Abstract
The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.
Highlights
Hematology, Centre Hospitalier Universitaire de Nice (France)
Quantitative analysis showed that the lack of 3BP2 dramatically reduced the number of tartrate-resistant acidic phosphatase (TRAP)-positive multinuclear osteoclasts and the number of nuclei/TRAP-positive cells induced upon RANKL
To examine the implication of 3BP2 in the osteoclast actin reorganization, sh3BP2 RAW264.7 cells and shLacZ control cells were cultured for 4 days with sRANKL and subjected to immunofluorescence analysis with fluorescent phalloidin to detect actin organization
Summary
Cell Line and Culture—RAW264.7 cells were purchased from American Type Culture Collection (Manassas, VA). After 8 days, the cells were analyzed for their morphology under a Zeiss Axiovert 40C light microscope or stained with fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies reactive to CD11c, CD80, or CD86 (all purchased from Becton-Dickinson), followed by flow cytometry analysis on a FACScan (Becton Dickinson). Following three washes with cold PBS, 1% bovine serum albumin, polymerized actin was measured by flow cytometry. Normalized luciferase activities were determined in triplicate and expressed as fold increase relative to the basal activities measured in control vector-transfected cells. The cells were transfected with mock retroviral expression vectors, constitutively active NFATc1 expression vector, or a combination of the SrcY527F expression vector and enhanced GFP expression vector, as described above. Effects with a p value less than 0.05 were considered statistically significant
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