NUCLEAR FACTOR OF ACTIVATED T CELLS 2 IS REQUIRED FOR OSTEOCLAST DIFFERENTIATION AND FUNCTION IN VITRO BUT NOT IN VIVO

Abstract

Nuclear factor of activated T cells (NFAT)c2 is important for the immune response and it compensates for NFATc1 for its effects on osteoclastogenesis, but its role in this process is not established. To study the function of NFATc2 in the skeleton, Nfatc2^loxP/loxP mice, where the Nfact2 exon2 is flanked by loxP sequences, were created and mated with mice expressing the Cre recombinase under the control of the Lyz2 promoter. Bone marrow-derived macrophage (BMM) from Lyz2^Cre/WT;Nfatc2^Δ/Δ mice cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) exhibited a decrease in the number and size of osteoclasts and a smaller sealing zone when compared to BMMs from Nfatc2^loxP/loxP littermate controls. Bone resorption was decreased in osteoclasts from Lyz2^Cre/WT;Nfatc2^Δ/Δ mice. This demonstrates that NFATc2 is necessary for optimal osteoclast maturation and function in vitro. Male and female Lyz2^Cre/WT;Nfatc2^Δ/Δ mice did not exhibit an obvious skeletal phenotype by microcomputed tomography (μCT) at either 1 or 4 months of age when compared to Nfatc2^loxP/loxP sex-matched littermates. Bone histomorphometry confirmed the μCT results, and conditional 4 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ mice did not exhibit changes in parameters of bone histomorphometry. In conclusion, NFATc2 is necessary for optimal osteoclastogenesis in vitro, but its downregulation in the myeloid lineage has no consequences in skeletal remodeling in vivo.

Nuclear factor of activated T cells (NFAT) are a family of five transcription factors (NFATc1 to c4 and NFAT5) that regulate the growth and differentiation of multiple cell lineages [Crabtree and Olson, 2002; Sitara and Aliprantis, 2010]. Initially studied for their role in the early immune response, NFATs were subsequently found to regulate cellular events in non-immune cells. Activation of NFATs requires dephosphorylation of specific serine residues in their regulatory domain by the phosphatase calcineurin. This results in the translocation of NFAT from the cytoplasm, where they reside in a phosphorylated state, to the nucleus where they regulate the transcription of target genes [Chow et al., 2008; Hogan et al., 2003; Okamura et al., 2000].

NFATc1 through c4 are expressed by skeletal cells, and NFATc1 plays an undisputable role in osteoclast differentiation [Aliprantis et al., 2008; Ikeda et al., 2006; Ikeda et al., 2004; Ishida et al., 2002; Matsuo et al., 2004; Takayanagi et al., 2002]. The functions of NFATc1 and c2 are not necessarily redundant, and the role of NFATc2 in osteoclast differentiation and function is less certain [Ikeda et al., 2006; Ranger et al., 1998]. This is in part because of limitations in the genetic manipulations used in former studies to explore the role of NFATs in skeletal cells [Hodge et al., 1996; Koga et al., 2005; Sun et al., 2005; Winslow et al., 2006; Yeo et al., 2007]. Whereas transgenic overexpression of constitutive active (ca)NFATc2 under the control of the Acp5 promoter results in increased osteoclastogenesis and bone resorption, the inactivation of Nfatc2 has led to conflicting results [Asagiri et al., 2005; Bauer et al., 2011; Ikeda et al., 2006; Koga et al., 2005]. Studies interrogating the function of NFATc2 in the skeleton have suffered from the use of a systemic gene inactivation strategy. Mice harboring the global inactivation of Nfatc2 were reported not to have a skeletal phenotype, to exhibit osteopenia due to reduced bone formation or osteopetrosis due to reduced osteoclast number [Asagiri et al., 2005; Bauer et al., 2011; Koga et al., 2005]. Nfatc2 null mutants display hyperproliferation of B, T and other immune cells and dysregulated expression of multiple cytokines, which may confound the interpretation of the skeletal phenotype [Hodge et al., 1996; Monticelli and Rao, 2002; Ranger et al., 2000]. Moreover, the Nfatc2 targeting vector used contains a Neo cassette, and retention of selectable marker cassettes, like phosphoglycerate kinase (PGK)-neo, can cause unexpected phenotypes by disrupting the expression of neighboring genes [Hodge et al., 1996; Olson et al., 1996; Pham et al., 1996]. Mouse models harboring either the activation or the downregulation of calcineurin also have generated conflicting skeletal phenotypes [Sun et al., 2005; Yeo et al., 2007], possibly because this approach does not discriminate between the independent effects of each NFAT isoform or other calcineurin-dependent signals.

The intent of the present study was to define the function of NFATc2 in osteoclast differentiation and function in vitro and in vivo. For this purpose, we created Nfatc2^loxP/loxP conditional mice. Nfatc2 was inactivated by Cre recombination directed by the Lyz2 gene expressed in cells of the myeloid lineage including osteoclast precursors [Clausen et al., 1999; Takeda et al., 1999]. The skeletal phenotype of Nfatc2 conditional null mice was determined by microcomputed tomography (μCT) and by bone histomorphometry, and by the study of osteoclast differentiation and resorption activity in vitro.

