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-FmsY697 is specifically required for osteoclastogenesis in the absence...
c-FmsY697 is specifically required for osteoclastogenesis in the absence of αvβ3. (a) Equal numbers of β3+/+ and β3–/– BMMs were retrovirally transduced with vector alone, nonmutated EpoR/c-Fms (WT), or EpoR/c-Fms carrying individual tyrosine-to-phenylalanine point mutation. Cells were selected in puromycin for 3 days and exposed for 3 days to RANKL and low-dose MCSF. The cells were then placed in serum-free medium for 2 hours, exposed to 25 U/ml Epo for varying times, and lysed. Equivalent expression of c-Fms and mutated forms of EpoR/c-Fms was established by immunoblot using a C-terminal anti–c-Fms antibody. (b) Puromycin-selected cells were cultured for 7 days in the optimal osteoclastogenic concentration of Epo (25 ng/ml) and RANKL (100 ng/ml). Osteoclastogenesis was measured by TRAP assay and expressed as a percentage of the value obtained with control (WT) EpoR/c-Fms transductants. While EpoR/c-FmsY697F was as effective as control in inducing β3+/+ osteoclastogenesis, the same mutant dampened the process 3.5-fold in β3–/– cells. In contrast, EpoR/c-FmsY721F did not impact the osteoclastogenic process. *P < 0.001 vs. vector; **P < 0.05 vs. vector.