture mineralization, although these effects were not observed in the context of  Binding of nuclear proteins to DNA was tested by electrophoretic mobility shift assay in Rosa Notch calvarial osteoblasts transduced with Ad-CMV-caNFATc2 (NFATc2) or control Ad-CMV-GFP (Control) and co-transduced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP). Cells were cultured to confluence, and nuclear extracts were incubated with [␥-32 P]ATP-labeled oligonucleotides. In A, a radiolabeled oligonucleotide containing a Csl consensus sequence from the EBNA2 promoter was used. Competition of binding reactions was performed in the presence of unlabeled Csl consensus and mutant oligonucleotides or unlabeled Nfatc oligonucleotides containing homologous or mutated consensus sequences from the Bnp promoter in 200-fold excess. In B, a radiolabeled oligonucleotide containing an Nfatc consensus sequence from the Bnp promoter was used. Competition of binding reactions was performed in the presence of unlabeled Nfatc consensus and mutant oligonucleotide or unlabeled Csl oligonucleotides containing homologous or mutated consensus sequences from the EBNA2 promoter in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis and visualized by autoradiography, and arrows indicate the position of complexes. Autoradiographs are representative of four independent experiments. NICD overexpression (Fig. 6C). NFATc2 inhibited osteopontin expression, whereas NICD opposed this effect, confirming previous observations demonstrating induction of osteopontin by Notch (40). These results demonstrate that Notch and NFATc2, with the exception of osteopontin, suppress the expression of osteoblast gene markers and inhibit mature osteoblastic function in vitro.

DISCUSSION
In this study, we investigated the effects of Notch on the expression of the four NFAT paralogs in primary calvarial osteoblasts. In previous work, we demonstrated that Notch suppresses NFAT transactivation by inhibiting NFATc1 expression (14). We confirmed that Notch suppresses Nfatc1 mRNA levels and reported that NICD overexpression in osteoblast induces Nfatc2 by post-transcriptional mechanisms (14). Accordingly, exposure of osteoblasts to the Notch ligand Dll1 induced Nfatc2 mRNA levels, confirming that activation of Notch signaling induces Nfatc2 expression. Transcript levels of Nfatc3 and Nfatc4 were not affected by NICD overexpression, indicating that in osteoblasts, Notch regulates NFAT signaling by suppressing Nfatc1 and inducing Nfatc2 expression. Although NFAT activity is controlled by cycles of phosphorylation and dephosphorylation, availability of NFATc constitutes an additional level of regulation in osteoclasts, and a sim-ilar mechanism may be operational in osteoblasts in the context of Notch induction (6,41).
To our knowledge, this is the first report demonstrating posttranscriptional control of Nfatc2 expression. Previously, we had shown that cortisol, an inducer of Notch1 and Notch2 expression in osteoblasts, stabilizes collagenase 3 transcripts by inducing the formation of cytosolic protein complexes with the 3Ј-untranslated region (UTR) of the collagenase 3 mRNA (42,43). Activation of Notch signaling might stabilize Nfatc2 mRNA levels by a similar mechanism. Post-transcriptional control of NFATc2 by Notch could involve the regulation of microRNAs, which are short RNA molecules that bind to target mRNAs and block protein translation and induce transcript degradation (44). It is important to note that NICD stabilized native Nfatc2 transcripts as well as transcripts derived from the caNFATc2 adenoviral vector transduced into osteoblasts, an effect that appears to be due to post-transcriptional mechanisms. 3 Because the cDNA transcribed from the adenoviral 3 S. Zanotti and E. Canalis, unpublished observations. FIGURE 5. NFATc2 opposes the association of CSL to the Hey2 promoter in osteoblasts. Binding of NFATc2 and CSL to the Hey2 promoter was tested by ChIP in Rosa Notch calvarial osteoblasts transduced with Ad-CMV-caNFATc2 tagged with HA (NFATc2) or control Ad-CMV-GFP (Control) and co-transduced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP). Cells were cultured to confluence, DNA cross-linked to associated proteins, and fragmented by sonication. DNA-protein complexes were obtained by immunoprecipitation (I.P.) with an HA (A) or a CSL (B) antibody or control IgG, and DNA was purified following reversal of the cross-linking reaction. Immunoprecipitated DNA and fragmented input DNA were amplified by qPCR with primers flanking Csl consensus sequences that regulate the activity of the Hey2 promoter. Data are expressed as enrichment of DNA by immunoprecipitation with the HA or CSL antibody relative to control IgG, corrected for input DNA levels. Values are means of three qPCR reactions. FIGURE 6. NICD and NFATc2 regulate osteoblast function. Rosa Notch calvarial osteoblasts were transduced with Ad-CMV-caNFATc2 (NFATc2, black bars) or control Ad-CMV-GFP (Control, white bars) and co-transduced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP), and cells were cultured for 7-14 days in conditions favoring osteoblast differentiation. In A, following 7 days of culture, total RNA was extracted and amplified by qRT-PCR. Data are expressed as ratio of Dlx5, Runx2, Alpl, Bsp, osteocalcin, Col1a1, and osteopontin copy number, corrected for Rpl38 expression, relative to corrected expression in control cells co-transduced with Ad-CMV-GFP. Values are means Ϯ S.E., n ϭ 4, and representative results from two independent experiments are shown. In B, cells were cultured for 7 days and extracted with Triton X-100 for determination of alkaline phosphatase activity, expressed as nanomoles of p-nitrophenol/min/g of total protein. Values are means from four independent experiments Ϯ S.E. In C, cells cultured for 14 days were fixed and stained with Alizarin Red, digital images were acquired, and the mineralized area was quantified with the measure function of ImageJ software. A representative culture from two independent experiments is shown. Values are means Ϯ S.E., n ϭ 6. *, significantly different between NFATc2 and control. ϩ, significantly different between CRE and GFP, p Ͻ 0.05. In A and B, selected data from control cells co-transduced with CRE or GFP were published previously (14).
