CHIP regulates skeletal development and postnatal bone growth

Abstract C terminus of Hsc70‐interacting protein (CHIP) is a chaperone‐dependent and U‐box containing E3 ubiquitin ligase. In previous studies, we found that CHIP regulates the stability of multiple tumor necrosis factor receptor‐associated factor proteins in bone cells. In Chip global knockout (KO) mice, nuclear factor‐κB signaling is activated, osteoclast formation is increased, osteoblast differentiation is inhibited, and bone mass is decreased in postnatal Chip KO mice. To determine the role of Chip in different cell types at different developmental stages, we created Chip flox/flox mice. We then generated Chip conditional KO mice Chip CMV and Chip OsxER and demonstrated defects in skeletal development and postnatal bone growth in Chip conditional KO mice. Our findings indicate that Chip conditional KO mice could serve as a critical reagent for further investigations of functions of CHIP in bone cells and in other cell types.


| INTRODUCTION
Skeletal development and endochondral bone formation is a complicated process involving chondrocyte proliferation, differentiation and hypertrophy, cartilage calcification, resorption of calcified cartilage, vascular invasion, and osteoblast differentiation. All of these steps are regulated precisely by multiple growth factors and signaling molecules. Nuclear factor-κB (NF-κB) signaling controls osteoclast formation (Boyce, Yao, & Xing, 2010) and is involved in the process of removal of calcified cartilage. In NF-κB p50/p65 double knockout (KO) mice, osteoclast formation is severely impaired leading to defects in the resorption of calcified cartilage and largely expanded hypertrophic zone at the postnatal stage (Xing, Chen, & Boyce, 2013).
NF-κB signaling plays an important role in bone biology. C terminus of Hsc70-interacting protein (CHIP) is a chaperonedependent and U-box containing E3 ubiquitin ligase. It targets the degradation of proteins critical for multiple cellular functions and signaling pathways. In previous studies, we found that CHIP controls the degradation of multiple tumor necrosis factor receptorassociated factor proteins and regulates NF-κB signaling in both osteoclasts and osteoblasts and NF-κB signaling is activated in Chip KO mice Wang et al., 2018). It is known that activation of NF-κB signaling leads to stimulation of osteoclast formation and inhibition of osteoblast differentiation (Abu-Amer, 2013;Boyce et al., 2010;Jimi et al., 2016;Otero, Chen, Zhang, & Abu-Amer, 2012;Park et al., 2007;Swarnkar, Zhang, Mbalaviele, Long, & Abu-Amer, 2014;Yao et al., 2014). However, the role of NF-κB signaling in skeletal development remains unclear. In the present studies, we investigated changes in skeletal development in

| Whole embryo alizarin red/alcian blue staining
Embryos at E14.5 and E18.5 were collected and the skin, viscera, and adipose tissues were carefully removed. Whole skeletons were fixed in 95% ethanol for 2 days followed by fixation in acetone for an additional day, and stained with 0.015% alcian blue and 0.005% alizarin red for 3 days. Images of the skeletons were taken when most of the soft tissue was digested in 1% potassium chloride.

| Microcomputed tomography analysis
We used a Scanco microcomputed tomography 35 scanner (Scanco Medical, Brüttisellen, Switzerland) with 55 kVp source and 145 μA current for formalin-fixed mouse legs with a resolution of 10 μm. The scanned images from each group were evaluated at the same thresholds to allow three-dimensional structural rendering of each sample.

| Histology
Tibiae were harvested and fixed in 10% neutral-buffered formalin for 3 days and decalcified for 14 days in 14% EDTA and then paraffinembedded. Three-micrometer sections were cut and alcian blue/H&E orange G staining and tartrate-resistant acid phosphatase (TRAP) staining were performed (Shu et al., 2013;Wang et al., 2017).

| Statistical analysis
Data are presented as the mean ± standard deviation. For experiments comparing two groups of data, unpaired Student's t test was performed. A value of p < .05 was considered to be significant.  (Figure 2i). In addition, we also found that ColX expression was reduced in the hypertrophic zone of E18.5 Chip CMV KO embryos (Figure 2j).

