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Comparative Study
. 2005 Apr 4;201(7):1169-77.
doi: 10.1084/jem.20041444.

IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation

Affiliations
Comparative Study

IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation

Hongmei Li et al. J Exp Med. .

Abstract

Osteoporosis is a serious problem worldwide; it is characterized by bone fractures in response to relatively mild trauma. Osteoclasts originate from the fusion of macrophages and they play a central role in bone development and remodeling via the resorption of bone. Therefore, osteoclasts are important mediators of bone loss that leads, for example, to osteoporosis. Interleukin (IL)-1 receptor (IL-1R)-associated kinase M (IRAK-M) is only expressed in cells of the myeloid lineage and it inhibits signaling downstream of IL-1R and Toll-like receptors (TLRs). However, it lacks a functional catalytic site and, thus, cannot function as a kinase. IRAK-M associates with, and prevents the dissociation of, IRAK-IRAK-4-TNF receptor-associated factor 6 from the TLR signaling complex, with resultant disruption of downstream signaling. Thus, IRAK-M acts as a dominant negative IRAK. We show here that mice that lack IRAK-M develop severe osteoporosis, which is associated with the accelerated differentiation of osteoclasts, an increase in the half-life of osteoclasts, and their activation. Ligation of IL-1R or TLRs results in hyperactivation of NF-kappaB and mitogen-activated protein kinase signaling pathways, which are essential for osteoclast differentiation. Thus, IRAK-M is a key regulator of the bone loss that is due to osteoclastic resorption of bone.

