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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Figure 9

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Exogenously added STAT6 blocks NF-κB activation and osteoclastogenesis. ...
Exogenously added STAT6 blocks NF-κB activation and osteoclastogenesis. (a) STAT6 knockout cells were incubated with 100 nM TAT-STAT6 for 1 hour in the absence or presence of 10 ng/ml IL-4. Cells were fixed and immunostained using anti-STAT6 antibody. (b) STAT6 knockout cells were incubated with TAT or TAT-STAT6 in the presence of IL-4 and RANKL. Cells were then fixed and stained with STAT6 and NF-κB antibodies. (c) EMSA for NF-κB was performed using control and RANKL-treated wild-type and STAT6 nuclear extracts in the presence of 2 μl TAT (lanes 2 and 5) or TAT-STAT6 (lanes 3 and 6) purified proteins (STAT6p). (d) STAT6 knockout cells were cultured with 100 nM TAT-STAT6 and RANKL in the absence or presence of 10 ng/ml IL-4 as described for Figure 1. Osteoclast cultures were fixed and TRAP-stained on day 4 of culture. TRAP+ MNC (more than three nuclei) cells were counted, and the average of triplicate wells from four independent experiments was recorded as follows: upper left panel, 226 ± 21; lower left panel, 247 ± 29; upper right panel, 186 ± 18 (P < 0.005); and lower right panel, 25 ± 11 (P < 0.0001). Note that cells treated with TAT-STAT6 appeared condensed and generally smaller, and fewer by approximately 30%, compared with controls.