MATERIALS AND METHODS

Generation of Nfatc2 Conditional Mice

To create a conditional allele of Nfatc2, 9.7 kilobase pair (kb) of Nfatc2 sequence were selected from a bacterial artificial chromosome library of C57BL/6J mouse genomic DNA (id RP24-223B7) and retrieved into a PL253 vector. The vector contains an MC1 driven herpes simplex virus-thymidine kinase (MC1-HSV-TK) cassette for negative selection of embryonic stem (ES) cells [Liu et al., 2003; Mansour et al., 1988]. A 5′ loxP site was introduced approximately 0.4 kb 5′ of exon 2, followed by the insertion of a PGK promoter-driven neomycin selection cassette flanked by flippase recognition target (Frt) sequences and a 3′ loxP site (Frt-PGKneo-Frt-LoxP) approximately 0.4 kb 3′ of exon 2 (Figure 1) [Buchholz et al., 1996] (Figure 1). The targeting vector containing 4.3 kb of 5′ homology arm and 3.5 kb of 3′ homology arm was linearized and electroporated into ES cells derived from F1 (129Svj/C57BL/6J) embryos. G418 and gancyclovir resistant colonies were isolated and screened by long range nested polymerase chain reaction (PCR) [Lay et al., 1998]. Targeted ES clones were used for aggregations to generate chimeric mice at the Center for Mouse Genome Modification at UConn Health [Eakin and Hadjantonakis, 2006; Gertsenstein et al., 2010; Pettitt et al., 2009; Pluck and Klasen, 2009]. Chimeras that were transmitters of ES-derived sperm were bred with mice expressing the Flp recombinase under the control of the Rosa promoter (Gt(ROSA)26Sor^tm2(FLP+)Sor, Jackson Laboratory, Bar Harbor, ME, Stock 007844) for the removal of the PGK-neo selection cassette flanked by Frt [Buchholz et al., 1998; Canalis et al., 2010a; Canalis et al., 2010b; Farley et al., 2000; Mallo, 2006; Raymond and Soriano, 2007]. The excision of the cassette was confirmed by PCR (Table 1), and the Flp recombinase transgene was segregated by mating with C57BL/6J wild type mice. Mice with the Nfatc2 allele flanked by loxP sites were backcrossed into a C57BL/6J background for 7 generations. Cre recombination excises exon 2, encoding for the regulatory domain of NFATc2, and leads to a frame shift and the creation of a STOP codon in exon 3 so that only 42 amino acids of the mature protein are expressed, lacking both the regulatory and the DNA binding domain (aa 400–680) [Okamura et al., 2000].

Figure 1. Engineering of the Nfatc2 conditional allele showing the wild type and Nfatc2 targeted alleles.

In the conditional allele exon 2 is flanked by loxP sites upstream a PGK-neo selection cassette flanked by Frt sites. The Nfatc2 targeted allele is shown prior to and after Flp and Cre recombination for the removal of the neo cassette and excision of exon 2, respectively.

Table 1.

Primers used for genotyping by PCR.

Allele	Strand	Sequence	Amplicon Size (bp)
Lyz2Cre	Forward1 Forward2 Reverse	5′-TTACAGTCGGCCAGGCTGAC-3′	Lyz2WT = 350 Lyz2Cre = 700
		5′-CCCAGAAATGCCAGATTACG-3′ 5′-CTTGGGCTGCCAGAATTTCTC-3′
Nfatc2loxP	Forward Reverse	5′-GGTGTCTCCCTCAAATGTTCA-3′ 5′-GCGAATATGAGGACCCAAGT-3′	WT = 160 Nfatc2loxP= 250
Frt and LoxP recombination	Strand	Sequence	Amplicon Size (bp)
Frt recombination of Nfatc2 targeted allele	Forward Reverse	5′-AGTATGTAACCTCTGCTTCC-3′ 5′-AGCACAATGCCCATGTTCAC-3′	WT = 250 Not recombined = no band Recombined = 351 (Nfatc2loxP)
LoxP recombination of Nfatc2loxP	Reverse Forward	5′-GGTGTCTCCCTCAAATGTTCA-3′ 5′-AGCACAATGCCCATGTTCAC-3′	Not recombined = 2342 (Nfatc2loxP) Recombined = 358 (Nfatc2Δ)

Deletion of Nfatc2 in the Myeloid Lineage

C57BL/6J mice where the Cre coding sequence was inserted into the endogenous Lyz2 locus (Lyz2^Cre; Jackson Laboratory, Stock 004781) were used to express the Cre recombinase in cells of the myeloid lineage [Clausen et al., 1999; Takeda et al., 1999]. To induce the deletion of Nfatc2 in osteoclast precursors, homozygous Nfatc2^loxP/loxP mice heterozygous for the Lyz2^Cre allele (Lyz2^Cre/WT;Nfatc2^loxP/loxP) were bred with Nfatc2^loxP/loxP mice to create Lyz2^Cre/WT;Nfatc2^Δ/Δ mice and Nfatc2^loxP/loxP control littermates.

