CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

Abstract

Objective

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling.

Methods

The bone phenotype of Chip^−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays.

Results

In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip^−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription.

Conclusion

CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases.

Bone tissue is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts (1–3). Abnormal osteoclastogenesis may result in bone disorders, such as osteoporosis, Paget’s disease and rheumatoid arthritis. Thus, the understanding of the mechanism of osteoclastogenesis is important for rational design of therapeutic approaches for the treatment of bone disorders. However, the regulatory mechanism of osteoclastogenesis is not fully understood. Nuclear factor of κB (NF-κB) plays a key role in osteoclast formation and bone resorption. During osteoclast differentiation, NF-κB is activated by RANKL, after RANKL association with its receptor, it initiates different cascades during activation of NF-κB pathways. A key event in NF-κB pathways is the activation of TRAF6, which functions as an E3 ligase. Once activated, TRAF6 synthesizes lysine 63-sepecific polyubiquitin chains leading to the activation of IKKβ (I-κB kinase), which phosphorylates I-κBα (the inhibitor of NF-κB), leading to its degradation. The degradation of I-κBα releases NF-κB from the I-κBα/NF-κB complex and allows NF-κB to enter the nucleus where it activates gene expression required for osteoclast formation (4–5). The importance of NF-κB in osteoclastogenesis has been highlighted by several mouse models. Mice lacking RANK, TRAF6, IKKβ and RelA/p65 (a subunit of NF-κB), lead to increased bone mass with absence of osteoclasts (6–9). Therefore, the TRAF6-NF-κB axis appears to be the critical molecular basis for the pathogenesis of abnormal osteoclastogenesis and bone diseases.

Recent studies suggest that TRAF6 is critical in osteoclast formation and bone remodeling. For instance, TRAF6 interacts with RANK, while RANKL binds to RANK, to activate NF-κB signaling which in turn regulates osteoclastogenesis (2, 10–12). Moreover, TRAF6 knockout (KO) mice develop severe osteopetrosis and failure of bone resorption due to impaired RANK signaling pathway (7, 13). The suppressor of RANK signaling pathway, FHL2, inhibits the activated osteoclasts through blocking RANK/TRAF6 interaction. FHL2^−/− osteoclasts are hyper-resorptive and FHL2^−/− mice undergo RANKL-stimulated bone loss (14). Collectively, TRAF6 functions as a major adaptor molecule for RANK signaling pathway associated with osteoclastogenesis. To date, several factors have been identified to regulate TRAF6, including deubiquitination enzymes, such as A20, CYLD and USP4 (15–17). In contrast, three E3 ligases, including Smurf1, NUMBL and TRIM38, have been reported to regulate TRAF6 protein ubiquitination and degradation in vitro (18–20). However, the importance of TRAF6 regulation in osteoclast formation in vivo and detail molecular mechanism of TRAF6 degradation remain undefined.

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is a U-box containing protein that interacts with Hsp70 and functions as an E3 ligase for several protein substrates. CHIP induces the ubiquitination of proteins, such as p53, Smads, ER-α, Runx2, Src-3, p65 and TLR4 for proteasome-dependent degradation (20–27). Although it is known that CHIP plays a critical role in immunology and in tumor growth and metastasis, the role of CHIP in skeletal growth and bone remodeling in vivo has not been reported. In this study, we report the role of CHIP in TRAF6 degradation and in regulation of bone mass.

Materials and Methods

Mice

Chip^−/− mice were obtained from National Institutes of Health. The first three coding exons were targeted by homologous recombination (NCBI accession: NM019719). Both wild-type and Chip^−/− mice were maintained in a C57BL/6 and 129SvEv background.

Plasmids and reagents

pCMV/Myc-TRAF6 was gifted from Dr. Lingqiang Zhang (State Key Laboratory of Proteomics, Beijing, China). pCMV/Flag-TRAF6 was kindly provided by Dr. Hiroyasu Nakano (Juntendo University, Tokyo, Japan). pEFNeo/HA-CHIP, pRKIM/Myc-CHIP, pRKIM/Myc-CHIP (K30A) and pRKIM/Myc-CHIP (H260Q), pGEX-5X-3/CHIP and-His-Ub were same as previously used (21).

