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 mice, where the Nfact2 exon 2 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 ;Nfatc2 mice cultured in the presence of macrophage-colony stimulating factor and receptor activator of NF-κB ligand exhibited a decrease in the number and size of osteoclasts and a smaller sealing zone when compared to BMMs from Nfatc2 littermate controls. Bone resorption was decreased in osteoclasts from Lyz2 ;Nfatc2 mice. This demonstrates that NFATc2 is necessary for optimal osteoclast maturation and function in vitro. Male and female Lyz2 ;Nfatc2 mice did not exhibit an obvious skeletal phenotype by microcomputed tomography (μCT) at either 1 or 4 months of age when compared to Nfatc2 sex-matched littermates. Bone histomorphometry confirmed the μCT results, and conditional 4-month-old Lyz2 ;Nfatc2 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.

Network analysis of transcriptomic diversity amongst resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system

“…Etv1 expression in macrophages has been implicated in functional polarisation in vitro and the response to altered mitochondrial membrane potential [ 99 ]. NFATC2 is required for osteoclast differentiation in vitro [ 100 ], but its roles in macrophage differentiation/function have not been explored. Tcf4 encodes a transcription effector of the Wnt/β-catenin pathway, which is implicated in responses to E-cadherin and other effectors in macrophage differentiation [ 101 ].…”

An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice

“…BMMs from Notch3 tm1.1Ecan mutants and control littermates were incubated with M‐CSF at 30 ng/ml for 3 days followed by the subsequent addition of RANKL at 10 ng/ml in conjunction with M‐CSF at 30 ng/ml, as reported (Canalis & Yu et al, ; Yu et al, ). The number of osteoclasts, defined as multinucleated cells that were positive for TRAP, was augmented by approximately 30% in Notch3 tm1.1Ecan cultures (Figure ).…”

“…M‐CSF was purified as described, and M‐CSF complementary DNA (cDNA) and expression vector were provided by D. Fremont (Washington University, St. Louis, MO; Lee et al, ). Cells were plated at a density of 300,000 cells/cm 2 on uncoated plastic petri dishes and cultured in the presence of M‐CSF for 3 days, as described (Canalis & Yu et al, ; Yu et al, ). The cell layer was then treated with 0.25% trypsin/EDTA for 5 min and cells recovered and seeded at a density of 47,000 cells/cm 2 on tissue culture plates in α‐MEM with 10% FBS, 30 ng/ml of M‐CSF, and 10 ng/ml of murine receptor activator of NF Kappa B ligand (RANKL) and anti‐Notch3 NRR or control antibodies at 20 μg/ml.…”

“…Microarchitectural analysis of femurs was conducted using a Scanco µCT 40 instrument (Scanco Medical AG, Bassersdorf, Switzerland), which underwent periodic calibrations using a manufacturer‐provided phantom (Scanco Medical AG; Bouxsein et al, ; Canalis, Kranz, & Zanotti, ; Glatt, Canalis, Stadmeyer, & Bouxsein, ; Yu, Zanotti, Schilling, & Canalis, ). Femoral bones were scanned in 70% ethanol at high resolution, energy level of 55 kVp, intensity of 145 µA, and integration time of 200 ms, as reported (Yu et al, ). One hundred slices at midshaft and 160 slices at the distal metaphysis were acquired at a thickness of 6 µm and isotropic voxel size of 216 µm 3 , and selected for analysis.…”

Transcriptional network analysis of transcriptomic diversity in resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system

“…Etv1 expression in macrophages has been implicated in functional polarization in vitro and the response to altered mitochondrial membrane potential [71]. Nfatc2 is required for osteoclast differentiation in vitro [72] but roles in macrophage differentiation/function have not been explored. Tcf4 encodes a transcription effector of Wnt/β-catenin pathway, implicated in responses to E-cadherin and other effectors in macrophage differentiation [73].…”