Culture of Bone Marrow-derived Macrophages (BMMs) and Osteoclast Formation

To obtain BMMs, the marrow from Lyz2^Cre/WT;Nfatc2^Δ/Δ and control Nfatc2^loxP/loxP littermates was removed by flushing with a 26 gauge needle and erythrocytes were lysed in 150 mM NH₄Cl, 10 mM KHCO₃ and 0.1 mM EDTA (pH 7.4) as described previously [Canalis et al., 2017]. Cells were centrifuged and the sediment suspended in α-minimum essential medium (α-MEM) in the presence of 10% fetal bovine serum (FBS; both from Thermo Fisher Scientific, Waltham, MA) and recombinant human macrophage-colony stimulating factor (M-CSF) at 30 ng/ml. M-CSF cDNA and expression vector were obtained from D. Fremont (St. Louis, MO) and M-CSF was purified as previously reported [Lee et al., 2006]. Cells were seeded on plastic petri dishes at a density of 300,000 cells/cm² and cultured for 3 to 4 days. Deletion of the Nfatc2 allele was documented by PCR of genomic DNA using primers specific for the Nfatc2^Δ/Δ allele (Table 1). For osteoclast formation, cells were collected following treatment with 0.25% trypsin/EDTA for 5 min and seeded on tissue culture plates at a density of 47,000 cells/cm² in α-MEM with 10% FBS, M-CSF at 30 ng/ml and recombinant murine receptor activator of NF-κB ligand (RANKL) at 10 ng/ml. Tnfsf11 encoding RANKL cDNA and expression vector were obtained from M. Glogauer (Toronto, Canada), and GST-tagged RANKL was expressed and purified as described [Wang et al., 2008]. Cultures were carried out until formation of multinucleated tartrate resistant acid phosphatase (TRAP)-positive cells. TRAP enzyme histochemistry was conducted using a commercial kit (Sigma-Aldrich, St. Louis, MO), in accordance with manufacturer’s instructions. TRAP-positive cells containing more than 3 nuclei were considered osteoclasts.

For Actin structure staining and bone resorption assay of osteoclasts in vitro, BMMs were seeded at a density of 4.7 × 10⁴ cells/cm² on bovine cortical bone slices and cultured in α-MEM with 10% FBS, M-CSF at 30 ng/ml and RANKL at 10 ng/ml. To visualize the sealing zone of osteoclasts on the bone slices, cells were fixed with 4% paraformaldehyde for 10 min and were permeabilized with 0.3% triton X-100 for 5 min. To block non-specific background staining, cells on bone discs were incubated with 2% BSA for 1 hour. Cells were stained with Alexa Fluor^™ 594 Phalloidin (Thermo Fisher Scientific) at a 1:40 dilution for 20 min. The sealing zones were viewed on a Leica fluorescence microscope (Model DMI6000B), and collected images were processed using the Leica Application Suite × 1.5.1.1387 (Leica Microsystem, Buffalo Grove, IL). After visualizing the sealing zone, cells were stained for TRAP to assess their morphology. To visualize bone resorption pits, the bone slices were sonicated to remove osteoclasts and stained with 1% toluidine blue in 1% sodium borate. To evaluate the ability of osteoclasts to resorb bone, the total resorption area/total bone area was measured on images acquired with an Olympus DP72 camera using cellSens Dimension software v1.6 (Olympus Corporation, Center Valley, PA). The total resorption area/total bone area was corrected for the total number of TRAP-positive multinucleated cells [Canalis et al., 2017].

Microcomputed Tomography

Femoral microarchitecture was determined using a microcomputed tomography instrument (Scanco μCT 40; Scanco Medical AG, Bassersdorf, Switzerland), which was calibrated periodically using a phantom provided by the manufacturer [Bouxsein et al., 2010; Glatt et al., 2007]. Femurs were scanned in 70% ethanol at high resolution, energy level of 55 kVp, intensity of 145 μA, and integration time of 200 ms. A total of 100 slices at midshaft and 160 slices at the distal metaphysis were acquired at an isotropic voxel size of 216 μm³ and a slice thickness of 6 μm, and chosen for analysis. Trabecular bone volume fraction (bone volume/total volume) and microarchitecture were evaluated starting approximately 1.0 mm proximal from the femoral condyles. Contours were manually drawn every 10 slices, a few voxels away from the endocortical boundary, to define the region of interest for analysis, whereas the remaining slice contours were iterated automatically. Total volume, bone volume, bone volume fraction, trabecular thickness, trabecular number, connectivity density, structure model index (SMI) and material density were measured in trabecular regions using a Gaussian filter (σ = 0.8) and user defined thresholds [Bouxsein et al., 2010; Glatt et al., 2007]. For analysis of cortical bone, contours were iterated across 100 slices along the cortical shell of the femoral midshaft, excluding the marrow cavity. Analysis of bone volume/total volume, porosity, cortical thickness, total cross sectional and cortical bone area, periosteal and endosteal perimeter and material density were conducted using a Gaussian filter (σ = 0.8, support = 1) with operator-defined thresholds.