Antibody and reagent

Phospho-IKKα/β (Ser176/180), I-κBα, phosphor-I-κBα (Ser32) and anti-α-tubulin (3873p) were purchased from Cell Signaling Technology (Beverly, MA). Anti-TRAF6 (H-274), anti-p65 (C-20), anti-ub (P4D1) and Sp1 (sc-59-G) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CHIP antibody was developed in our lab (21). MG132 was purchased from Millipore (Billerica, MA). Cyclohexanone (CHX) was purchased from Amresco (AMRESCO Inc., OH).

Cell culture

HEK293T and RAW264.7 cells were cultured in DMEM supplemented with 10% FBS at 37°C under 5% CO₂. For proteasome inhibition assays, cells were treated with MG132 (12.5 μM) 6 h before cells were harvested.

Immunoprecipitation, Immunostaining and Western blotting

Immunoprecipitation, immunostaining and western blotting were performed as previously described (21).

GST-pull down assay

GST and GST-CHIP proteins were expressed in the E. Coli strain BL21 and purified by affinity chromatography. Myc-TRAF6 expressed in HEK293T cells was first immunoprecipitated by anti-myc antibody and then incubated with purified GST-CHIP or GST protein for 4 h and then with Glutathione Sepharose beads for another 2 h at 4°C. Beads were washed with cell lysis buffer and the precipitant was analyzed by western blotting.

Real-time PCR

Total RNA was isolated from cells using Trizol reagent (Invitrogen). Reverse transcription was done using Quantscript RT Kit (Bio-rad laboratorie Inc, California, USA). Real time PCR was performed using SYBR Green kit (Bio-rad laboratorie Inc, California, USA) and carried out on a Bio-Rad iCycler. The primers for the genes listed in supplemental table 1. The relative expression level of genes was analyzed using ΔΔCt method. All the experiments were performed in triplicate.

Luciferase assay

RAW.264.7 cells were transiently transfected with the indicated plasmids using FuGENE6 (Promega, IN). Briefly, 0.2 μg pGL3/NF-κB-luc reporter and 10 ng pRL-TK were transfected into the cells cultured in a 24-well plate. Luciferase activity was assayed 24 hrs after transfection using a Dual-Luciferase reporter assay system (Promega, IN). The luciferase activity was normalized against renilla luciferase activity and presented as mean ± standard deviation (SD) (27).

Nuclear and cytoplasm extraction

Nuclear and cytoplasmic extraction was performed with the NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce biotechnology, IL), according to the manufacture’s instruction. Cell lysates were analyzed by immunoblotting for the indicated proteins.

Histology

Tibiae from 1-month-old male and female wild-type and Chip^−/− mice were fixed in 10% formalin for 3 days, decalcified in 14% EDTA for 14 days and embedded in paraffin. Serial midsagittal sections (3-μm thick) were cut and stained with H&E and TRAP staining (28–29).

Micro-computed tomography (μCT)

Femurs of 1-month-old female wild-type and Chip^−/− mice were subjected for analysis of changes in bone structure using a Scanco VivaCT 40 cone-beam scanner (Scanco Medical AG, Bassersdorf, Switzerland). Briefly, scanning in the femurs began approximately at the lower growth plate and extended proximally for 350 slices (10 μm thick for each slice). Morphometric analysis was performed on 100 slices extending proximally, beginning with the first slice in which the tibial condyles had fully merged. The trabecular bone was segmented from the cortical shell manually on key slices using a contouring tool, and the contours were morphed automatically to segment the trabecular bone on all slices. The three-dimensional structure and morphometry were reconstructed and analyzed (29).

In vitro osteoclast differentiation assay

Bone marrow (BM) cells were isolated from femur and tibia of 1-month-old wild-type and Chip knockout mice. Non-adherent bone marrow macrophages were isolated from total bone marrow cells after 48 h culture. BM cells were cultured with DMEM supplemented with 10% FBS and treated with M-CSF (10 ng/ml) for 3 days and then switched to the differentiation medium (M-CSF 10 ng/ml and RANKL 50 ng/ml) for 7 days. TRAP staining was performed and the numbers of TRAP-positive multinucleated cells (MNCs: defined as having 3 or more nuclei per cell) were measured (29–31).

