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Cited by 202Bone Metabolism and DiseasesCytokine Signaling Pathways and InteractionsRheumatoid Arthritis Research and Therapies

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Interleukin-4 Reversibly Inhibits Osteoclastogenesis via Inhibition of NF-κB and Mitogen-activated Protein Kinase Signaling

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Wei, Shi, Wang, Michael W.-H., Teitelbaum, Steven L., Ross, F. Patrick·2002·Journal of Biological Chemistry
DOI 10.1074/jbc.m104957200
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Abstract

To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colony-stimulating factor (M-CSF), receptor for activation of NF-kappa B ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-kappa B, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-kappa B and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing I kappa B phosphorylation and thus NF-kappa B activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways.

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Figures

FIG. 1 IL-4 inhibits RANKL-induced osteoclastogenesis. BMMs were purified from wild type mice, and osteoclasts were generated using M-CSF and RANKL as described under ' Experimental Procedures. ' IL-4 was added at the initiation of culture at the indicated concentrations. TRAP stain was performed on day 4. Characteristic OCs ( asterisks ) are seen only in the absence of IL-4.
FIG. 1 IL-4 inhibits RANKL-induced osteoclastogenesis. BMMs were purified from wild type mice, and osteoclasts were generated using M-CSF and RANKL as described under ' Experimental Procedures. ' IL-4 was added at the initiation of culture at the indicated concentrations. TRAP stain was performed on day 4. Characteristic OCs ( asterisks ) are seen only in the absence of IL-4.CC-BY · From the PDF
FIG. 2 IL-4 targets only early osteoclast precursors. Osteoclasts were generated from wild type BMMs without or with IL-4 (5 ng/ml) added from day 0 through day 4, and TRAP stain was performed on day 5. IL-4 is inhibitory only if added on days 0 or 1.
FIG. 2 IL-4 targets only early osteoclast precursors. Osteoclasts were generated from wild type BMMs without or with IL-4 (5 ng/ml) added from day 0 through day 4, and TRAP stain was performed on day 5. IL-4 is inhibitory only if added on days 0 or 1.CC-BY · From the PDF
FIG. 3 IL-4 inhibition of osteoclastogenesis is reversible. Osteoclasts were generated in the absence ( top left panel ) or presence of IL-4 (5 ng/ml). IL-4 was kept in the culture or withdrawn on day 3 or 5 by medium change. TRAP stain was performed on day 4 ( left panel ) or 4 days after withdrawal of IL-4 ( right panel ).
FIG. 3 IL-4 inhibition of osteoclastogenesis is reversible. Osteoclasts were generated in the absence ( top left panel ) or presence of IL-4 (5 ng/ml). IL-4 was kept in the culture or withdrawn on day 3 or 5 by medium change. TRAP stain was performed on day 4 ( left panel ) or 4 days after withdrawal of IL-4 ( right panel ).CC-BY · From the PDF
FIG. 4 IL-4 does not alter M-CSF signaling in BMMs. BMMs were maintained in the absence or presence of IL-4 (5 ng/ml) for 3 days. Cells were then stimulated with M-CSF (10 ng/ml) as indicated. Cells were lysed, fractionated by SDS-PAGE, and analyzed by Western blotting as described under ' Experimental Procedures. ' Cells treated with TNF and processed in parallel represent positive controls for JNK, p38, and I 𝛋 B 𝛂 signals ( right-hand lane ).
FIG. 4 IL-4 does not alter M-CSF signaling in BMMs. BMMs were maintained in the absence or presence of IL-4 (5 ng/ml) for 3 days. Cells were then stimulated with M-CSF (10 ng/ml) as indicated. Cells were lysed, fractionated by SDS-PAGE, and analyzed by Western blotting as described under ' Experimental Procedures. ' Cells treated with TNF and processed in parallel represent positive controls for JNK, p38, and I 𝛋 B 𝛂 signals ( right-hand lane ).CC-BY · From the PDF
FIG. 5 IL-4 does not alter RANK or TRAF6 levels. BMMs were maintained in the absence or presence of IL-4 (5 ng/ml) for 3 days and then stimulated with RANKL as indicated. Cells were lysed, fractionated by SDS-PAGE, and applied for Western blotting as described under ' Experimental Procedures. '
FIG. 5 IL-4 does not alter RANK or TRAF6 levels. BMMs were maintained in the absence or presence of IL-4 (5 ng/ml) for 3 days and then stimulated with RANKL as indicated. Cells were lysed, fractionated by SDS-PAGE, and applied for Western blotting as described under ' Experimental Procedures. 'CC-BY · From the PDF
Image 6
CC-BY · From the PDF
Image 7
CC-BY · From the PDF
FIG. 8 IL-4, when present for at least 3 days, reversibly inhibits MAP kinase activation pathways. BMMs were maintained with IL-4 (5 ng/ml) for 1 or 24 h ( A ) or 3 days ( B and C ) and then stimulated with RANKL as indicated. In parallel, IL-4 was withdrawn for 24 h before stimulation from cells pretreated with the cytokine for 3 days ( + 3 -), followed by RANKL stimulation ( B ). Cells were lysed and analyzed by Western blotting.
FIG. 8 IL-4, when present for at least 3 days, reversibly inhibits MAP kinase activation pathways. BMMs were maintained with IL-4 (5 ng/ml) for 1 or 24 h ( A ) or 3 days ( B and C ) and then stimulated with RANKL as indicated. In parallel, IL-4 was withdrawn for 24 h before stimulation from cells pretreated with the cytokine for 3 days ( + 3 -), followed by RANKL stimulation ( B ). Cells were lysed and analyzed by Western blotting.CC-BY · From the PDF
FIG. 9 IL-4 does not alter RANKL-induced activation of Akt. BMMs were pretreated without or with IL-4 (5 ng/ml) for 3 days and then stimulated with RANKL as indicated. Cells were then lysed and analyzed by Western blotting.
FIG. 9 IL-4 does not alter RANKL-induced activation of Akt. BMMs were pretreated without or with IL-4 (5 ng/ml) for 3 days and then stimulated with RANKL as indicated. Cells were then lysed and analyzed by Western blotting.CC-BY · From the PDF
FIG. 10 IL-4 selectively inhibits TNF-induced signaling and enhances IL-1-induced signaling. BMMswerepretreated without or with IL-4 (5 ng/ml) for 3 days, and then stimulated with TNF 𝛂 ( A ) or IL-1 𝛂 ( B ) as indicated. Cells were then lysed and analyzed by Western blotting.
FIG. 10 IL-4 selectively inhibits TNF-induced signaling and enhances IL-1-induced signaling. BMMswerepretreated without or with IL-4 (5 ng/ml) for 3 days, and then stimulated with TNF 𝛂 ( A ) or IL-1 𝛂 ( B ) as indicated. Cells were then lysed and analyzed by Western blotting.CC-BY · From the PDF
FIG. 11 ERK activation is required for osteoclastogenesis. BMMs were cultured with M-CSF plus RANKL in the absence or presence either PD98059 or U0126, two MEK inhibitors, as indicated. TRAP stain ( A ) or TRAP solution assays ( B ) were performed on day 4. In parallel, inhibitors were added to OC-generating cultures for 3 days and withdrawn for 2 days by medium change before TRAP solution assays were performed ( C ). The values shown are mean ± S.D. of six determinations. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (inhibitors versus vehicles ( B , veh ) or withdrawal versus with inhibitors ( C ) by unpaired t test).
FIG. 11 ERK activation is required for osteoclastogenesis. BMMs were cultured with M-CSF plus RANKL in the absence or presence either PD98059 or U0126, two MEK inhibitors, as indicated. TRAP stain ( A ) or TRAP solution assays ( B ) were performed on day 4. In parallel, inhibitors were added to OC-generating cultures for 3 days and withdrawn for 2 days by medium change before TRAP solution assays were performed ( C ). The values shown are mean ± S.D. of six determinations. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (inhibitors versus vehicles ( B , veh ) or withdrawal versus with inhibitors ( C ) by unpaired t test).CC-BY · From the PDF
FIG. 12 IL-4 inhibition of osteoclastogenesis and NF𝛋 B and MAP kinase activation is STAT6-dependent but not SHIP1-dependent. A , BMMs were purified from STAT6- or SHIP1-deficient mice, and IL-4 was added at the initiation of osteoclast-generating culture at 5 ng/ml. TRAP stain was performed on day 4. B and C , BMMs from wild type and STAT6-deficient mice ( B ) or SHIP1 heterozygous and deficient mice ( C ) were maintained without/with IL-4 (5 ng/ml) for 3 days, stimulated with RANKL as indicated, and lysed. Western blotting was performed with indicated antibodies.
FIG. 12 IL-4 inhibition of osteoclastogenesis and NF𝛋 B and MAP kinase activation is STAT6-dependent but not SHIP1-dependent. A , BMMs were purified from STAT6- or SHIP1-deficient mice, and IL-4 was added at the initiation of osteoclast-generating culture at 5 ng/ml. TRAP stain was performed on day 4. B and C , BMMs from wild type and STAT6-deficient mice ( B ) or SHIP1 heterozygous and deficient mice ( C ) were maintained without/with IL-4 (5 ng/ml) for 3 days, stimulated with RANKL as indicated, and lysed. Western blotting was performed with indicated antibodies.CC-BY · From the PDF