| Bone mass was decreased in Chip OsxER conditional KO mice
In previous studies, we found bone loss phenotype in Chip global KO mice. However, it is not known if CHIP exerts its effect directly through bone cells or CHIP acts on bone through an indirect mechanism. To determine the specific effect of Chip on postnatal bone growth, we generated Chip OxsER conditional KO mice by breeding Chip flox/flox mice with Osx-CreER transgenic mice, which target osteoblast precursor cells (Maes et al., 2010). Tamoxifen was administered into 2-week-old Chip OxsER mice and Cre-negative littermates (Chip flox/flox mice). The results of a significant decrease in body weight and slightly reduced body size were found in Chip OxsER KO mice (Figure 4a-c). Results of μCT analysis showed that bone volume, BMD, trabecular numbers, and connectivity density were significantly reduced and trabecular separation was significantly increased in Chip OxsER KO mice (Figure 4d-i). We also analyzed bone morphological changes with histological method in 4-week-old Chip OxsER KO mice. Trabecular bone loss phenotype was found in Chip OxsER KO mice ( Figure 4j). These findings suggest that bone mass and bone quality were reduced in Chip OsxER KO mice and indicate that local produced CHIP could directly regulate bone mass. In contrast, we did not observe significant changes in growth plate cartilage (Figure 4k). To further determine changes in bone formation in Chip OsxER KO mice, we performed calcein/calcein double labeling assay. We found that mineral apposition rates and bone formation rates were significantly decreased in Chip OsxER KO mice (Figure 4l-n). Consistent with these findings, results of von Kossa staining showed that mineralized trabecular bone volume was significantly reduced in Chip OsxER KO mice (Figure 4o).
In previous studies, we demonstrated that NF-κB signaling is activated in Chip global KO mice (Wang et al., 2018). In this study, we performed TRAP staining in subchondral bone areas and in trabecular bone areas underneath the growth plate. We found that osteoclast formation was significantly increased in Chip OxsER KO mice (Figure 5a-c), suggesting that osteoclast formation was also affected by Chip OxsER KO mice.