Bone resorption assay

Bone marrow cells were seeded at 1 x 10⁴ cells per well in 200 μl DMEM medium containing 50 ng/ml RANKL and 10 ng/ml MCSF in Corning Osteo Assay surface 96-well plates (Corning Life Science, 3988). The condition medium was changed every 3 days, and cells were incubated for 7 days. Then cells were removed by 10% bleach solution and the plates were airdried prior to imaging.

Results

Chip KO mice develop osteopenic phenotype

We observed that both male and female Chip KO mice had much smaller size and lower body weight than their wild-type (WT) littermates (Figure 1A–D). 1-month-old female Chip^+/−, Chip^−/− mice and WT littermates were subjected to X-ray radiographic analysis. The results showed that long bones and vertebral bodies of Chip^−/− mice were more radiolucent and reduced in length compared to Chip^+/− and WT littermates (Figure 1E). The long bones of Chip^−/− mice showed a reduced bone density (Figure 1F). We then analyzed femurs of 1-month-old female WT and Chip^−/− mice by μCT. The Chip^−/− mice exhibited significant loss of trabecular bone (Figure 1G). Consistently, analysis of trabecular architecture by μCT showed that Chip^−/− mice had a significant reduction in bone volume (%, BV/TV), bone mineral density (BMD), trabecular number (Tb.N.) and trabecular thickness (Tb.Th.) (Figure 1H–K). In contrast, trabecular separation (Tb.Sp.) was increased (Figure 1L). Structure model index (SMI) is scored from 1 to 3 for indication of increased fragility. Higher SMI numbers and lower connectivity density were observed in Chip^−/− mice, suggesting more fragile bone in Chip^−/− mice (Figure 1M, N). Furthermore, μCT analysis of femurs also demonstrated that cortical BV and cortical BMD were significantly decreased in Chip^−/− mice (Figure 1O–Q). Collectively, these results suggest that loss of Chip in mice develops bone loss phenotype.

Figure 1. Chip KO mice develop osteopenic phenotype.

(A, C) Gross appearance of WT and Chip^−/− mice. The ruler indicates size in centimeters. Chip^−/− mice have smaller size than WT mice. (B, D) The bar graphs represent mean body weight, showing Chip^−/− mice have reduced body weight compared to WT littermates (B, Male: WT, n=14, Chip^−/−, n=9; D, Female: WT, n=7, Chip^−/−, n=3, *p<0.01). (E, F) X-ray radiographic analysis of the bone density of 1-month-old female WT (^+/+), Chip heterozygosis (^+/−) and Chip KO (^−/−) mice. (G) μCT scanning of the skeletons of mice. 1-mon-old female WT and Chip^−/− mice were subjected to μCT analysis. Representative images of 3D μCT reconstruction of femurs showed the severe bone loss in Chip^−/− mice. (H–N) Parameters of trabecular bone, including bone volume (BV/TV, %), bone mineral density (BMD), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), structure model index (SMI) and connectivity density (Conn.D.). (O–Q) Parameters of cortical bone mass, including bone volume (BV/TV, %) and bone mineral density (BMD) were decreased in Chip^−/− mice. *p<0.05 versus WT. Scale bar: 200 μm.

Osteoclast formation and TRAF2/TRAF6 protein levels are increased in Chip KO mice

Similar to the μCT analysis, the results in H&E staining of tibial sections showed a significant reduction in trabecular bone in Chip^−/− mice than that in WT littermates (Figure 2A). TRAP staining of tibiae revealed that TRAP-positive osteoclasts were significantly increased in Chip^−/− mice (Figure 2B). Histomorphometric analysis showed that osteoclast numbers and osteoclast surfaces were increased in Chip^−/− mice (Figure 2C and D). These results suggest that one of the major reasons for osteopenic phenotype observed in Chip^−/− mice might be due to increased osteoclast formation. To further confirm the effect of Chip deficiency on osteoclast formation, bone marrow cells derived from WT and Chip^−/− mice were cultured with M-CSF and RANKL for 7 days, followed by TRAP staining. The result showed that there were more osteoclasts in Chip^−/− mice and the size of osteoclasts is larger in Chip^−/− mice compared to WT mice (Figure 2E and F). In addition, we also found that osteoclastic bone resorption was also significantly increased in cultures of bone marrow cells derived from Chip^−/− mice (Figure 2G). As expected, the expression of osteoclast marker genes, including Cathepsin K, Mmp9, Trap and Nfatc1, was significantly increased after RANKL treatment in BM cells derived from Chip^−/− mice compared to those from WT mice (Figure 2H–K).

