Abstract
Osteoclast differentiation from hematopoietic precursors is controlled by the tumor necrosis factor family member tumor necrosis factor-related activation-induced cytokine (TRANCE) via induction of various transcription factors, including nuclear factor of activated T cells (NFAT) c1. During osteoclast differentiation, NFATc1 is further activated via calcium signaling when costimulatory receptors expressed on osteoclast precursors, such as osteoclast-associated receptor (OSCAR), are stimulated. Here we show that NFATc1 expression precedes that of OSCAR during TRANCE-mediated osteoclastogenesis and that inhibition of NFATc1 by cyclosporin A abolishes TRANCE-induced OSCAR expression and subsequent osteoclast differentiation. Moreover, we show that the 1.0-kb promoter region of the OSCAR gene contains three potential NFATc1-binding sites. Induction of an OSCAR promoter-luciferase reporter is significantly increased when transiently transfected into 293T cells in combination with NFATc1 expression plasmid. Deletion and site-directed mutant constructs confirmed that NFATc1-binding sites are both functional and NFATc1-specific. Furthermore, NFATc1 synergistically activates an OSCAR reporter construct together with microphthalmia transcription factor and PU.1, transcription factors previously shown to be critical for osteoclast differentiation. In addition, a plasmid expressing constitutively active MAP kinase kinase 6 enhances the transactivation activity of NFATc1/microphthalmia transcription factor/PU.1 on the OSCAR promoter. Taken together, our results indicate that NFATc1 is an important transcription factor in the induction of OSCAR during osteoclastogenesis. Elucidation of NFATc1 as a transcription factor for OSCAR expression implies the presence of a positive feedback circuit of TRANCE-induced activation of NFATc1, involving NFATc1-mediated OSCAR expression and its subsequent activation of NFATc1, necessary for efficient differentiation of osteoclasts.
Highlights
Bone is continuously remodeled by osteoclasts and osteoblasts, and their balanced activity is important for maintaining bone density
Gene Expression of NFATc1 and osteoclast-associated receptor (OSCAR) during Osteoclastogenesis Mediated by tumor necrosis factor-related activation-induced cytokine (TRANCE)—NFATc1 is a key regulator in the late phase of TRANCE-induced osteoclastogenesis and acts upon osteoclast-specific genes such as TRAP and calcitonin receptor [13,14,15, 22, 23]
Cyclosporin A inhibited the expression of NFATc1 as well as of OSCAR, which would normally be induced by TRANCE stimulation (Fig. 1B)
Summary
M-CSF, macrophage colony-stimulating factor; TRANCE, tumor necrosis factor-related activation-induced cytokine; RANK, receptor activator of nuclear factor B; TRAP, tartrate-resistant acid phosphatase; Mitf, microphthalmia transcription factor; OSCAR, osteoclast-associated receptor; NFAT, nuclear factor of activated T cells; DAP, DNAX-activating protein; FcR␥, Fc receptor common ␥ chain; ITAM, immunoreceptor tyrosine-based activation motif; BMM, bone marrow-derived macrophage-like cells; EMSA, electrophoretic mobility shift assay; MAP, mitogen-activated protein; RT, reverse transcription; HPRT, hypoxanthine ribosyltransferase; ChIP, chromatin immunoprecipitation; MKK, MAP kinase kinase. It has been shown that the expression of NFATc1 is induced by TRANCE and that NFATc1 and c-Fos synergistically induce the expression of osteoclast-specific genes [14, 22, 23]. NFATc1 plays a central role in regulating terminal differentiation of osteoclasts mediated by TRANCE and in inducing the expression of osteoclast-specific genes such as TRAP and calcitonin receptor, a role for NFATc1 in the regulation of OSCAR gene expression has not yet been described. We report here that NFATc1 directly induces OSCAR gene expression. OSCAR expression is synergistically regulated by the combined activity of Mitf, PU., and NFATc1. This study indicates that NFATc1, cooperating with other transcription factors, plays a role in the up-regulation of its target genes that include the costimulatory receptor OSCAR. Our data suggest a potentially important molecular mechanism of osteoclast differentiation via a positive feedback circuit involving TRANCE, NFATc1, and OSCAR
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