| DISCUSSION
In previous studies, we demonstrated that CHIP controls NF-κB signaling and regulates postnatal bone growth. In Chip global KO mice, osteoclast F I G U R E 4 Bone mass was decreased in Chip OsxER conditional KO mice. Chip flox/flox mice were bred with Osx-CreER transgenic mice and resultant Chip OsxER mice were administered with tamoxifen (1 mg/10 g body weight, i.p. injection, x 5 days) in 2-week-old Chip flox/flox (Crenegative control) mice and Chip OsxER mice. Chip OsxER KO mice were then killed at 4 weeks of age. (a and b) Changes in body size and body weight were observed in Chip OsxER KO mice. (c) In contrast, the body length was not significantly changed. (d-i) Bone mass was analyzed by µCT. The μCT images of bone structure and bone morphology showed reduced (d) bone mass, (e) bone volume, (f) bone mineral density (BMD), (g) trabecular number (Tb.N.), and (i) connectivity density (Conn.D.) were significantly reduced in Chip OsxER KO mice. (h) In contrast, trabecular separation (Tb.Sp.) was significantly increased in Chip OsxER KO mice. (j and k) Consistent with µCT analysis, histological results showed bone mass decrease in Chip OsxER KO mice (trabecular bone is indicated by yellow arrowheads). In contrast, no obvious changes in growth plate cartilage morphology were observed in Chip OsxER KO mice. (l-n) We also performed calcein/calcein double labeling assay and results showed that mineral apposition rates (MAR) and bone formation rates (BFR) were significantly reduced Chip OsxER KO mice. (o) Results of von Kossa staining showed that the mineralized bone formation was also reduced in Chip OsxER KO mice. BV/TV, bone volume/total volume; KO, knockout; µCT, microcomputed tomography formation and bone resorption were increased and osteoblast differentiation was decreased Wang et al., 2018). In the present studies, we generated Chip flox/flox mice and then created Chip CMV and Chip OsxER conditional KO mice. We analyzed changes in skeletal development in Chip CMV KO embryos and we also analyzed changes in postnatal bone growth and bone mass in 1-month-old Chip CMV and Chip OsxER conditional KO mice. Our findings suggest that CHIP plays an important role in skeletal development and postnatal bone growth and regulates bone mass. Our findings indicate that we have successfully generated Chip conditional KO mice and confirmed that Chip is expressed in Osx-expressing osteoblast precursor cells and plays a specific role in the regulation of bone mass.
Our previous study demonstrated that CHIP regulates NF-κB signaling through inducing the degradation of multiple TRAF proteins, including TRAF2, TRAF5, and TRAF6 Wang et al., 2018). It is known that NF-κB controls growth plate cartilage development. In NF-κB p50/p65 double KO mice, osteoclast formation is severely impaired leading to defects in the resorption of calcified cartilage and largely expanded hypertrophic cartilage zone at the postnatal stage (Xing et al., 2013). In this study, we found that growth plate cartilage development relatively normal in postnatal Chip CMV and Chip OsxER KO mice. These findings suggest that the activation of NF-κB signaling may not be able to significantly affect postnatal growth plate cartilage development although osteoclast formation is increased in Chip KO mice.
Bone is an endocrine organ and bone remodeling is a dynamic process that is active throughout the entire life. To determine the specific role of CHIP at different cell populations and at the different developmental stages of life, it requires the generation of inducible Chip conditional KO mice. As a long-term goal of this project, we will determine the roles of Chip in osteoclast and osteoblast lineage cells at different developmental stages. We have recently generated Chip flox/flox mice and Chip conditional KO mice which allow us to perform tissue-specific and longitudinal studies to investigate the role of CHIP in bone remodeling.
CHIP may play an important role in aging. Sirtuin 6 (SirT6) is a stress-responsive protein deacetylase and mono-ADP ribosyltransferase enzyme encoded by the SirT6 gene (Min et al., 2008). Studies in mice have revealed that Sirt6 is essential for postnatal development and survival. SirT6 functions in multiple molecular pathways related to aging, including DNA repair, telomere maintenance, glycolysis, and inflammation (Frye, 2000). SirT6 promotes resistance to DNA damage and oxidative stress, the defects closely related to age-associated diseases (Beauharmois, Bolivar, & Welch, 2013). CHIP prevents proteasome-dependent degradation of SirT6 and SirT6 stability is increased in Chip-deficient cells. These results suggest that CHIP protects proteasomal degradation of SirT6. To study the roles of CHIP in SirT6 and other proteins associated with aging, we need to create Chip flox/flox mice and Chip conditional KO mice since Chip global KO mice died at postnatal or early adult stages. We have recently F I G U R E 5 TRAP-positive osteoclast formation was increased in Chip OsxER conditional KO mice. Chip flox/flox mice were bred with Osx-CreER transgenic mice and resultant Chip OsxER KO mice were administered with tamoxifen in 2-week-old Chip flox/flox (Cre-negative control) mice and Chip OsxER mice and the mice were killed at 4 weeks of age. TRAP staining was performed. Significant increases in the numbers of TRAP-positive cells were detected in subchondral bone areas (a) and in trabecular bone areas underneath the growth plate (b and c) in Chip OsxER KO mice. KO, knockout; TRAP, tartrate-resistant acid phosphatase generated Chip CMV and Chip OsxER conditional KO mice, which showed bone loss phenotype as we observed in Chip global KO mice Wang et al., 2018), suggesting that Chip flox/flox mice could be used for long-term and longitudinal studies.
The roles of CHIP in physiological functions and disease initiation and progression have been extensively investigated in recent years. For example, CHIP plays a critical role in neurodegenerative diseases, inflammation, and cardiovascular diseases. Generation and making the availability of Chip flox/flox mice to other fields will make studies of CHIP functions in other organ systems easier.

ACKNOWLEDGMENTS
This study was supported by National Institutes of Health Grants R01AR054465 and R01AR070222 to D. C. This study was also partially supported by the National Natural Science Foundation of China (NSFC) Grants 81874011, 81572104, and 81301531 to T. W.

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
W. W., J. L., F. C. K., X. Z., Y. Q., and R. S. L. carried out experiments. T. W. and D. C. prepared the manuscript, contributed to the experimental design, data interpretation, and finalized the manuscript. D. R. S. revised the manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The animal protocol of this study has been approved by the IACUC of the Rush University Medical Center and all experimental methods and procedures were carried out in accordance with the approved guidelines.