Figure 2. Osteoclast formation and TRAF6 protein levels are increased in Chip^−/− mice.

(A) Representative images of H&E-stained sections of tibiae from 1-month-old male WT and Chip^−/− mice. (B) TRAP staining was performed on tibial sections. Increased TRAP-positive osteoclasts were found in Chip^−/− mice. (C and D) Histomorphometric analyses of TRAP staining showed that osteoclast numbers and osteoclast surfaces were significantly increased in 1-month-old male and female Chip^−/− mice (each group n≥3). (E, F) Bone marrow macrophages derived from 1-month-old male and female WT and Chip^−/− mice were cultured in the presence of M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 7 days, followed by TRAP staining. The numbers of multinucleated TRAP-positive osteoclasts increased (MNCs: defined as having 3 or more nuclei per cell). (G) Bone marrow macrophages were cultured on Corning Osteo Assay surface plates in the presence of M-CSF and RANKL for 7 days. Ostoeclastic bone resorption (white area) was significantly increased in Chip^−/− bone marrow cells. (H–K) mRNA levels of osteoclast marker genes were examined in bone marrow cells from WT and Chip^−/− mice after stimulation with RANKL.

CHIP promotes TRAF6 ubiquitination and degradation

CHIP has been reported to function as an E3 ligase and mediates the ubiquitination and proteasome degradation of several protein substrates (21, 24–25, 32–33). To determine the mechanism of CHIP-regulated osteoclastogenesis, we examined changes in steady-state protein levels of p65 (33) and TRAF2 (32) which are known to be targeted by CHIP and are involved in osteoclast formation. In addition, we also examined changes in TRAF6 which belongs to the TRAF family and plays a critical role in RANK-induced osteoclast formation. Among these candidate proteins, TRAF2 and TRAF6 protein levels were significantly increased, while the mRNA levels were not changed in RAKNL-induced Chip^−/− osteoclasts (Figure 3A–C). No change in protein levels for p65 (Figure 3A). Therefore, TRAF6 appears to be a novel substrate of CHIP which may regulates osteoclastogenesis. To further confirm the effect of CHIP on TRAF6 protein levels, we performed western blot analysis and found that the protein levels of TRAF6 were decreased when CHIP expression was increased in 293T cells. In contrast, the mutant CHIP (H260Q) which lacks the E3 ligase activity had no effect on TRAF6 protein levels (Figure 3D). In a parallel experiment, we observed that decreased TRAF6 protein levels were partially rescued by proteasome inhibitor MG132 (Figure 3E), suggesting that CHIP-mediated TRAF6 degradation is proteasome-dependent.

Figure 3. CHIP promotes TRAF6 ubiquitination and proteasome degradation.

(A) TRAF6 and TRAF2 protein levels increased in Chip^−/− bone marrow macrophage after treatment with RANKL for 6 days. (B, C) No changes in mRNA levels of TRAF6 and TRAF2 were found. (D) Myc-TRAF6 was co-transfected with increasing concentrations of Myc-CHIP or Myc-CHIP H260Q. WT but not H260Q CHIP promotes TRAF6 degradation. (E) Myc-TRAF6 and Myc-CHIP were co-transfected into 293T cells treated with MG132. MG132 inhibited CHIP-induced TRAF6 degradation. (F, G) 293T cells were transfected with the indicated plasmids and treated with CHX for described time periods. CHIP acceleratesTRAF6 turnover. (H, I) WT and Chip^−/− bone marrow cells were treated with CHX after RANKL induction and harvested at described time points. Chip deficiency stabilized TRAF6 protein. (J) Flag-TRAF6 and His-ubiquitin were co-transfected with increasing amounts of Myc-CHIP in 293T cells. Total ubiquitinated proteins were precipitated by Ni-NTA resin followed by immunoblotting with the anti-Flag antibody. CHIP facilitates TRAF6 ubiquitination. (K) Purified Flag-TRAF6, E1, E2 were incubated with bacteria-expressed His-CHIP and His-Ub for ubiquitination assays. CHIP facilitates TRAF6 ubiquitination in vitro. (L) WT and Chip^−/− bone marrow cells were immunoprecipitated with the anti-Ub antibody followed by immunoblotting with the anti-TRAF6 antibody. Chip deficiency decreases TRAF6 ubiquitination.

