Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway

Abstract Objectives To clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC). Materials and Methods Tooth extraction models of Ctsk knockout mice (Ctsk ‐/‐) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK‐8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk‐/‐ JBMMSC was sent to RNA‐seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis. Results We found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up‐regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA‐seq results showed that coding genes of key enzymes in glycolysis were significantly up‐regulated in Ctsk‐/‐ JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC’s regeneration was blocked subsequently. Conclusions Our findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.


| INTRODUC TI ON
Cathepsin K (CTSK) is a key enzyme in bone organic matrix degradation. In the beginning, it was thought to be specifically expressed in osteoclasts and played a critical role in bone resorption. 1,2 In recent years, expression of CTSK in bone formation related cells, such as fibroblasts, osteoblasts and mesenchymal stem cells (MSC), was also confirmed. [3][4][5] It was found that inhibition of endogenous CTSK could promote the expression of sclerostin in periodontal ligament (PDL) fibroblasts, thereby inhibiting Wnt/β-Catenin pathway, 4 indicating that CTSK may be related to osteogenic activity of PDL fibroblasts.
However, the exact role of CTSK in these cells remains unclear.
Whether CTSK can regulate the proliferation and differentiation of JBMMSC, the most potential seed cells in the field of alveolar bone regeneration, 6,7 has not been reported, let alone its regulatory mechanism.
Glycocatabolism, the main way to obtain energy, plays a critical role in regulating the regeneration of MSC. It has been reported that hypoxia can increase the pluripotency and self-renewal of stem cells by enhancing their anaerobic oxidation. 8,9 It was confirmed that Wnt/LRP5 pathway could promote osteogenic differentiation of ST2 cells by promoting glycolysis, 10 while canonical Notch pathway could inhibit osteogenic differentiation of BMMSC and ST2 cells by inhibiting glycolysis. 11 However, there are few reports on the regulation of glycolysis by CTSK.
Yang et al 12 reported that compared with WT mice, the expression of Glut4 in adipose tissue of Ctsk -/mice was significantly increased, and glucose metabolism was enhanced. But Dauth et al 13 found that lack of CTSK in astrocytes appeared not to affect their metabolic supply functions. According to the latest research, selective inhibition of CTSK by ODN can increase the production of reactive oxygen species (ROS) in mitochondria of human renal carcinoma Caki cells, promote mitochondrial fusion and finally enhance tumour cell apoptosis. 14 However, the role and mechanism of CTSK in regulating glycolysis is still unclear.
In this study, we used tooth extraction models of Ctsk -/and WT littermates to study the role of CTSK in alveolar bone regeneration.
We further investigated the effects of Ctsk deficiency or pharmacal inhibition on the proliferation and osteogenic differentiation of JBMMSC in vitro. At last, we explored that whether endogenous Ctsk deficiency promote JBMMSC regeneration via glycolysis.

| Animals and tooth extraction
Ctsk −/− mice were generated by Shanghai Model Organisms Center, Inc (Shanghai, China). Eight-week-old Ctsk −/− mice and their WT littermates were used to extract the bilateral maxillary first molars.
At the end of each experimental period (3, 7, 10 and 14 days after tooth extraction), three mice were sacrificed with an excessive dose of anaesthetic to collect their maxillae. All mice were bred and maintained in the SPF Laboratory Animal Center of the Fourth

| Micro-computed tomography
All maxillae were fixed in 4% paraformaldehyde. Next, all specimens were scanned by Micro-computed tomography (Micro-CT) (Siemens Inveon Micro-CT, Siemens AG) and performed at a voltage of 80 kV, a current of 500 μA and a resolution of 10 μm.
Subsequently, three-dimensional images were reconstructed using the Inveon Research Workplace (Siemens AG). The region of interest included the whole three tooth extraction sockets.
Osteoclasts were defined as multinuclear TRAP-positive cells, and the number of osteoclasts per bone surface (N. Oc/BS) of the distobuccal socket was determined.

| Immunohistochemistry staining
For immunohistochemistry, slides were preincubated with 3% H 2 O 2 for 10 minutes. Goat serum was used to block the nonspecific binding, and then, sections were incubated overnight at 4°C with rabbit anti-cathepsin K polyclonal antibody (Abcam, Cat#ab19027) or rabbit anti-Osx polyclonal antibody (Abcam, Cat#ab209484).
Subsequently, sections were incubated with horseradish peroxidaseconjugated secondary antibody for 30 minutes at 37°C. Colour was developed using DAB substrate kit (Boster, Cat#13J25J14J1022), and haematoxylin was used for counterstaining. The number of positive cells per unit area in the distal socket was calculated.

