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. 2001 Jun;107(11):1375-85.
doi: 10.1172/JCI10530.

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

Y Abu-Amer  1
Affiliations

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

Y Abu-Amer. J Clin Invest. 2001 Jun.

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-kappaB. NF-kappaB activation is manifested by nuclear translocation and binding to DNA, events secondary to phosphorylation and dissociation of IkappaBalpha. It is shown here that IL-4 reduces NF-kappaB nuclear translocation by inhibiting IkappaB phosphorylation, thus markedly inhibiting NF-kappaB DNA binding activity and blocking osteoclastogenesis entirely. Residual translocation of NF-kappaB in the presence of IL-4, however, suggests that nuclear mechanisms must primarily account for inhibition of NF-kappaB DNA binding and blockade of osteoclastogenesis. To address this issue, this study examined whether IL-4-induced STAT6 transcription factor blocks NF-kappaB transactivation. The results show that excess unlabeled consensus sequence STAT6, but not its mutated form, inhibits NF-kappaB binding. Furthermore, exogenously added STAT6 protein inhibits NF-kappaB/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-kappaB activation in a STAT6-dependent manner.

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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
IL-4 inhibits NF-κB DNA binding activity. (a) Pure population of bone marrow macrophages was grown to confluence for 4 days in the presence of M-CSF. Cells were then treated with 10 ng/ml mIL-4 for 1 hour followed by 20 ng/ml RANKL as indicated. At the end of treatment, cells were harvested, nuclear extracts were prepared, and NF-κB EMSA was performed as described in Methods. Similar results represent three independent experiments. Incomplete inhibition of RANKL-induced NF-κB by IL-4 in lanes 5 and 6 correlates with basal expression of NF-κB under IL-4–treated conditions (lane 4), which does not support osteoclastogenesis. Extracts from 10- and 20-minute TNF-treated cells (10 ng/ml) were included as a positive control. (b) Identity of the NF-κB band was confirmed by binding with various NF-κB subunit antibodies (1 μl/reaction) as indicated or by a representative nonimmune IgG (lane 2). (c) Specificity of NF-κB binding was assessed by the addition of 50-fold (50×) unlabeled or mutated oligonucleotides, resulting in complete displacement or lack of displacement of the labeled probe, respectively.
Figure 3
Figure 3
IL-4 inhibits IκB phosphorylation and NF-κB nuclear translocation. Cells were grown as described for Figure 2 and treated with RANKL for the amounts of time indicated in the absence or presence of pretreatment with IL-4. Cells were then lysed, and cytoplasmic and nuclear fractions were separated. Cytosols were analyzed for IκB, p50, p52, and p65 NF-κB by immunoblots. Anti-IκB antibody raised against the carboxy terminus of the protein that recognizes both native and phosphorylated IκB was used. Results are representative of two independent experiments.
Figure 4
Figure 4
Residual levels of NF-κB subunits are present in nuclei under basal and IL-4–treated conditions. Nuclear extracts from cells used in Figure 3 were mixed with sample buffer and assayed for NF-κB by immunoblots.
Figure 5
Figure 5
IL-4 and RANKL induce endogenous STAT6 and NF-κB interaction. Macrophages were grown in tissue culture coverslips for 3 days. Cells were then treated with RANKL (20 minutes) and/or IL-4 (60 minutes) or left untreated, followed by fixation with 0.25% glutaraldehyde. Cells were immunostained with STAT6 (red) and p52 NF-κB (green) antibodies alone or in combination as shown. Colocalization (d, yellow) is indicated.
Figure 6
Figure 6
STAT6 is required for IL-4 inhibition of NF-κB activation. Nuclear extracts were prepared from wild-type (BALB/c) or STAT6 knockout mice treated with IL-4, with or without RANKL. NF-κB EMSA was performed as described previously.
Figure 7
Figure 7
STAT6 consensus sequence inhibits NF-κB DNA-binding activity. Nuclear extracts were prepared from control and RANKL-stimulated cells as described. EMSA was carried out with 32P-ATP–labeled NF-κB oligonucleotide in the absence or presence of excess 10-fold (10×) unlabeled consensus sequence (CS) or point mutated STAT6 oligonucleotide. Specificity of the NF-κB bands is documented in Figure 2c.
Figure 8
Figure 8
Endogenous STAT6 is required for IL-4 inhibition of osteoclastogenesis in vitro. Osteoclast precursors harvested from STAT6 knockout mice were plated as described for Figure 1. Cells were left untreated or treated with RANKL in the absence or presence of IL-4. Osteoclast cultures were then fixed and stained for TRAP activity. TRAP+ MNC cells were counted, and the average of five independent experiments was recorded as: Control = 0, IL-4 = 0, RANKL = 210 ± 28, IL-4 + RANKL = 231 ± 17 with no statistical difference between the last two groups.
Figure 9
Figure 9
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.
Figure 10
Figure 10
IL-4 inhibits RANKL-induced JNK activation. Wild-type and STAT6-null osteoclast precursor cells were treated for 20 minutes with RANKL (20 ng/ml) in the absence or presence (1 hour) of 10 ng/ml IL-4. C-Jun N-terminal kinase (JNK) assay was performed using c-Jun as substrate (cell signaling). Equal amounts of protein from the initial cell lysates were analyzed for total JNK protein content.
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