Bone Histomorphometry

Bone histomorphometry was carried out in 1 and 4 month old mice injected with calcein 20 mg/kg and demeclocycline 50 mg/kg at a 2 or 7 day interval, respectively, and sacrificed 2 days after demeclocycline administration. Femurs were dissected, fixed in 70% ethanol and embedded in methyl methacrylate. For cancellous bone analysis, bones were sectioned at a thickness of 5 μm along the sagittal plane on a Microm microtome (Richards-Allan Scientific, Kalamazoo, MI), and stained with 0.1% toluidine blue. Static and dynamic parameters of bone morphometry were measured in a defined area between 0.35 mm and 2.16 mm from the growth plate at a magnification of 10× using an OsteoMeasure morphometry system (Osteometrics, Atlanta, GA). Stained sections were used to draw bone tissue and to measure trabecular separation, number and thickness, osteoid and eroded surface, as well as to count osteoblast and osteoclast surface and number. Mineralizing surface per bone surface and mineral apposition rate were measured on unstained sections visualized under UV light and a triple diamidino-2-phenylindole/fluorescein/Texas red set long pass filter, and bone formation rate was calculated [Dempster et al., 2013].

RNA Integrity and Quantitative Reverse Transcription-PCR (qRT-PCR)

Total RNA was extracted from BMMs with the RNeasy kit (Qiagen, Valencia, CA) and from homogenized bones with the micro RNeasy kit (Qiagen), in accordance with manufacturer’s instructions. The integrity of the RNA extracted from bones was assessed by microfluidic electrophoresis on an Experion system (BioRad, Hercules, CA), and RNA with a quality indicator number equal to or higher than 7.0 was used for subsequent analysis. Equal amounts of RNA were reverse-transcribed using the iScript RT-PCR kit (BioRad) and amplified in the presence of specific primers (all primers from Integrated DNA Technologies, IDT, Coralville, IA; Table 2) with the SsoAdvanced^™ Universal SYBR Green Supermix (BioRad) at 60°C for 40 cycles. Transcript copy number was estimated by comparison with a serial dilution of cDNA for Nfatc1 (Addgene plasmid 11793), Nfatc2 (Addgene plasmid 11791, both created by A. Rao, La Jolla, CA), Acp5, Ctsk and Calcr (Thermo Fisher Scientific). Tnfrsf11a copy number was estimated by comparison to a serial dilution of a 152 base pair (bp) synthetic DNA template (IDT) cloned into pcDNA3.1(−) (Thermo Fisher Scientific) by isothermal single reaction assembly using commercially available reagents (New England BioLabs, Ipswich, MA).

Table 2.

Primers used for qRT-PCR determinations. GenBank accession numbers identify transcript recognized by primer pairs.

Gene	Strand	Sequence	GenBank Accession Number
Acp5	Forward Reverse	5′-GACAAGAGGTTCCAGGAGAC-3′ 5′-TTCCAGCCAGCACATACC-3′	NM_001102404; NM_001102405; NM_007388
Calcr	Forward Reverse	5′-TCTGAGAAACTGCAAAATGCGTAC-3′ 5′-AGCAACCAAAGCAGCAATCG-3′	NM_007588
Ctsk	Forward Reverse	5′-AGATATTGGTGGCTTTGGAA-3′ 5′-AACGAGAGGAGAAATGAAACA-3′	NM_007802
Nfatc1	Forward Reverse	5′-GCGCAAGTACAGTCTCAATGGCC-3′ 5′-GGATGGTGTGGGTGAGTGGT-3′	NM_198429; NM_001164110; NM_001164111; NM_001164112; NM_00116641091; NM_016791
Nfatc2	Forward Reverse	5′-AGAACAACATGAGAGCCACCATC-3′ 5′-AGCTCGATGTCAGCGTTTCG-3′	NM_010899
Rpl38	Forward Reverse	5′-AGAACAAGGATAATGTGAAGTTCAAGGTTC-3′ 5′-CTGCTTCAGCTTCTCTGCCTTT-3′	NM_001048057; NM_001048058; NM_023372
Tnfrsf11a	Forward Reverse	5′-GATGCCTCACAGTTAAGAAGAAGA-3′ 5′-GAGCTGGAGTTACATGGAGTTG-3′	NM_009399.3

Amplification reactions were conducted in CFX96 qRT-PCR detection systems (BioRad), and fluorescence was monitored during every PCR cycle at the annealing step. Data are expressed as copy number corrected for Rpl38 expression estimated by comparison with a serial dilution of Rpl38 (from American Type Culture Collection, ATCC, Manassas, VA) [Kouadjo et al., 2007].

Immunoblotting

Osteoclasts from control or Lyz2^Cre/WT;Nfatc2^Δ/Δ mice were extracted in buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 1 mM EDTA, 0.5% Triton X-100, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenyl methyl sulfonyl fluoride and a protease inhibitor cocktail (all from Sigma Aldrich, St. Louis, MO). Quantified total cell lysates (50 μg of total protein) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) in 8% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA). The blots were probed with anti-NFATc2 (4389) and anti-β-Actin (3700) antibodies, both from Cell Signaling Technology (Danvers, MA), and anti-NFATc1 (556602; BD Bioscience, San Jose, CA) antibodies. The 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 (BioRad). The bands were detected by ChemiDoc^™ XSR+ molecular imager (BioRad) with Image Lab^™ software (version 5.2.1) [Zanotti et al., 2013].

Statistics

Data are expressed as means ± S.D. Statistical differences were determined by Student’s t test or two-way analysis of variance with Holm-Šídák post-hoc analysis for pairwise or multiple comparisons, respectively.

