c-Fms and the αvβ3 integrin collaborate during osteoclast differentiation
Roberta Faccio, Sunao Takeshita, Alberta Zallone, F. Patrick Ross, Steven L. Teitelbaum
Roberta Faccio, Sunao Takeshita, Alberta Zallone, F. Patrick Ross, Steven L. Teitelbaum
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Article Bone biology

c-Fms and the αvβ3 integrin collaborate during osteoclast differentiation

Abstract

β3 integrin–null osteoclasts are dysfunctional, but their numbers are increased in vivo. In vitro, however, the number of β3–/– osteoclasts is reduced because of arrested differentiation. This paradox suggests cytokine regulation of β3–/– osteoclastogenesis differs in vitro and in vivo. In vitro, additional MCSF, but not receptor activator of NF-κB ligand (RANKL), completely rescues β3–/– osteoclastogenesis. Similarly, activation of extracellular signal-regulated kinases (ERKs) and expression of c-Fos, both essential for osteoclastogenesis, are attenuated in β3–/– preosteoclasts, but completely restored by additional MCSF. In fact, circulating and bone marrow cell membrane-bound MCSFs are enhanced in β3–/– mice, correlating with the increase in the osteoclast number. To identify components of the MCSF receptor that is critical for osteoclastogenesis in β3–/– cells, we retrovirally transduced authentic osteoclast precursors with chimeric c-Fms constructs containing various cytoplasmic domain mutations. Normalization of osteoclastogenesis and ERK activation, in β3–/– cells, uniquely requires c-Fms tyrosine 697. Finally, like high-dose MCSF, overexpression of c-Fos normalizes the number of β3–/– osteoclasts in vitro, but not their ability to resorb dentin. Thus, while c-Fms and αvβ3 collaborate in the osteoclastogenic process via shared activation of the ERK/c-Fos signaling pathway, the integrin is essential for matrix degradation.

Authors

Roberta Faccio, Sunao Takeshita, Alberta Zallone, F. Patrick Ross, Steven L. Teitelbaum

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c-Fos overexpression by β3–/– cells rescues osteoclastogenesis but not m...
c-Fos overexpression by β3–/– cells rescues osteoclastogenesis but not matrix resorption. β3+/+ BMMs and β3–/– BMMs retrovirally transduced with pBabe vector (MOCK) or pBabe/c-Fos were selected in puromycin for 5 days, and resistant cells were used in the indicated experiments. (a) An equal number of BMMs were plated in 96-well plates and cultured in the presence of RANKL and low-dose MCSF. After 7 days, cells were stained for TRAP activity. While osteoclastogenesis remained arrested in MOCK-transduced β3–/– cells, those overexpressing c-Fos generated OCs indistinguishable from WT. (b) Equal amounts of protein were loaded in each lane, and c-Fos content was assessed by immunoblot. β3–/– OCs retrovirally transduced with pBabe/c-Fos vector expressed the same level of c-Fos protein as did β3+/+ cells (arrow). (c) β3+/+, MOCK β3–/–, and c-Fos β3–/– pre-OCs were plated on dentin slices with RANKL and low-dose MCSF. MOCK β3–/– cells were also cultured with RANKL and high-dose MCSF. After four days, dentin slices were stained for TRAP activity (+ Cells), or the cells were removed to visualize resorptive pits (– Cells). β3+/+ cells differentiated into OCs with a characteristic resorptive phenotype and excavated many large, well-demarcated lacunae. MOCK-transduced β3–/– cells formed few OCs in the presence of low-dose MCSF and generated poorly defined, small pits. High-dose MCSF and c-Fos overexpression yielded numerous multinucleated TRAP-expressing OCs that exhibited a nonresorbing phenotype and also generated poorly defined, small pits. Indicated are the mean numbers of pits ± SEM from three different fields per variable. ×10.