We then determined if CHIP affects the half-life of TRAF6 protein. Protein translation was inhibited by cycloheximide (CHX). The results showed that the half-life of TRAF6 protein was greatly reduced by WT CHIP but not by the mutant CHIP (H260Q) (Figure 3F and G). We next examined the stability of TRAF6 protein in RANKL-induced osteoclasts. The steady-stably protein levels of TRAF6 were markedly increased in Chip^−/− osteoclasts (Figure 3H and I). These results suggest that CHIP negatively regulates the stability of TRAF6.

We also examined whether CHIP regulates TRAF6 protein levels through a ubiquitin-dependent mechanism. Flag-TRAF6 and His-ubiquitin were transfected with Myc-CHIP into 293T cells. Ubiquitinated proteins were precipitated from cell lysates under a denatured condition and analyzed by immunoblotting with the anti-Flag antibody. The results showed that the ubiquitination of TRAF6 was markedly increased when CHIP was expressed in a dose-dependent manner (Figure 3J). Next, we determined if CHIP directly promotes TRAF6 ubiquitination, we reconstituted the ubiquitination in vitro using purified E1 and UbcH5a and bacteria-expressed CHIP protein. We detected auto-ubiquitination of TRAF6 (Figure 3K, lane 2). Addition of CHIP augmented the ubiquitination of TRAF6 (Figure 3K, lane 3, 4). To further determine whether CHIP mediates ubiquitination of endogenous TRAF6, we detected ubiquitination of TRAF6 in wild-type and Chip^−/− bone marrow cells. The result showed that ubiquitinated TRAF6 was immunoprecipitated by the anti-ubiquitin antibody from lysates of WT cells (Figure 3L, lane2) and the TRAF6 ubiquitination was decreased in Chip^−/− cells (Figure 3L, last lane). These results suggest that CHIP mediates TRAF6 ubiquitination.

CHIP interacts with TRAF6

Next, we examined whether CHIP directly interacts with TRAF6 by IP assay. The results demonstrated that CHIP was precipitated by Flag-TRAF6 in 293T cells (Figure 4A). Reciprocally, we observed that TRAF6 was also precipitated by Myc-CHIP (Figure 4B). Meanwhile, the result showed that TRAF6 was immunoprecipitated by either WT or H260Q mutant CHIP, but not by K30A mutant CHIP which lacks chaperone binding ability (34) (Figure 4B). These results demonstrate that the residue K30 of CHIP is required for the interaction of CHIP with TRAF6. It appears that there is a direct interaction between CHIP and TRAF6 since TRAF6 was specifically precipitated by purified GST-CHIP in vitro (Figure 4C). Finally, we further examined whether CHIP interacts with endogenous TRAF6. The results showed that CHIP was precipitated by endogenous TRAF6 in bone marrow cells, while normal mouse IgG failed to do so (Figure 4D). Reciprocally, an antibody against CHIP could pull down TRAF6 protein from the same cells (Figure 4E). These results indicate that CHIP interacts with TRAF6 both in vitro and in vivo.

Figure 4. CHIP interacts with TRAF6.

(A) Myc-CHIP was immunoprecipitated by Flag-TRAF6. Myc-CHIP and Flag-TRAF6 were co-transfected into 293T cells. Cell lysates were incubated with the anti-Flag antibody and immunoprecipitates were subjected to western blotting. (B) TRAF6 was immunoprecipitated by WT and K30A mutant CHIP, but not H260Q mutant CHIP. 293T cells were co-transfected with Flag-TRAF6, Myc-CHIP, Myc-CHIP K30A as well as Myc-CHIP H260Q. Cells lysates were immunoprecipitated by the anti-Myc antibody. (C) CHIP interacted with TRAF6 in vitro. 293T cell lysats expressing Myc-TRAF6 was first immunoprecipitated by anti-Myc antibody and then incubated with purified GST-CHIP protein. (D, E) CHIP interacts with TRAF6 in vivo. The cell lysates of bone marrow macrophage cells were incubated with the anti-TRAF6 (D) or anti-CHIP (E) antibody, respectively.