RESULTS

Nfatc2 mRNA and Protein Expression during Osteoclastogenesis

To verify Nfatc2 gene expression during osteoclast differentiation, we checked mRNA and protein levels of Nfatc2 in RANKL-stimulated BMMs undergoing differentiation into multinucleated osteoclasts. Cellular extracts were collected prior to, and after 2 and 4 days following RANKL stimulation for the determination of total RNA and protein. Nfatc2 mRNA levels were decreased, whereas NFATc2 protein levels were increased by RANKL during osteoclast differentiation (Figure 2).

Figure 2. NFATc2 expression during osteoclast differentiation.

BMMs were cultured for 4 days in the presence of M-CSF at 30ng/ml and RANKL at 10ng/ml. The cells were collected at the indicated times for extraction of total RNA and proteins. (A) Nfatc2 mRNA levels were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 4 biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different compared to day 0, p < 0.05. (B) 50 μg of total proteins were separated by SDS-PAGE and NFATc2 protein levels were detected by using anti-NFATc2 antibodies. β-Actin levels were detected using anti-β-Actin antibodies and served as a loading control in the same blot.

Conditional Inactivation of Nfatc2 in Cells of the Myeloid Lineage including Osteoclast Precursors

To establish the inactivation of Nfatc2 in cells of the myeloid lineage, the Nfatc2^loxP allele (Figure 1) was introduced into Lyz2^Cre/WT heterozygous mice. Subsequently, Lyz2^Cre/WT;Nfatc2^loxP/loxP mice were crossed with Nfatc2^loxP/loxP mice for the creation of Lyz2^Cre/WT;Nfatc2^Δ/Δ experimental mice and Nfatc2^loxP/loxP littermate controls. Lyz2^Cre/WT;Nfatc2^Δ/Δ mice appeared healthy and their weight was not different from that of Nfatc2^loxP/loxP littermate controls (Figure 3). Lyz2^Cre-mediated recombination was demonstrated in genomic DNA from Lyz2^Cre/WT;Nfatc2^Δ/Δ BMM cultures with the consequent decrease in Nfatc2 mRNA and NFATc2 protein levels. Nfatc2 transcripts were decreased by 80% in Lyz2^Cre/WT;Nfatc2^Δ/Δ BMM cultures when compared to control cultures, and NFATc2 protein levels were virtually undetectable in Lyz2^Cre/WT;Nfatc2^Δ/Δ cultures, documenting downregulation of Nfatc2 in osteoclast precursor BMMs (Figure 3).

Figure 3. Deletion of the Nfatc2 allele in Lyz2-expressing cells.

Weight, documentation of DNA recombination of the Nfatc2^loxP allele by Cre and Nfatc2 deletion in BMMs. (A) Body weight of 1 and 4 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ and sex-matched control Nfatc2^loxP/loxP littermates. Values are means ± SD; n = 10 control and n = 7 Nfatc2^Δ/Δ for 1 month old male mice; n = 8 control and n = 9 Nfatc2^Δ/Δ for 4 month old male mice; n = 6 control and n = 6 Nfatc2^Δ/Δ for 1month old female mice; n = 6 control and n = 9 Nfatc2^Δ/Δ for 4 month old female mice. (B) Genomic DNA from BMMs obtained from 1 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ mice and respective controls was isolated. Nfatc2 deletion by DNA recombination was demonstrated by gel electrophoresis of PCR amplification products obtained with primers for the Nfatc2^loxP and Nfatc2^Δ alleles. The arrow head indicates the position of the 2.3 kilobase pair (kb) amplicon verifying the Nfatc2^loxP allele, and the arrow indicates a 0.36 kb amplicon verifying the Nfatc2^Δ allele. n = 5 control and n = 3 Nfatc2^Δ/Δ biological replicates. (C) Nfatc2 transcript levels were measured by qRT-PCR in total RNA from BMMs of Lyz2^Cre/WT;Nfatc2^Δ/Δ and respective controls. Transcript levels are reported as copy number corrected for Rpl38. Values are means ± SD; n = 4 biological replicates for control and Nfatc2^Δ/Δ. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05. (D) 50 μg of total proteins of BMMs from 1 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ and respective controls were separated by SDS-PAGE and NFATc2 protein levels were detected by using anti-NFATc2 antibodies. β-Actin levels served as a loading control in the same blot.

NFATc2 Is Required for the Differentiation, Maturation and Resorption of Osteoclasts in vitro

To determine the direct effects of NFATc2 on osteoclast differentiation and function, BMMs derived from Lyz2^Cre/WT;Nfatc2^Δ/Δ and control Nfatc2^loxP/loxP littermates were cultured in the presence of M-CSF and RANKL. Cultured BMMs from Lyz2^Cre/WT;Nfatc2^Δ/Δ mice exhibited a 30% decrease in osteoclast number in comparison to cells from littermate controls (Figure 4). In addition, TRAP and Phalloidin staining of osteoclasts from Lyz2^Cre/WT;Nfatc2^Δ/Δ mice cultured on bone discs revealed smaller cells with smaller sealing zones than littermate controls. Accordingly, Lyz2^Cre/WT;Nfatc2^Δ/Δ osteoclasts exhibited a 50% decrease in total bone resorption area. When corrected for osteoclast number, bone resorption area was 30% lower in Lyz2^Cre/WT;Nfatc2^Δ/Δ than controls, indicating decreased osteoclast resorptive activity. These findings demonstrate that NFATc2 is necessary for optimal osteoclast maturation and function in vitro (Figure 4).