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Figures

Figure 1.
Figure 1.
IRAK-M–deficient mice are smaller than wild-type mice and have reduced total bone mineral density. (A) Male and female IRAK-M−/− mice are smaller than IRAK-M+/+ mice; n = 10. (B) microCT analysis of distal femurs from 4-mo-old male IRAK-M−/− and IRAK-M+/+ mice. Note the paucity of the trabeculae inside the bone in IRAK-M−/− mice. The wider diameter of the bone sections measures ∼3 mm. Bar, 1 mm. (C) Toluidine blue staining of sections from epiphyseal bones from 4-mo-old male IRAK-M−/− and IRAK-M+/+ mice. Bar, 1 mm. (D) TRAP staining of sections from epiphyseal bones from 4-mo-old male IRAK-M−/− and IRAK-M+/+ mice. Bar, 200 μm.
Figure 1.
Figure 1.
IRAK-M–deficient mice are smaller than wild-type mice and have reduced total bone mineral density. (A) Male and female IRAK-M−/− mice are smaller than IRAK-M+/+ mice; n = 10. (B) microCT analysis of distal femurs from 4-mo-old male IRAK-M−/− and IRAK-M+/+ mice. Note the paucity of the trabeculae inside the bone in IRAK-M−/− mice. The wider diameter of the bone sections measures ∼3 mm. Bar, 1 mm. (C) Toluidine blue staining of sections from epiphyseal bones from 4-mo-old male IRAK-M−/− and IRAK-M+/+ mice. Bar, 1 mm. (D) TRAP staining of sections from epiphyseal bones from 4-mo-old male IRAK-M−/− and IRAK-M+/+ mice. Bar, 200 μm.
Figure 2.
Figure 2.
Absence of IRAK-M alters osteoclastogenesis in response to M-CSF and RANKL. (A) Bone marrow cells from IRAK-M−/− and IRAK-M+/+ male mice were cultured in the presence of M-CSF (5 ng/ml) for 12–18 h. Nonadherent cells were cultured further for the indicated times in 96-well dishes and their numbers determined in terms of OD450nm (n = 5; see Materials and methods for details). Standard deviations are too small to show. P < 0.001 for IRAK-M+/+ versus IRAK-M−/− macrophages treated for 3, 4, and 5 d; P < 0.01 for IRAK-M+/+ versus IRAK-M−/− macrophages treated for 6 d. (B) Cells from (A) were stained for TRAP. Bar, 100 μm. (C) Nonadherent cells, prepared as in (A), were treated further with 30 ng/ml M-CSF for 2 d, replated in 96-well dishes (2 × 105 cells/ml), and treated with 300 ng/ml RANKL plus with increasing concentrations of M-CSF for 3 d. The surface area occupied by TRAP+ multinucleated osteoclasts was recorded (n = 4). Standard deviations are too small to show. P < 0.001 for IRAK-M+/+ versus IRAK-M−/− macrophages treated with 7.5, 15, 30, and 60 ng/ml M-CSF. (D) Photographs of cells in (C). Bar, 100 μm. (E) Nonadherent cells, prepared as in (A), were treated further with 30 ng/ml M-CSF for 2 d, replated in 96-well dishes (2 × 105 cells/ml), and stimulated with 30 ng/ml M-CSF plus with increasing concentrations of RANKL for 3 d. The surface area occupied by TRAP+ multinucleated cells was recorded (n = 4). Standard deviations are too small to show. P < 0.001 for IRAK-M+/+ versus IRAK-M−/− macrophages at each RANKL concentration. (F) Cells from (E) were stained for TRAP. Bar,100 μm. (G) Osteoclasts were generated as in (E) but were treated with 300 ng/ml RANKL and 30 ng/ml M-CSF for the indicated times. Bar, 100 μm. Each experiment was repeated at least three times with similar results.
Figure 3.
Figure 3.
Osteoclasts express IRAK-M, and demonstrate hyperactivation of signaling molecules downstream of IL-1R/TLR when IRAK-M is absent. (A) Bone marrow macrophages from wild-type mice were cultured in the presence of M-CSF (5 ng/ml) for 12–18 h. Nonadherent cells were cultured further for 2 d in 24-well dishes (macrophages) and treated for the indicated times with 20 ng/ml IL-1α or 1 mg/ml LPS (macrophages), or cultured for an additional 3 d in the presence of 30 ng/ml M-CSF and 300 ng/ml RANKL (osteoclasts, OC) before activation with IL-1α or LPS. Cells were lysed in Laemmli sample buffer supplemented with inhibitors of proteases and phosphatases and subjected to Western blotting analysis with anti–IRAK-M and anti-GADPH antibodies. (B) Bone marrow macrophages from wild-type mice were treated with 300 ng/ml RANKL for the indicated times, bone marrow macrophages from IRAK-M-deficient mice were treated similarly for 24 h and used as a negative control. Cells were analyzed as in (B). (C) IRAK-M+/+ and IRAK-M−/− osteoclasts were starved for 2 h, and then stimulated with 20 ng/ml IL-1α, IL-1β, or 1 mg/ml LPS for the indicated times. (D) IRAK-M+/+ and IRAK-M−/− osteoclasts were stimulated with 1 mg/ml LPS for the indicated times. The right lane shows a positive control for the phosphorylation of JNK. Each experiment was repeated three times with similar results. P-p38, phosphorylated p38.
Figure 4.
Figure 4.
Hypothetical scheme for the role of IRAK-M in the differentiation and activation of osteoclasts. Ligation of c-Fms (1) stimulates the production of IL-1, which activates IL-1R. Ligation of RANK (2) leads to TRAF6-mediated activation of NF-κB (3), and induction of expression of IRAK-M, (4) IL-1 activates IL-1R (5), which stimulates osteoclastogenesis and the osteoclastic resorption of bone, and extends the half-life of osteoclasts. When IRAK-M is present, it prevents IL-1R/TLR-mediated downstream signaling and the activation of NF-κB. Thus, IRAK-M mediates a negative feedback mechanism that interrupts signaling downstream of IL-1R. In the absence of IRAK-M, the IL-1 cycle continues without interruption, leading to increased osteoclastogenesis, with the hyperactivation and prolonged half-life of osteoclasts.

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