IL-4 abrogates osteoclastogenesis through STAT6-dependent inhibition of NF-κB
Yousef Abu-Amer
Yousef Abu-Amer
Published June 1, 2001
Citation Information: J Clin Invest. 2001;107(11):1375-1385. https://doi.org/10.1172/JCI10530.
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Article

IL-4 abrogates osteoclastogenesis through STAT6-dependent inhibition of NF-κB

Abstract

IL-4, an anti-inflammatory cytokine, inhibits osteoclast differentiation, but the basis of this effect has been unclear. Osteoclastogenesis requires activation of RANK, which exerts its biologic effect via activation of NF-κB. NF-κB activation is manifested by nuclear translocation and binding to DNA, events secondary to phosphorylation and dissociation of IκBα. It is shown here that IL-4 reduces NF-κB nuclear translocation by inhibiting IκB phosphorylation, thus markedly inhibiting NF-κB DNA binding activity and blocking osteoclastogenesis entirely. Residual translocation of NF-κB in the presence of IL-4, however, suggests that nuclear mechanisms must primarily account for inhibition of NF-κB DNA binding and blockade of osteoclastogenesis. To address this issue, this study examined whether IL-4–induced STAT6 transcription factor blocks NF-κB transactivation. The results show that excess unlabeled consensus sequence STAT6, but not its mutated form, inhibits NF-κB binding. Furthermore, exogenously added STAT6 protein inhibits NF-κB/DNA interaction. Further supporting a role for STAT6 in this process are the findings that IL-4 fails to block osteoclastogenesis in STAT6–/– mice but that this blockade can be restored with addition of exogenous STAT6. Thus, IL-4 obliterates osteoclast differentiation by antagonizing NF-κB activation in a STAT6-dependent manner.

Authors

Yousef Abu-Amer

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IL-4 blocks RANKL-mediated osteoclastogenesis by bone marrow macrophages...
IL-4 blocks RANKL-mediated osteoclastogenesis by bone marrow macrophages. Osteoclast precursor cells were isolated from the bone marrow of 4- to 6-week-old mice as described in Methods. Pure (∼90%) marrow macrophages were plated in 48-well plates at 1 × 106 cells/ml using α-MEM supplemented with 10% heat-inactivated (HI) FCS and 10 ng/ml M-CSF. Cells were treated with PBS, 10 ng/ml mIL-4 for 30–60 minutes, 20 ng/ml soluble RANKL for 4 days, or a combination of IL-4 and RANKL (as shown). Cultures were placed at 37°C in a 5% CO2 incubator and supplemented with an additional dose of M-CSF and RANKL on the third day of culture. Osteoclasts developed on days 4–5, after which they were washed, fixed, and stained for TRAP activity following manufacturers’ directions. TRAP-positive (purple) mono- and multinucleated large cells are osteoclasts and their committed precursors. The average number of osteoclasts in RANKL-treated cultures was 182 ± 22/cm2 compared with no osteoclasts in all other conditions. Results represent average number of quadruplicate wells from three independent experiments. ×20 taken by light microscope.