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  1. McNally, 1995, Am. J. Pathol., 147, 1487

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Metadata

Authors
Wei, S., Wang, M. W.-H., Teitelbaum, S. L., & Ross, F. P.
Affiliations
Washington University in St. Louis (US)
Corresponding author
Ross, F. Patrick
ORCID
Teitelbaum, Steven L.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2002-02
Volume
277
Issue
8
Pages
6622-6630
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m104957200
OpenAlex
W1998128158
Cited by
202
References
55
Citations by year
2026: 3 · 2025: 3 · 2024: 4 · 2023: 7 · 2022: 4 · 2021: 3 · 2020: 15 · 2019: 3 · 2018: 9 · 2017: 5 · 2016: 7 · 2015: 6 · 2014: 3 · 2013: 10 · 2012: 5
FWCI
5.55
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
  • Health Sciences › Medicine › Rheumatology › Rheumatoid Arthritis Research and Therapies
Keywords
IL-4, osteoclastogenesis, NF-κB signaling, mitogen-activated protein kinase, STAT6, SHIP1, RANKL, macrophage colony-stimulating factor, TNF signaling, IL-1 signaling, MEK inhibitors, bone marrow-derived macrophages
MeSH terms
Animals, Bone Marrow Cells, Carrier Proteins, Cells, Cultured, Gene Expression Regulation, Kinetics, Macrophages, Membrane Glycoproteins, Mice, Inbred C57BL, Osteoclasts, Osteogenesis, Signal Transduction, Interleukin-4, Macrophage Colony-Stimulating Factor, NF-kappa B, Cell Culture Techniques, Reverse Transcriptase Polymerase Chain Reaction, MAP Kinase Signaling System, Mice, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B

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