CHIP suppresses TRAF6-mediated NF-κB signaling pathway

Since CHIP interacts with TRAF6 and promotes its degradation, we reasoned that CHIP might affect IKK-β and I-κBα phosphorylation and p65 nuclear translocation. To test this hypothesis, we examined changes in IKK-β activity and I-κBα phosphorylation upon RANKL stimulation. The results showed that the phosphorylation of IKK-β and I-κBα were markedly increased in bone marrow cells derived from Chip ^−/− mice compared to that from WT mice (Figure 5A). Meanwhile, the total I-κBα protein levels were decreased in Chip ^−/− bone marrow cells. This is probably because the phosporylation-induced I-κBα degradation is increased after RANKL stimulation in Chip^−/− cells. These results suggest that NF-κB signaling was constitutively activated in Chip^−/− Bone marrow cells after treatment with RANKL.

Figure 5. CHIP suppresses TRAF6-mediated NF-κB signaling pathway.

(A) Bone marrow cells derived from 1-month-old male and female WT and Chip^−/− mice were cultured under serum starvation condition overnight and then stimulated with 100ng/ml RANKL for the indicated time periods. The levels of endogenous and phosphorylated proteins were examined. Chip deficiency increases the phosphorylation of IKK-β and I-κBα. (B) After treatment with RANKL (100ng/ml) in WT and Chip^−/− bone marrow cells, p65 protein levels in the cytoplasm/nucleus were detected. Deletion of Chip enhances p65 nuclear translocation. (C) NF-κB luciferase reporter was transfected with HA-CHIP or empty vectors into RAW264.7 cells which were either treated with RANKL or transfected with TRAF6 expression plasmids. Over-expression of CHIP significantly inhibited RANKL or TRAF6-induced NF-κB activity (*p<0.05, n=3).

To further confirm the effect of CHIP on NF-κB signaling, we performed western blot analysis using the fractions of the cytoplasm and nuclear proteins to examine changes of p65 localization. The results showed that deletion of CHIP significantly enhanced p65 nuclear translocation when bone marrow cells were treated with RANKL for 40 min (Figure 5B, lane 8 vs lane 7). The protein levels of p65 in nucleus in Chip ^−/− cells were much higher than that in WT cells even without RANKL treatment (Figure 5B, lane 6 vs lane 5). These results suggest that CHIP reduces RANKL-induced IKK-β and I-κBα phosphorylation and NF-κB activation.

We further determined whether CHIP affects RANKL or TRAF6-mediated NF-κB activity. NF-κB luciferase reporter was transfected with HA-CHIP or empty vectors into RAW264.7 cells which were either treated with RANKL or transfected with TRAF6 expression plasmids. The results showed that NF-κB reporter activity was significantly increased by the treatment of RANKL and transfection with TRAF6. However, over-expression of CHIP significantly inhibited RANKL or TRAF6-induced NF-κB activity (Figure 5C). These results suggest that CHIP suppresses NF-κB signal pathway via inhibiting phosphorylation of IKK-β/I-κBα and p65 translocation into the nucleus.

Discussion

Bone tissue undergoes continuous remodeling throughout life. This dynamic process involves a balance between bone-forming osteoblasts, derived from mesenchymal progenitor cells, and bone-resorbing osteoclasts, derived from hematopoietic stem cells (35). A balance of the activity of these cells is required for maintaining normal bone homeostasis (36). The key signaling pathway regulating osteoclast formation is RANKL/RANK pathway. RANKL is produced from bone marrow stromal cells (37–39), osteocytes (40–41) and hypertrophic chondrocytes (41). RANKL is localized on the surface of these cells (37) and osteoclast formation requires direct cell-cell contact between these cells and osteoclast precursor cells which contain RANK receptor on their cell surface. The key downstream molecule of RANKL-RANK signaling is NF-κB (37).