Figure 4. Number, size and resorption activity are decreased in Lyz2^Cre;Nfatc2^Δ/Δ osteoclasts.

BMMs derived from 1 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ mice and Nfatc2^loxP/loxP control littermates were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml in cell culture-coated plates (A and B) or bone discs (C and D). (A) After 4 days, cells were stained with TRAP and representative images of TRAP-stained multinucleated cells were shown (4× magnification). (B) TRAP-positive cells with more than 3 nuclei were counted as osteoclasts and values are means ± SD; n = 4 biological replicates for control and Nfatc2^Δ/Δ. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05. (C) Cultured osteoclasts on bone discs were stained by Alexa Fluor 594 Phalloidin (middle, 10X magnification) and TRAP (upper, 4× magnification). To stain for resorption pits, cells were removed by sonication and bone discs were stained by Toluidine blue (bottom, 10× magnification). n = 4 biological replicates for control and Nfatc2^Δ/Δ and representative images for TRAP, Phalloidin and Toluidine blue are shown. (D) Data are expressed as total resorption pit area (%, upper) and total resorption pit area corrected for number of osteoclasts (%, lower). Values are means ± SD; n = 4 biological replicates for control and Nfatc2^Δ/Δ. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05.

Nfatc2 was downregulated specifically since neither Nfatc1 mRNA nor NFATc1 protein levels were affected in Lyz2^Cre/WT;Nfatc2^Δ/Δ osteoclasts compared to control (Figure 5). Tnfrsf11a encoding for RANK, the specific RANKL receptor was not affected in either BMMs or osteoclasts from Lyz2^Cre/WT;Nfatc2^Δ/Δ. Expression of gene markers representative of osteoclasts, such as Acp5 encoding for TRAP, Ctsk, encoding for Cathepsin K, and Calcr, encoding for the Calcitonin receptor, were all significantly decreased in Lyz2^Cre/WT;Nfatc2^Δ/Δ osteoclasts compared to cultures from control littermates (Figure 5).

Figure 5. Acp5, Ctsk and Calcr mRNA levels are decreased in Lyz2^Cre;Nfatc2^Δ/Δ Osteoclasts.

BMMs derived from 1 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ mice and Nfatc2^loxP/loxP control littermates were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml. The cells were collected at the indicated times for extraction of total RNA and proteins. (A) Nfatc2 and Nfatc1 mRNA levels were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 5 control and n = 3 Nfatc2^Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05. (B) 50 μg of total protein were separated by SDS-PAGE and NFATc1 and NFATc2 levels were detected by using immunoblot anti-NFATc1 and anti-NFATc2 antibodies, respectively. β-Actin levels served as a loading control in the same blot. (C) Tnfrs11a, Acp5, Ctsk and Calcr mRNA levels in total RNA were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38. Values are means ± SD; n = 5 control and n = 3 Nfatc2^Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05.

Conditional Inactivation of Nfatc2 in Lyz2-expressing Cells Does Not Cause a Skeletal Phenotype In Vivo

In preliminary experiments, we documented that 1 and 4 month old Lyz2^Cre and 1 and 4 month old Nfatc2^loxP/loxP mice do not have a skeletal phenotype as determined by μCT of distal femurs, when compared to wild type controls (data not shown). Femoral microarchitecture of 1 and 4 month old male and female Lyz2^Cre/WT;Nfatc2^Δ/Δ mice revealed no differences compared to Nfatc2^loxP/loxP sex-matched littermate controls (Tables 3 and 4). Bone histomorphometry of 4 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ male mice confirmed the absence of a skeletal phenotype when compared to Nfatc2^loxP/loxP littermate sex-matched controls (Table 5).

Table 3.

Femoral microarchitecture assessed by μCT of 1 and 4 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ male mice (Nfatc2^Δ/Δ) and sex-matched littermate Nfatc2^loxP/loxP controls.

	1 Month		4 Months
	Control n = 10	Nfatc2Δ/Δ n = 7	Control n = 8	Nfatc2Δ/Δ n = 9
Distal Femur Trabecular Bone
Bone Volume/Total Volume (%)	7.2 ± 2.5	8.6 ± 2.2	7.8 ± 3.0	6.8 ± 3.2
Trabecular Separation (μm)	226 ± 40	216 ± 21	257 ± 13	249 ± 23
Trabecular Number (1/mm)	4.6 ± 0.7	4.7 ± 0.5	3.9 ± 0.2	4.0 ± 0.4
Trabecular Thickness (μm)	28 ± 3	29 ± 2	38 ± 9	34 ± 6
Connectivity Density (1/mm3)	330 ± 190	426 ± 162	140 ± 45	135 ± 56
Structure Model Index	2.6 ± 0.3	2.4 ± 0.2	2.1 ± 0.4	2.3 ± 0.5
Density of Material (mg HA/cm3)	946 ± 27	942 ± 28	1012 ± 14	1004 ± 20
Femoral Midshaft Cortical Bone
Bone Volume/Total Volume (%)	82.6 ± 4.2	82.6 ± 2.5	89.6 ± 0.6	89.4 ± 0.5
Porosity (%)	17.4 ± 4.2	17.4 ± 2.5	10.4 ± 0.6	10.6 ± 0.5
Cortical Thickness (μm)	95 ± 17	94 ± 7	171 ± 8	170 ± 9
Total Area (mm2)	1.6 ± 0.1	1.6 ± 0.1	2.3 ± 0.2	2.1 ± 0.2
Bone Area (mm2)	0.5 ± 0.1	0.5 ± 0.1	1.0 ± 0.1	0.9 ± 0.1
Periosteal Perimeter (mm)	4.4 ± 0.2	4.4 ± 0.1	5.3 ± 0.3	5.2 ± 0.2
Endocortical Perimeter (mm)	3.7 ± 0.1	0.6 ± 0.1	4.0 ± 0.2	3.9 ± 0.2
Density of Material (mg HA/cm3)	1049 ± 19	1051 ± 14	1235 ± 25	1224 ± 22