| Immunocytochemistry
The JBMMSC were seeded on the glass slides until 80% confluence. Then Lyso Tracker (KeyGEN, Cat#KGMP006) was added and incubated with cells at 37°C for 1 hour. After that, cells were fixed in 4% paraformaldehyde. Cells were permeabilized in PBS containing 0.5% Triton X-100 for 20 minutes and blocked with normal goat serum for 30 minutes at room temperature. Then, the cells were incubated overnight at 4°C with a rabbit anti-CTSK polyclonal antibody (Abcam, Cat#ab19027). After that, the sections were incubated with goat anti-rabbit IgG (H + L) (Proteintech, Cat#SA0013-2) at 37°C for 30 minutes. Nuclei were counterstained with DAPI.

| Osteogenic differentiation
Jaw bone marrow mesenchymal stem cells were cultured under osteogenic culture conditions in medium containing DMEM complete medium, 10 mmol/L β-glycerol phosphate, 50 μmol/L ascorbate and 10 −7 M dexamethasone (Cyagen, Cat#MUBMX-90021). The medium was changed every 3 days. After osteogenic induction for 7 days, Western blot was performed to analyse the osteogenesisrelated proteins (ALP and Runx2). Fifteen days after induction, alizarin red staining was used to assess matrix mineralization. Briefly, cells were incubated with 10 μL CCK-8 solutions for 90 minutes at 37°C, and then, absorbance of each well at 450 nm was recorded.

| RNA-seq and bioinformatics analysis
Total RNA was extracted from WT and Ctsk -/-JBMMSC and subjected to RNA sequencing by Beijing Genomics Institute (ShenZhen, China).
In brief, mRNA sequencing was performed using BGISEQ-500 platform and the high-quality reads were aligned to the mouse reference genome (GRCm38). Gene expression was established by the number of fragments per kilobase of exon per million fragments mapped reads by Expectation Maximization. Differentially expressed genes were defined by both the fold change (FC ≥ 1.2) and statistical difference (P < .05).

| Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from cells with the TRIzol reagent™ (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using PrimeScript™ RT Master Mix (Takara, Cat# RR036A). RT-qPCR was performed by TB Premix Ex Taq™ II kit (Takara, Cat# RR820A) and then detected on the CFX96 Real-Time System (Bio-Rad). The mRNA levels were calculated using 2 − ΔΔCt method after normalization to the expression of β-actin. The primers were described in Table S1 | 5 of 12 ZHANG et Al.

| Extracellular acidification rate
Cells were plated at 4 × 10 4 cells/well in a Seahorse XF24 Cell Culture Microplate (Agilent). Twenty-four hours later, WT cells were treated with 1 μmol/L ODN or 1 μmol/L DMSO for 48 hours.
After that, the medium was switched to Seahorse XF Base Medium

| Glucose consumption and lactate production
For glucose consumption measurements, cells were seeded into sixwell plates at a density of 2 × 10 5 cells/well. Twenty-four hours later,

| Statistical analysis
All quantitative data were presented as means ± SD. Statistical analysis was performed using GraphPad Prism Software. Comparisons between two groups were analysed by two-tailed, unpaired Student's t tests. Comparisons between more than two groups were performed by one-way ANOVA. Statistical significance was defined as: *P < .05, **P < .01, ***P <.001.

| Ctsk deficiency accelerates osteoblast activity during the process of alveolar bone filling
There are two processes, including bone filling and bone remodelling, after tooth extraction. In mice, the bone filling stage was during the  Figure 2D).
These findings indicated that Ctsk knockout could accelerate the osteogenic capability during alveolar bone filling process.

