Cherubism Mice Also Deficient in c-Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts

Abstract

Currently, it is believed that osteoclasts positive for tartrate-resistant acid phosphatase (TRAP+) are the exclusive bone-resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2^KI/KI ) with a homozygous gain-of-function mutation in the SH3-domain binding protein 2 (SH3BP2) was shown to develop auto-inflammatory joint destruction. Here, we demonstrate that Sh3bp2^KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c-Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C-terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross-linked C-telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2^KI/KI mice deficient in c-Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c-Fos-deficient Sh3bp2^KI/KI mice. After activation of the NF-κB pathway, macrophage colony-stimulating factor (M-CSF)-dependent macrophages from c-Fos-deficient Sh3bp2^KI/KI mice expressed increased amounts of MMP14 compared with wild-type macrophages. Interestingly, receptor activator of NF-κB ligand (RANKL)-deficient Sh3bp2^KI/KI mice failed to show notable bone erosion, whereas c-Fos deletion did restore bone erosion to the RANKL-deficient Sh3bp2^KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss-of-function of c-Fos and gain-of-function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in inflammatory bone disease and suggest that MMP14 is a key mediator conferring pathological bone-resorbing capacity on c-Fos-deficient Sh3bp2^KI/KI macrophages. In summary, the paradigm that osteoclasts are the exclusive cells executing inflammatory bone destruction may need to be reevaluated based on our findings with c-Fos-deficient cherubism mice lacking osteoclasts. © 2017 American Society for Bone and Mineral Research.

Keywords: C-FOS; CHERUBISM; MMP14; NON-CANONICAL OSTEOCLASTS/OSTEOLYTIC MACROPHAGES; OSTEOCLAST-INDEPENDENT INFLAMMATORY BONE DESTRUCTION; SH3BP2.

Fig. 1. c-Fos-deficient Sh3bp2^KI/KI mice develop inflammatory bone erosion

(A) μCT images of the ankle joint at 12 weeks old. (B) Quantitative measurement of eroded bone surface area at the distal tibia. (C) In vivo μCT images of the distal tibia from the same c-Fos^−/− Sh3bp2^KI/KI mouse at different ages. Arrows indicate a location of bone erosion that appeared after 12 weeks old. (D) H&E staining of the distal tibia showing inflammatory lesions invading into marrow spaces (arrow). 12 weeks old. Bar = 200 μm. (E) Serum TNF-α levels quantitated by ELISA at 12 weeks old. (F) qPCR analysis of Tnf mRNA expression levels in ankle joint tissue at 12 weeks old. Average expression level in wild-type mice is set as 1. (G) Histomorphometric analysis of liver lesions in 12-week-old mice. (H) Transmitted electron microscope images on the surface of the distal tibia. 12 weeks old. Arrow and arrowhead indicate collagen fibers and ruffled border, respectively. Bar = 5 μm. Data are presented as mean ± SD. *p < 0.05. ANOVA with Tukey-Kramer post-hoc test. NS = not significant.

Fig. 2. c-Fos-deficient Sh3bp2^KI/KI mice develop no TRAP+ osteoclasts

(A) TRAP staining of inflammatory lesions at the distal tibia. 12 weeks old. Bar = 100 μm. (B) ELISA of serum TRAP5b at 12 weeks old. (C) qPCR analysis of osteoclast-associated genes in ankle joint tissue at 12 weeks old. Average expression levels in wild-type mice are set as 1. Data are presented as mean ± SD. *p < 0.05. ANOVA with Tukey-Kramer post-hoc test. NS = not significant. #: p < 0.05 with two-tailed t-test (B, C).

Fig. 3. M-CSF-dependent macrophages from c-Fos-deficient Sh3bp2^KI/KI mice fail to differentiate to TRAP+ osteoclasts

(A) TRAP staining of M-CSF-dependent macrophages from fetal liver cultured in the presence of RANKL or RANKL/TNF-α for 5 days. Bar = 100 μm. (B) Number of TRAP+ multinucleated cells per well of cultures in (A). n = 6. (C) qPCR analysis of osteoclast-associated genes in fetal liver-derived M-CSF-dependent macrophages stimulated with RANKL or RANKL/TNF-α. Average expression levels in wild-type mice at 0 hour are set as 1. n = 3. (D) Phalloidin staining of actin rings. Fetal liver-derived M-CSF-dependent macrophages were stimulated with RANKL/TNF-α for 5 days. Bar = 100 μm. (E) Western blotting analysis of NFATc1 with cell lysates from fetal liver-derived M-CSF-dependent macrophages cultured in the presence of RANKL or RANKL/TNF-α. Data are presented as mean ± SD. *p < 0.05 vs. c-Fos-sufficient mice. ANOVA with Tukey-Kramer post-hoc test.