μCT was performed on distal femurs for trabecular bone and midshaft for cortical bone. Values are means ± SD.

Table 4.

Femoral microarchitecture assessed by μCT of 1 and 4 month old Lyz2^Cre/WT;Nfatc2^δ/δ female mice (Nfatc2^δ/δ) and sex-matched littermate Nfatc2^loxP/loxP controls.

	1 Month		4 Months
	Control n = 6	Nfatc2Δ/Δ n = 6	Control n = 6	Nfatc2Δ/Δ n = 9
Distal Femur Trabecular Bone
Bone Volume/Total Volume (%)	3.8 ± 0.5	3.8 ± 0.4	3.9 ± 1.2	3.1 ± 0.6
Trabecular Separation (μm)	285 ± 22	276 ± 20	314 ± 18	326 ± 18
Trabecular Number (1/mm)	3.6 ± 0.2	3.7 ± 0.2	3.2 ± 0.2	3.1 ± 0.1
Trabecular Thickness (μm)	26 ± 1	25 ± 1	37 ± 3	35 ± 3
Connectivity Density (1/mm3)	79 ± 36	89 ± 27	58 ± 27	52 ± 18
Structure Model Index	2.8 ± 0.2	2.8 ± 0.1	2.9 ± 0.3	3.0 ± 0.2
Density of Material (mg HA/cm3)	978 ± 10	973 ± 16	1014 ± 8	1009 ± 13
Femoral Midshaft Cortical Bone
Bone Volume/Total Volume (%)	82.0 ± 3.6	81.5 ± 3.8	88.1 ± 0.6	87.6 ± 0.6
Porosity (%)	18.0 ± 3.6	18.5 ± 3.8	11.9 ± 0.6	12.4 ± 0.6
Cortical Thickness (μm)	91 ± 11	86 ± 13	171 ± 6	166 ± 4
Total Area (mm2)	1.6 ± 0.1	1.5 ± 0.2	1.8 ± 0.1	1.7 ± 0.1
Bone Area (mm2)	0.5 ± 0.1	0.5 ± 0.1	0.9 ± 0.1	0.8 ± 0.1
Periosteal Perimeter (mm)	4.4 ± 0.2	4.4 ± 0.2	4.7 ± 0.1	4.7 ± 0.1
Endocortical Perimeter (mm)	3.7 ± 0.2	3.6 ± 0.2	3.4 ± 0.1	3.4 ± 0.1
Density of Material (mg HA/cm3)	1060 ± 17	1058 ± 24	1269 ± 13	1262 ± 9

μCT was performed on distal femurs for trabecular bone and midshaft for cortical bone. Values are means ± SD.

Table 5.

Cancellous bone histomorphometry of 4 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ male mice (Nfatc2^Δ/Δ) and sex-matched littermate Nfatc2^loxP/loxP controls.

	Control n = 6	Nfatc2Δ/Δ n = 6
Distal Femur Trabecular Bone
Bone Volume/Tissue Volume (%)	10.7 ± 3.2	9.7 ± 3.8
Trabecular Number (1/mm)	2.5 ± 0.3	2.4 ± 0.6
Trabecular Thickness (μm)	42.8 ± 11.7	40.5 ± 10.5
Osteoblasts/Bone Perimeter (1/mm)	7.2 ± 2.5	6.9 ± 3.5
Osteoid Surface/Bone Surface (%)	0.4 ± 0.3	0.6 ± 0.6
Osteoclasts/Bone Perimeter (1/mm)	2.1 ± 0.6	2.2 ± 0.6
Eroded Surface/Bone Surface (%)	1.6 ± 0.6	1.1 ± 0.6
Mineral Apposition Rate (μm/day)	1.0 ± 0.2	0.9 ± 0.1
Mineralizing Surface/Bone Surface (%)	1.7 ± 0.6	2.9 ± 1.8
Bone Formation Rate (μm3/μm2/day)	0.02 ± 0.01	0.03 ± 0.01

Bone histomorphometry was performed on saggital sections of distal femurs sagittal sections. Values are means ± SD.

