RIP140 in monocytes/macrophages regulates osteoclast differentiation and bone homeostasis

Abstract

Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage-specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases.

Figure 1. Femoral and vertebral trabecular bone is significantly decreased in mice with reduced RIP140 expression.

(A) Representative μCT images of the femoral diaphysis and metaphysis illustrating differences in cancellous bone volume fraction in 9-week-old male WT and monocyte/macrophage–specific RIP140-knockdown (mϕRIP140KD) mice. (B) μCT analysis of cortical bone in the femur diaphysis: cross-sectional volume, cortical volume, marrow volume, cortical thickness, and polar moment of inertia. (C) μCT analysis of cancellous bone in the distal femur metaphysis: bone volume fraction; connectivity density; trabecular number, thickness, and spacing. (D) μCT analysis of cancellous bone in 5th lumbar (L5) vertebra: bone volume fraction; connectivity density; trabecular number, thickness, and spacing. Data are presented as means ± SD. *Denotes significance at P < 0.05, n = 8–10/group.

Figure 2. Loss of RIP140 results in increased osteoclast activity, reduced osteoblast activity, and reduced bone formation.

(A) Photomicrographs illustrating differences in fluorochrome label incorporation (left) and osteoclast/osteoblast perimeters (right) in 9-week-old-male WT and monocyte/macrophage–specific RIP140-knockdown (mϕRIP140KD) mice. Scale bar: 50 μm. (B) Indices of bone formation (mineralizing perimeter, bone formation rate, mineral apposition rate, osteoblast perimeter), and bone resorption (osteoclast perimeter) in control and mϕRIP140KD mice at 9 weeks of age. Data are presented as mean ± SD. *Denotes significance at P < 0.05, n = 9–10/group. (C) Tartrate-resistant acid phosphatase (TRAP) activity in bone marrow–derived osteoclasts from WT and mϕRIP140KD mice (n = 3 mice/group) after RANKL and M-CSF treatment for 5 days (left). Alkaline phosphatase (ALP) activity in serum samples from WT and mϕRIP140KD mice (n = 9/group). Data are representative of 3 experimental repeats (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

Figure 3. Loss of RIP140 in osteoclasts results in increased osteoclast differentiation and reduced osteoblast markers.

(A) Representative images and results of tartrate-resistant acid phosphatase (TRAP)–stained osteoclast (OC) differentiated from bone marrow from WT and monocyte/macrophage–specific RIP140-knockdown (mϕRIP140KD) mice (n = 3) after 5 days of differentiation with RANKL and M-CSF treatment. Cell quantification data were normalized to WT values and are representative of 3 experimental repeats (mean ± SD), n = 3–4. *P < 0.05 by Student’s t test. Scale bar: 0.1 mm. (B) qRT-PCR analyses of osteoblast markers in primary osteoblasts incubated with conditioned medium of osteoclasts differentiated (5 days) from bone marrow cells in WT and mϕRIP140KD (n = 4 mice/group). Primary osteoblasts derived from mouse long bone (femur and tibia) were incubated with conditioned medium from osteoclasts cultured with or without 50 μg/ml ascorbic acid (AA) treatment for 3 days. qRT-PCR data are representative of 3 experimental repeats (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. Additional statistical significance shown in B was determined by 2-way ANOVA.

Figure 4. TR4/RIP140 complex formation and repressive activity in osteoclast differentiation.

(A) Silver staining of FLAG-RIP140–associated proteins in RAW264.7 cells. (B) Western blot analysis of RIP140/TR4 complex in RAW264.7 cells. (C) Coimmunoprecipitation (IP) of FLAG-RIP140/HA-TR4 from HEK293T. (D) qRT-PCR and Western analyses of TR4 after RANKL stimulation for 2 days. (E) qRT-PCR analysis of osteoclast marker genes in control (GFP KD) versus TR4-knockdown (TR4 KD) RAW264.7 cells with or without RANKL treatment for 24 hours. Additional statistical significance shown in E was determined by 2-way ANOVA. (F) ChIP assay of TR4 on osteoclast marker gene promoters in RAW264.7 cells treated with RANKL stimulation for 30 minutes. RIP140 overexpression was induced by doxycycline pretreatment (250 ng/ml) for 6 hours. Additional statistical significance was determined by 1-way ANOVA (Nfatc1 promoter P = 0.0014, Ctsk promoter P < 0.0001, Acp5 promoter P < 0.0001). Data are representative of 3 experimental repeats (mean ± SD). For all graphs, Student’s t test (n = 3) was used unless otherwise specified. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. RIP140 represses osteoclast differentiation and is degraded by RANKL treatment in osteoclast differentiation.

(A) qRT-PCR analyses of osteoclast markers in control (FLAG) or FLAG-RIP140–overexpressing RAW264.7 cells treated with RANKL for 2 days. (B) Western blot analyses of RIP140 in RAW264.7 cells treated with RANKL for indicated time periods with or without Syk (nonreceptor tyrosine kinase) inhibitor pretreatment. (C) qRT-PCR analyses of RIP140 in RAW264.7 cells with or without RANKL treatment for 4 days. NS, not significant. (D) qRT-PCR analyses of osteoclast markers in RAW264.7 cells treated with RANKL for 2 days with or without Syk inhibitor pretreatment. (E) qRT-PCR analyses. Bone marrow–derived osteoclasts were differentiated with M-CSF and RANKL (RK+M-CSF) for 3 days following transduction with lentiviruses carrying control (Ctrl), WT RIP140, or nondegradable mutant RIP140 (Y3F). Data are representative of 3 experimental repeats (mean ± SD). For all graphs, Student’s t test (n = 3) was used unless otherwise specified. *P < 0.05, **P < 0.01, ***P < 0.001. Additional statistical significance shown in A, D, and E was determined by 2-way ANOVA.

Figure 6. RIP140 suppresses RANKL signaling and transcription activation of osteoclastogenic genes.

(A) Western blot analyses of Flag-HA-RIP140 and MAPK/NF-κB signaling components in whole-cell lysates from control versus RIP140-overexpressing RAW264.7 cells. RIP140 overexpression was induced by doxycycline for 16 hours and cells were treated with RANKL at indicated times. (B) Tartrate-resistant acid phosphatase (TRAP) activity in doxycycline-induced (Dox) RIP140-expressing RAW264.7 cells. Statistical significance was determined with Student’s t test (n = 3), and each treated sample was compared to unstimulated one. (C) ChIP assay of HA-RIP140, TR4, NFATc1, PU.1, H3Ac, and H3K9me3 on the Nfatc1 promoter in doxycycline-induced RIP140-expressing RAW264.7 cells after RANKL treatment (0.5 hours for H3Ac and H3K9me3, 6–24 hours for others). Additional statistical significance in C was determined by 2-way ANOVA. Data are representative of 3 experimental repeats (mean ± SD). For all graphs, Student’s t test (n = 3) was used unless otherwise specified. *P < 0.05, **P < 0.01, ***P < 0.001.