NF-κB activity is strictly regulated by RANKL to maintain bone homeostasis. Upon ligand binding, RANK recruits TRAF family proteins to transduce the signal and TRAF2, TRAF3 and TRAF5 bind to the distal domain in the cytoplasmic tail of RANK, while TRAF6 binds to the proximal domain of RANK (10). It has been reported that the RANK binding to TRAF2, TRAF3 and TRAF5 proteins are dispensable for osteoclastogenesis, whereas TRAF6 binding to RANK is required for osteoclast formation (42–43). The significance of TRAF6 in the maintenance of normal bone architecture is also demonstrated in TRAF6 knockout mice, which display a defect in NF-κB signaling and consequently develop osteopetrosis (7, 13). Therefore, regulation of TRAF6 is critical for osteoclastogenesis. To date, it has been reported that CYLD, A20, and FHL2 negatively regulate RANK-mediated NF-κB signaling and osteoclastogenesis via inhibiting TRAF6 activity or blocking receptors/TRAF6 association (14, 17, 44). Our current study is aiming to investigate the regulatory mechanism of TRAF6 protein degradation during osteoclast formation and bone remodeling.

As an E3 ligase, CHIP has been reported to promote ubiquitination and degradation of several substrates involving in different signaling pathways (21, 24). In vivo findings demonstrated that Chip deficiency causes increased sensitivity to stress associated hyperthermia (45), leading to a markedly reduced life span in mice (46). In addition, Chip^−/− mice developed severe defects in heart and neural tissues (47–48). Moreover, Chip^−/− mice exhibit defects in motor sensory, cognitive, and reproductive function (49). However, to date the physiologic roles of CHIP in bone remodeling has not been elucidated. In this study, we showed that Chip^−/− mice develop osteopenic phenotype. Our study further demonstrates that Chip deficiency enhances osteoclastogenesis by negatively regulating TRAF6 and TRAF2 protein levels. Considering that TRAF2 has been reported as a substrate of CHIP in breast cancer cells, we decided to focus our efforts on characterizing the regulatory mechanism of TRAF6 protein degradation by CHIP during osteoclast formation. Furthermore, our results revealed that CHIP directly interacts with TRAF6 to promote its ubiquitination and proteasome degradation. Consequently, CHIP represses p65 nuclear translocation and attenuates NF-κB activation. This study reveals that CHIP is a novel negative regulator of osteoclastogenesis via induction of TRAF6 degradation.

Our previous studies demonstrated that CHIP induces Runx2 degradation in pre-osteoblasts (21). However, Chip^−/− mice show phenotype of increased osteoclast formation and reduced bone mass. In addition, we didn’t find significant changes in Runx2 protein levels in bone marrow stromal cells derived from Chip^−/− mice, indicating that the osteopenic phenotype observed in Chip^−/− mice is not due to regulation of Runx2 in osteoblasts. The expression level of CHIP is high in preosteoblast, but is down-regulated during osteoblast differentiation (20, 50). Therefore, CHIP might not function as a critical regulator in mature osteoblast in vivo. In contrast to the osteoblast, we found that CHIP protein level was increased in osteoclast precursor cells after treatment with RANKL (Figure 5A, lane1 vs lane 2–6, and Figure 5B, lane1 vs lane 3). Moreover, no significant changes in Chip mRNA expression were observed during osteoclast formation (data not shown). These results suggest that CHIP protein level is up-regulated by RANKL stimulation in osteoclast precursor cells. Taken together, CHIP might play a more important role in osteoclast formation than in osteoblast differentiation in vivo.

In summary, we provide the novel evidence that CHIP is an E3 ligase which induces TRAF6 ubiquitin-proteasome degradation and subsequently inhibits NF-κB signaling in osteoclasts. Given the essential role of TRAF6 in osteoclastogenesis, our study demonstrates that Chip^−/− mice have increased osteoclast differentiation and develop osteopenic phenotype. Since abnormal osteoclastogenesis has been reported in many metabolic bone diseases, such as osteoporosis, Paget’s disease and rheumatoid arthritis, our present study suggests that CHIP may be a novel candidate for drug target to treat bone loss associated diseases.