DISCUSSION

In this study, the direct effects of NFATc2 on osteoclast differentiation and function were explored by the conditional inactivation of Nfatc2 in cells of the myeloid lineage. The present data revealed that Nfatc2 mRNA levels are decreased under the RANKL stimulation, but NFATc2 protein level is moderately increased during osteoclastogenesis, suggesting a possible post-translational regulation of NFATc2 expression during osteoclast differentiation. Previous studies reported that NFATc2 is recruited to the Nfatc1 promoter to promote amplification of NFATc1 by cooperating with NF-κB [Asagiri et al., 2005]. In the present study, Nfatc1 mRNA and NFATc1 protein levels were not affected by the downregulation of NFATc2 possibly because NFATc1 is sufficient for its own autoamplification suggesting that NFATc2 is dispensable for the induction of NFATc1. Although NFATc1 levels were not different between Nfatc2^Δ/Δ and control osteoclasts, Nfatc2^Δ/Δ osteoclasts exhibited a small sealing zone and decreased mRNA levels of osteoclast differentiation markers, such as Acp5, Ctsk and Calcr. Accordingly, bone resorptive activity of Nfatc2^Δ/Δ osteoclasts was reduced suggesting a direct role of NFATc2 in osteoclast maturation and bone resorption. However, this effect was not translated in vivo and inactivation of Nfatc2 in Lyz2-expressing cells did not cause an obvious skeletal phenotype. This would indicate that NFATc2 is dispensable for the differentiation or function of cells of the osteoclast lineage in vivo. These findings are consistent with previous in vivo work demonstrating that the global inactivation of Nfatc2 does not alter osteoclast number or bone resorption in vivo [Asagiri et al., 2005]. It is likely that the absence of a phenotype following the deletion of Nfatc2 in vivo is because of genetic compensation by Nfatc1 which is considered essential for osteoclastogenesis [Asagiri et al., 2005].

Previous work has demonstrated that the delivery of caNFATc2 to cells of the osteoclast lineage can induce osteoclastogenesis and rescue the Nfatc1 null phenotype confirming that in the absence of NFATc1, NFATc2 has the capacity to induce osteoclast differentiation [Asagiri et al., 2005]. A similar role of NFATc2 in osteoclastogenesis and bone resorption was observed following the transgenic expression of caNFATc2 under the control of the Acp5 promoter. Acp5-Nfatc2 transgenic mice exhibit osteopenia due to increased osteoclast number and bone resorption [Ikeda et al., 2006]. The mechanism responsible for the increase in osteoclast maturation seemed to involve the induction of c-Src. It is possible that caNFATc2 targets signals similar to those targeted by NFATc1 to induce osteoclastogenesis, but its downregulation in the presence of NFATc1 results in a modest reduction in osteoclast differentiation.

Although NFATc1 and NFATc2 have important interactions with Notch signaling in osteoblasts, there is no evidence at present that these occur in the myeloid lineage [Zanotti et al., 2013]. Notch induces Nfatc2 expression in osteoblasts by post-transcriptional mechanisms, and NFATc2 competes for binding to DNA with the Notch transcriptional complex resulting in the downregulation of Notch signaling in what seems to be a negative feed-back regulatory loop. However, we have not detected consistent increases in Nfatc2 mRNA expression in the myeloid lineage in models of Notch2 gain-of-function (E. Canalis et al., unpublished observations).

Similar to the results observed with the delivery of caNFATc2 to cells of the myeloid lineage, the transgenic delivery of caNFATc2 under the control of the 3.6 kb Col1a1 promoter to cells of the osteoblast lineage results in a biological response. Col1a1-Nfatc2 transgenic mice exhibit osteopenia secondary to decreased bone formation [Zanotti and Canalis, 2015]. NFATc1 and NFATc2 also play an important role in chondrogenesis, and disruption of Nfatc1/c2 is associated with osteoarthritis, and alterations in NFAT signaling may explain bone and cartilage disorders associated with inflammation [Greenblatt et al., 2013; Zanotti and Canalis, 2013]. NFATc1 and NFATc2 oppose osteoarthritis progression, NFATc1 inhibit chondrogenesis, and the deletion of Nfatc1 and Nfatc2 result in entheseal osteochondromas reflecting a restrictive role of these transcription factors on osteochondral growth [Ge et al., 2016; Greenblatt et al., 2013; Zanotti and Canalis, 2013].

In conclusion, our studies reveal that the inactivation of Nfatc2 in osteoclast precursors decreases osteoclast differentiation and bone resorption in vitro but this effect is not translated in vivo possibly because of compensation by NFATc1.

ABBREVIATIONS

The abbreviations used are

α-MEM

α-minimum essential medium

bp

base pair

BMM

bone marrow-derived macrophage

ca

constitutive active

ES

embryonic stem

FBS

fetal bovine serum

Frt

flippase recognition target

kb

kilobase pair

MC1-HSV-TK

MC1 driven herpes simplex virus-thymidine kinase

M-CSF

macrophage colony stimulating factor

μCT

microcomputed tomography

NFAT

Nuclear factor of activated T cells

PGK

phosphoglycerate kinase

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

qRT-PCR

quantitative reverse transcription-PCR

RANKL

receptor activator of NF-κB ligand

SDS

sodium dodecyl sulfate

SMI

structure model index

TRAP

tartrate resistant acid phosphatase