construct lacks the native Nfatc2 3Ј-UTR, stabilization of the Nfatc2 mRNA is dependent, at least in part, on Nfatc2 sequences present in the coding region. However, this does not exclude the presence of additional regulatory elements in the 3Ј-UTR.
NFATc2 suppressed Hey1 and Hey2 expression, and inhibition of the 12ϫCSL-Luc reporter suggested that this effect is mediated by suppression of Notch canonical signaling. Results obtained by EMSA suggested that NFATc2 bound to Csl consensus sequences and that NFATc2 and CSL interact with similar DNA sequences. ChIP analysis of fragments of the Hey2 promoter containing Csl consensus sequences confirmed these results and suggested that NFATc2 reduced binding of CSL to the Hey2 promoter. Therefore, NFATc2 appears to suppress Notch canonical signaling by competing with CSL for the interaction with Csl consensus sequences that regulate the expression of Notch target genes. Although NFATc2 displaced CSL from the Hey2 promoter, either in the context of NICD overexpression or under basal conditions, association of NFATc2 with the Hey2 promoter was not observed under conditions of Notch stimulation. This apparent discrepancy may be due to the formation of Notch transcriptional complexes that modify chromatin organization of Notch target genes and prevent detection of NFATc2 associated to the Hey2 promoter. Suppression of Notch canonical signaling by NFATc2 is reminiscent of the inhibitory effects of NFATc1 on Notch transactivation, and due to the extensive conservation of protein sequences among the NFAT family members, it is conceivable that inhibition of Notch signaling is a common function of NFAT proteins (13).
In contrast to the results from EMSA, ChIP analysis indicated that in the context of Notch induction, the CSL antibody immunoprecipitated Hey2 promoter sequences to a greater extent than under basal conditions. Although these results are not consistent with the most widely accepted model of Notch canonical signaling, the data are in agreement with previous reports indicating that NICD increases CSL binding to DNA (45)(46)(47). As an alternative, association of NICD with CSL may have exposed epitopes not previously available for binding by the CSL antibody, resulting in enhanced immunoprecipitation of Hey2 promoter sequences in the presence of NICD.
We confirmed the inhibition of osteoblastic function by Notch, and we reported that constitutive activation of NFATc2 had a similar but less pronounced effect (48,49). However, the role played by NFAT transcription factors in osteoblasts is not clear (13). In mice, the osteoblast-specific conditional deletion of calcineurin, an activator of NFAT signaling, enhances osteoblastic function and trabecular bone volume, suggesting that NFAT signaling inhibits osteoblast differentiation and function (12,15,50). Similarly to the in vitro effects of NFATc1 in osteoblasts, we report that constitutive activation of NFATc2 suppresses osteoblast function, strengthening the notion that calcineurin/NFAT signaling suppresses osteoblast function (14,15). In contrast, global Nfatc2 null mice are osteopenic due to suppressed osteoblastic function (11,17). However, Nfatc2 null mice display hyperproliferation of B-and T-lymphocytes and increased systemic levels of interleukin 4 and other inflamma-tory molecules, which probably contribute to the osteopenic phenotype (18).
In conclusion, Notch induces Nfatc2 by post-transcriptional mechanisms, overexpression of NFATc2 suppresses Notch canonical signaling, and Notch and NFATc2 are inhibitors of osteoblast function.