The Tec Family Tyrosine Kinase Btk Regulates RANKL-induced Osteoclast Maturation

Daewon Jeong,Seoung Hoon Lee,Taesoo Kim,Nacksung Kim,Yongwon Choi

doi:10.1074/jbc.m708935200

Abstract

A spontaneous mutation in Bruton's tyrosine kinase (Btk) induces a defect in B-cell development that results in the immunodeficiency diseases X-linked agammaglobulinemia in humans and X-linked immunodeficiency (Xid) in mice. Here we show an unexpected role of Btk in osteoclast formation. When bone marrow cells derived from Xid mice were stimulated with receptor activator of NF-kappaB ligand, an osteoclast differentiation factor, they did not completely differentiate into mature multinucleated osteoclasts. Moreover, we found that the defects appeared to occur at the stage in which mononuclear preosteoclasts fuse to generate multinucleated cells. Supporting this notion, macrophages from Xid mice also failed to form multinucleated foreign body giant cells. The fusion defect of the Xid mutant osteoclasts was caused by decreased expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclast differentiation, as well as reduced expression of various osteoclast fusion-related molecules, such as the d2 isoform of vacuolar H(+)-ATPase V0 domain and the dendritic cell-specific transmembrane protein. This deficiency was completely rescued by the introduction of a constitutively active form of NFATc1 into bone marrow-derived macrophages. Our data provide strong evidence that Btk plays a critical role in osteoclast multinucleation by modulating the activity of NFATc1.

Highlights

Cally deficient for dendritic cell-specific transmembrane protein (DC-STAMP) or the d2 isoform of the vacuolar Hϩ-ATPase V0 domain (Atp6v0d2) exhibit osteopetrotic phenotypes due to defects in osteoclast fusion during osteoclastogenesis [4, 5]
The binding of RANKL to receptor activator of NF-␬B induces the expression of transcription factors, including nuclear factor of activated T cells c1 (NFATc1), NF-␬B, c-Fos, Mitf, and PU.1; these transcription factors have been shown to be important for osteoclastogenesis in vitro and in vivo [2, 3, 6]
When wild-type cells were treated with Cyclosporin A (CsA), the level of NFATc1 mRNA was significantly reduced to approximately the level observed in the RANKL-treated Bruton’s tyrosine kinase (Btk)-deficient cells, rather than that measured in untreated wild-type or Btk mutant cells. These results suggest that Btk deficiency inhibits the PLC␥2-dependent positive feedback loop governing NFATc1 expression, leading to decreased levels of NFATc1 mRNA and protein (Fig. 4, A and B). These results suggest that the defect in osteoclast fusion induced by the Btk mutation was a result of reduced NFATc1 activity

Summary

The abbreviations used are

M-CSF, macrophage colony-stimulating factor; BMM, bone marrow-derived macrophage; Btk, Bruton’s tyrosine kinase; Xid, X-linked immunodeficiency; RANKL, receptor activator of NF-␬B ligand; NFATc1, nuclear factor of activated T cells c1; Ca-NFATc1, constitutively active NFATc1; TRAP(ϩ) MNCs, tartrate-resistant acid phosphatasepositive multinucleated cells; ITAM, immunoreceptor tyrosine-based activation motif; PLC␥, phospholipase C␥; MAPK, mitogen-activated protein kinase; CsA, cyclosporin A; IL-3, interleukin 3; PGE2, prostaglandin E2; HA, hemagglutinin. Activated Tec family kinases are recruited to the receptor signaling complexes through interactions with adapter proteins, such as LAT, SLP-76, and BLNK, that recruit downstream effectors, including phospholipase C␥ (PLC␥). Recent evidence suggests that components of these ITAM-mediated signaling pathways participate in immune cell processes that are unrelated to adaptive immune responses, including collagen-induced activation of platelets [22], NK cells, and myeloid phagocytes [18, 19]. Preosteoclasts from Xid mice, which possess a natural mutation in the Btk gene, exhibit a deficiency in cell-cell fusion and decreased NFATc1 expression during RANKL-induced osteoclastogenesis. This study demonstrates that Btk plays a critical role in the processes underlying osteoclast multinucleation

EXPERIMENTAL PROCEDURES

Findings

DISCUSSION