Fig. 4. MMP2 and MMP14 expression levels are increased in joint tissue of c-Fos-deficient Sh3bp2^KI/KI mice

(A) ELISA for serum ICTP at 12 weeks old. (B) ELISA for serum CTX at 12 weeks old. (C) qPCR analysis of collagenotylic MMPs in joint tissue at 12 weeks old. Mmp1 expression was not analyzable due to low expression levels in both SYBR green and Taqman assays (Supplemental Table 1). Average expression levels in wild-type mice are set as 1. (D) Western blotting analysis for MMP2 and MMP14 with cell lysates from the ankle joints. (E, F) Immunohistochemical staining of MMP2 and MMP14 with tissue sections of the distal tibia. Bar = 100 μm. Data are presented as mean ± SD. *p < 0.05 with two-tailed t-test (A, B) or ANOVA with Tukey-Kramer post-hoc test (C). NS = not significant. #: p < 0.05 with two-tailed t-test.

Fig. 5. Macrophages expressing MMP14 are responsible for bone erosion in c-Fos-deficient Sh3bp2^KI/KI mice

(A) Immunohistochemical staining of inflammatory lesions at the distal tibia from c-Fos-deficient Sh3bp2^KI/KI mouse with anti-F4/80 antibody. 12 weeks old. Bar = 100 μm. (B) qPCR analysis of macrophage marker gene expression in the ankle joints. (C) μCT images of the distal tibia at 12 weeks old. (D) Quantitative measurement of eroded bone surface area at the distal tibia. (E) qPCR analysis of Tnf expression in the ankle joints. (F) qPCR analysis of Mmp2 and Mmp14 expression in the ankle joints. (G) Western blotting analysis of MMP2 and MMP14 with cell lysates from the ankle joints. (H) μCT images of the distal tibia from Sh3bp2^KI/KI mice treated with or without an MMP14 inhibitor NSC405020 (50 mg/kg/day) from 4 to 10 weeks old. (I) Quantitative measurement of eroded bone surface area at the distal tibia of Sh3bp2^KI/KI mice treated with or without NSC405020 from 4 to 10 weeks old. (J) Comparison of Mmp14 expression levels in fetal liver-derived M-CSF-dependent macrophages by qPCR. n = 3. (K) Measurement of collagenolytic activity of fetal liver-derived M-CSF-dependent macrophages. n = 6. (L) Measurement of collagenolytic activity of fetal liver-derived M-CSF-dependent macrophages in the presence or absence of NSC405020. n = 6. (M) Measurement of mineral resorption capacity of fetal liver-derived M-CSF-dependent macrophages. n = 6. Macrophages were cultured for 7 days on the plates coated with europium-labeled type I collagen fibers (K, L) or on the plates coated with fluoresceinamine-labeled calcium phosphate (M). Average expression levels in wild-type mice are set as 1 (B, E, F). Average expression level in wild-type mice at day 2 is set as 1 (J). Data are presented as mean ± SD. *p < 0.05 with two-tailed t-test (D, E, F, I) or ANOVA with Tukey-Kramer post-hoc test (B, J, K, L, M). NS = not significant.

Fig. 6. NF-κB pathway is responsible for increased MMP14 expression in c-Fos-deficient Sh3bp2^KI/KI macrophages

(A) Comparison of Mmp14 expression levels in fetal liver-derived M-CSF-dependent macrophages by qPCR. Non-adherent fetal liver cells were cultured with M-CSF for 2 days, then further cultured with M-CSF in the presence or absence of BMS-345541 for 2 days. Average expression level in wild-type mice with no inhibitor is set as 1. n = 3. Data are representative of three independent experiments. (B) Western blotting analysis of MMP14 in M-CSF-dependent macrophages treated with or without BMS-345541. Non-adherent fetal liver cells were cultured with M-CSF for 2 days, then further cultured with M-CSF in the presence or absence of BMS-345541 for 4 days (day 3–6). (C) Western blotting analysis of phosphorylated IKKα/β. Non-adherent fetal liver cells were cultured with M-CSF for 4 days, then M-CSF-dependent macrophages were starved for serum and M-CSF for 6 hours followed by re-stimulation with M-CSF (25 ng/ml). *p < 0.05 decreased vs. 0 μM in each group. ANOVA with Tukey-Kramer post-hoc test.

Fig. 7. Both c-Fos deficiency and SH3BP2 gain-of-function are required for osteolytic transformation of macrophages

(A, E) H&E staining of the distal tibia showing inflammatory lesions (arrows). 12 weeks old. Bar = 200 μm. (B, F) TRAP staining images of inflammatory lesions at the distal tibia at 12 weeks old. Bar = 200 μm. (C, G) μCT images of the distal tibia at 12 weeks old. (D, H) Quantitative measurement of eroded bone surface area at the distal tibia. (I) Mmp2 and Mmp14 expression levels in the ankle joints. Average expression levels in wild-type mice are set as 1. (J) Western blotting analysis of MMP2 and MMP14 with cell lysates from ankle joint tissue. Data are presented as mean ± SD. *p < 0.05. ANOVA with Tukey-Kramer post-hoc test. NS = not significant.