| CTSK is expressed in MSC-like cells and osteoblasts during the process of alveolar bone filling
To explore how Ctsk deficiency accelerates the osteogenic capability during the process of alveolar bone filling, the expression of CTSK

| Endogenous Ctsk deficiency promotes JBMMSC proliferation and osteogenic differentiation
Jaw bone marrow mesenchymal stem cells from Ctsk -/and WT mice were used to determine the influences of Ctsk deletion or inhibition F I G U R E 4 Endogenous Ctsk deficiency promotes JBMMSC proliferation and osteogenic differentiation. JBMMSC from Ctsk -/mice and their WT littermates were cultured. A, Flow cytometry was used to detect the expression of JBMMSC surface markers. B, The expression of endogenous CTSK in JBMMSC and its deficiency in JBMMSC from Ctsk -/mice were confirmed by Western blot. C, CTSK was mainly located in lysosomal in WT JBMMSC by immunofluorescence. D, Influence of Ctsk knockout or inhibition by ODN on proliferation of JBMMSC was assessed by CCK-8 (* represent WT compared to Ctsk -/group; # represent WT compared to ODN group). E, Expressions of osteogenic-related proteins of ALP and Runx2 were detected by Western blot after osteogenic induction for 7 d, and quantitative analyses were shown in (F). G, Mineralized nodules of JBMMSC were assayed by alizarin red staining after osteogenic induction for 15 d. H, Alizarin red staining was quantified with a spectrophotometer after dissolving by 10% cetylpyridinium chloride. The statistical analysis was shown: *P < .05;**P < .01; ***P < .001; #P < .05; ###P < .001 on their biological characteristics. Firstly, JBMMSC were positive (>95%) for MSC surface markers (CD105, CD90, CD44) and negative for hematopoietic surface markers (CD45, CD34) ( Figure 4A).
And the expression of endogenous CTSK in JBMMSC and its deficiency in JBMMSC from Ctsk -/mice were confirmed by Western blot ( Figure 4B). Then, the location of CTSK in WT JBMMSC was determined by immunofluorescence. The result indicated that CTSK was mainly expressed in lysosomes, but also in cytoplasm ( Figure 4C).
Further research indicated that both knocking out Ctsk and inhibiting its activity with ODN could promote the proliferation of JBMMSC ( Figure 4D), the expression of ALP and Runx2 ( Figure 4E,F), as well as the matrix mineralization ( Figure 4G,H). In other words, endogenous Ctsk deficiency promotes JBMMSC proliferation and osteogenic differentiation.

F I G U R E 5
Ctsk deficiency or inhibition promotes glycolysis. JBMMSC from Ctsk -/mice and their WT littermates were cultured and differentially expressed genes were selected by RNA-seq. A, Five differentially expressed genes of key enzymes in glycolysis were detected by RNA-seq. B, Expression levels of the five differentially expressed genes were confirmed by RT-qPCR. C, WT and Ctsk -/-JBMMSC were stimulated with 1 μmol/L ODN or 1 μmol/L DMSO for 48 h and then detected extracellular acidification rate by Seahorse. Glucose consumption (D) and lactate production (E) were performed to study the effect of Ctsk deficiency or inhibition on glycolysis. The statistical analysis was shown: *P < .05; **P < .01; ***P < .001
Additionally, there were 80 differentially expressed genes related to ATP synthesis, glycolysis, tricarboxylic acid cycle and ROS production between the two groups (FC ≥ 1.2, P < .05). Further analysis showed that HK1, Pfkfb3, Pfkl and Eno1 were the key driving genes in changing mitochondrial metabolism related genes ( Figure S3).
Subsequently, we confirmed that Ctsk deficiency or inhibition by decreased significantly, indicating the increased glucose intake (P < .05) ( Figure 5D). At the same time, increased lactate production was also detected in Ctsk -/or ODN group (P < .05) ( Figure 5E).
To confirm Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation by up-regulating glycolysis, 3PO (a specific inhibitor of Pfkfb3) was used to inhibit glycolysis in Ctsk -/or ODN treated JBMMSC. As a result, the ability of ODN or Ctsk deficiency to promote the proliferation of JBMMSC was blocked (P < .05) by 3PO ( Figure 6A and Figure S4A). On the other hand, compared with the Ctsk -/or ODN treated JBMMSC, the expression of Runx2 and ALP ( Figure 6B,C and Figure S4B,C) as well as the mineral node formation ( Figure 6D,E and Figure S4D,E) decreased in 3PO+ODN or Ctsk -/-+3PO group (P < .05). These results suggested that Ctsk deficiency or inhibition could promote JBMMSC proliferation and osteogenic differentiation by up-regulating glycolysis.

