Contribution of Nuclear Factor of Activated T Cells c1 to the Transcriptional Control of Immunoreceptor Osteoclast-associated Receptor but Not Triggering Receptor Expressed by Myeloid Cells-2 during Osteoclastogenesis

Abstract

Bone homeostasis depends on the coordination of osteoclastic bone resorption and osteoblastic bone formation. Receptor activator of NF-B ligand (RANKL) induces osteoclast differentiation through activating a transcriptional program mediated by the key transcription factor nuclear factor of activated T cells (NFAT) c1. Immunoreceptors, including osteoclast-associated receptor (OSCAR) and triggering receptor expressed by myeloid cells (TREM)-2, constitute the co-stimulatory signals required for RANKL-mediated activation of calcium signaling, which leads to the activation of NFATc1. However, it remains unknown whether the expression of immunoreceptors are under the control of NFATc1. Here we demonstrate that the expression of OSCAR, but not that of TREM-2, is up-regulated during osteoclastogenesis and markedly suppressed by the calcineurin inhibitor FK506, suggesting that OSCAR is transcriptionally regulated by NFATc1. NFATc1 expression results in the activation of the OSCAR promoter, which was found to be further enhanced by co-expression of PU.1 and microphthalmia-associated transcription factor (MITF). We further provide evidence that NFATc1 specifically regulates OSCAR by chromatin immunoprecipitation assay and quantification of OSCAR and TREM-2 mRNA in NFATc1 Bone is continuously remodeled by a series of cellular actions of boneresorbing osteoclasts and bone-forming osteoblasts. Osteoclasts are the primary cells that resorb bone, and our knowledge concerning the mechanisms of osteoclast differentiation has been increasing rapidly (1-4). The finding that bone marrow monocyte/macrophage lineage cells (BMMs) 2 can differentiate into osteoclasts in vitro when co-cultured with osteoblasts (5) suggested that osteoblast-derived factors induce osteoclast differentiation. Receptor activator of NF-B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) are provided by osteoblasts and essential for osteoclastogenesis (1-4). In addition, we discovered recently that the immunoreceptor-mediated calcium signals are also critical for osteoclastogenesis (6).In the osteoclast differentiation program, transcription factor nuclear factor of activated T cells (NFAT) c1 is induced significantly by RANKL (7). NFATc1 is involved in the transcriptional regulation of osteoclastspecific genes, such as ACP5 (encoding tartrate-resistant acid phosphatase (TRAP)) (7), calcitonin receptor (7,8), and cathepsin K (9). Moreover, NFATc1-deficient embryonic stem cells failed to differentiate into osteoclasts in vitro, and ectopic expression of NFATc1 caused induction of osteoclast differentiation even in the absence of RANKL (7). Thus, NFATc1 can be referred to as the master transcription factor for osteoclastogenesis.The activity of NFATc1 is controlled by the phosphatase calcineurin, which plays a critical role in the coupling of calcium signals with cellular responses (10 -13). Once activated by an increase of the intracellular calcium concentration, calcineurin induces the translocation of NFATc1 into the nucleus, where it activat...

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“…Consistent with previous results (19,25), cotransfection of the OSCAR reporter construct and NFATc1 resulted in approximately a 4-fold increase in promoter activity (Fig. 4D).…”

The R740S mutation in the V-ATPase a3 subunit increases lysosomal pH, impairs NFATc1 translocation, and decreases in vitro osteoclastogenesis

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“…A deletion mutant of pTA-Il23r-luc, which lacks a putative Maf binding site (MARE-like element) was constructed as described previously (18). DNA transfection and luciferase assays were performed as described previously (19,20). Briefly, the reporter plasmid was transfected along with a c-Maf expression vector (21), a GATA-3 expression vector (22), and/or the control vector pcDNA3.1 into HEK293T cells using FuGENE 6 (Roche Applied Science).…”