| D ISCUSS I ON
The main findings of the present study include (a) Ctsk deficiency could regulate alveolar bone regeneration by promoting JBMMSC proliferation and differentiation; (b) Ctsk deficiency or inhibition promotes JBMMSC proliferation and osteogenic differentiation by up-regulating glycolysis.
As a key regulator of bone metabolism, CTSK can regulate both bone resorption 1,2 and bone immunity. 15,16 So more and more studies focus on the dual regulation of CTSK on bone immunity and bone remodelling. [17][18][19] Silencing of Ctsk by AAV-RNAi was used to inhibit the development of periapical periodontitis in mice. 20 Silencing of Ctsk or inhibiting the function of CTSK by ODN was also used to inhibit inflammation and bone loss caused by periodontal diseases. 17,21 These studies confirmed the preventive effect of Ctsk deficiency/ inhibition on alveolar bone resorption secondary to odontogenic inflammatory diseases. However, whether Ctsk deficiency/inhibition could promote alveolar bone regeneration and then be used to treat alveolar bone defects remains unclear. In this study, tooth extraction models were used to investigate the role of CTSK in alveolar bone regeneration. As a result, we found that Ctsk knockout could significantly promote the new bone formation in the early stage of the socket healing and accelerate the regeneration of alveolar bone.
During this period, neither the number nor the morphology of osteoclasts showed significant difference between Ctsk -/and WT mice.
Contrarily, Osx-positive cells in Ctsk -/mice peaked earlier. The aim of this study is not to overturn the classical theory that CTSK regulates bone regeneration through osteoclasts. However, the fact that CTSK is expressed not only in osteoclasts but also osteoblast cells during the healing process after tooth extraction suggests that there may be other pathways for CTSK regulating bone regeneration. In other words, the regulation of CTSK on early alveolar bone healing and regeneration may depend on JBMMSC mediated bone formation.
It has been reported that CTSK can indirectly regulate bone formation by regulating osteoclasts. Briefly, inhibition of CTSK could increase the number of preosteoclasts and the endogenous levels of platelet-derived growth factor-BB, which could increase CD31(hi) Emcn(hi) vessel number and stimulate BMMSC proliferation and migration in mice. 22,23 However, CTSK was also expressed in osteoblasts from different bone 24,25 and different kinds of MSC. [3][4][5] The function of endogenous CTSK in these cells remains unclear. Using

CTSK knockdown experiments, Whitty et al 4 showed that CTSK
actively controlled sclerostin levels, subsequently affected Wnt/βcatenin pathway in PDL fibroblasts through a lysosomal mechanism. These results indicated that endogenous CTSK in these cells may be related to osteogenic activity. In this study, we confirmed the expression and location of CTSK in JBMMSC both in vitro and in vivo, and revealed that knocking out Ctsk or inhibiting its activity with ODN could promote the proliferation and osteogenic differentiation of JBMMSC in vitro.
In order to detect how CTSK regulates the regeneration of JBMMSC, RNA-seq was carried out in JBMMSC from WT and Ctsk -/mice. Bioinformatics analysis indicated the effect of CTSK on glycolysis. Glycolysis is the common stage of all the three pathways for the oxidative decomposition of glucose, including anaerobic oxidation, aerobic oxidation and pentose phosphate pathway. It has been reported that cells in proliferative tissues tend to convert up to 85% glucose to lactate regardless of whether oxygen is present. 26 It has been confirmed that glycolysis plays an important role in regulating the proliferation and osteogenic differentiation of BMMSC. 10,11 However, there are few reports on the role of CTSK on glycolysis.
There are a few studies on the relationship between CTSK and glucose metabolism. Yang et al 12  we not only confirmed that Ctsk deficiency/inhibition could promote the glycolysis of JBMMSC, but also revealed that Ctsk deficiency/ inhibition promoted JBMMSC proliferation and osteogenic differentiation by up-regulating glycolysis.
In summary, we have demonstrated that both knockout and inhibition of CTSK could promote JBMMSC proliferation and osteogenic differentiation by up-regulating glycolysis, thereby promote alveolar bone regeneration. Though these results could provide some evidence for promoting the regeneration of JBMMSC by inhibiting CTSK for jaw bone regeneration in different environments, how CTSK regulate glycolysis needs further research, and mice with conditional knockout of CTSK in BMMSC should be generated, too.