CRISPR/Cas9 knockout of MTA1 enhanced RANKL‐induced osteoclastogenesis in RAW264.7 cells partly via increasing ROS activities

Abstract Metastasis‐associated protein 1 (MTA1), belonging to metastasis‐associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. However, the effect of MTA1 on osteoclastogenesis is unknown. Currently, the regulation of MTA1 in osteoclastogenesis was reported for the first time. MTA1 knockout cells (KO) were established by CRISPR/Cas9 genome editing. RAW264.7 cells with WT and KO group were stimulated independently by RANKL to differentiate into mature osteoclasts. Further, western blotting and quantitative qRT‐PCR were used to explore the effect of MTA1 on the expression of osteoclast‐associated genes (including CTSK, MMP9, c‐Fos and NFATc1) during osteoclastogenesis. Moreover, the effects of MTA1 on the expression of reactive oxygen species (ROS) in osteoclastogenesis was determined by 2′, 7′ ‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining. Nuclear translocation of Nrf2 was assessed by immunofluorescence staining and western blotting. Our results indicated that the MTA1 deletion group could differentiate into osteoclasts with larger volume and more TRAP positive. In addition, compared with WT group, KO group cells generated more actin rings. Mechanistically, the loss of MTA1 increased the expression of osteoclast‐specific markers, including c‐Fos, NFATc1, CTSK and MMP‐9. Furthermore, the results of qRT‐PCR and western blotting showed that MTA1 deficiency reduced basal Nrf2 expression and inhibited Nrf2‐mediated expression of related antioxidant enzymes. Immunofluorescence staining demonstrated that MTA1 deficiency inhibited Nrf2 nuclear translocation. Taken together, the above increased basal and RANKL‐induced intracellular ROS levels, leading to enhanced osteoclast formation.


| INTRODUC TI ON
Bone is a kind of dynamic yet stable connective tissue that remodels constantly throughout life, which is influenced by the activities of two main cell populations, osteoclast-mediated bone resorption and osteoblast-mediated bone formation. 1 Absorption and formation are stable under physiological conditions. However, when the balance between bone formation and resorption is disturbed, which will lead to a disorder of bone homeostasis. Osteoblasts (OBs) are mainly derived from mesenchymal progenitor cells in the inner and outer periosteum and bone marrow matrix, which can specifically secrete a variety of bioactive substances to regulate and influence the process of bone formation and reconstruction. 2 Osteoclasts (OCs) originate from haematopoietic stem cells (HSCs), are a type of multinucleated giant cells (MNCs) that specializes in absorbing organic collagen and mineralized matrix. 3 Excess osteoclastic activity is a major cause of bone skeletal metabolic diseases, such as osteoporosis (OP), periodontitis, rheumatoid arthritis and other osteolysis-related diseases.
Clinical anti-osteolysis drugs such as bisphosphonates, oestrogens, serotonin and calcitonin, but long-term treatment with these drugs may cause many restrictions and side effects such as impaired fracture healing, increased risk of breast cancer, osteonecrosis of the jaw, the risk of venous thromboembolism and neurological reactions. 4,5 In addition, most US Food and Drug Administration (FDA)approved drugs treat osteolysis by inhibiting osteoclasts are still limited. 6,7 Therefore, it is important to elucidate intricate molecular mechanisms that regulate osteoclast differentiation and function to improve the treatment of the pathogenesis of bone diseases.
OCs are regulated by multiple hormones and local cytokines.
However, receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) are the key molecules of OCs differentiation. 8 The RANKL/RANK interaction with M-CSF/c-Fms triggers the activation of multiple downstream signalling pathways that are essential for osteoclast formation, such as nuclear factor κB (NF-κB) signalling, mitogen-activated protein kinase (MAPK) signalling, PI3K-protein kinase B (AKT) signalling, Ca 2+ -calcineurin-NFATc1 signalling and reactive oxygen species (ROS) signalling, etc. 6 Further activating the master transcription factors c-Fos and nuclear factor of activated T-cells 1 (NFATc1), which can induce the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metallopeptidase-9 (MMP-9) and osteoclast stimulatory transmembrane protein (OC-STAMP) contributes to osteoclast activation and maturation. 9 Normal metabolism of the body can produce reactive oxygen species, containing superoxide anion radical (O 2 ·− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (−OH) and nitric oxide (NO). 10 In addition, recent studies have shown that ROS are important components in RANKL-mediated osteoclast differentiation and maturation. 11 Metastasis-associated protein 1 (MTA1), belonging to metastasis-associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. 12 MTA1 is mainly studied in cancer, and it plays a role in the transformation, invasion, survival, DNA repair, angiogenesis, hormone dependence and treatment resistance of cancer. 13 Besides strong correlation between MTA1 upregulation and cancer, growing evidence strongly suggests that MTA1 could regulate divergent cell pathways by modifying status of crucial target genes under both pathological and physiological statuses. 14 It has been confirmed that MTA1 is involved in normal physiological processes such as embryonic development, spermatogenesis during reproduction, cell ageing, nervous system and photosensory system. 15 It has been reported that MTA1 can stimulate the growth of osteoblasts under hypoxia, but inhibit the differentiation of MC3T3 cells, which may promote the accumulation of osteoblasts in bone remodelling. 16 However, physiological characteristics of MTA1 have still been poorly addressed, especially the effects of MTA1 on osteoclast differentiation has not been reported so far. CRISPR/Cas 9 is a method for selectively editing target genes by Cas 9 proteins directed by RNA at the genomic level. Due to the simple operation and less time, applications are becoming more and more widespread. It plays an important role in gene knockdown, regulated expression levels of genes and gene editing therapy for diseases. 17 In this study, we constructed RAW264.7 macrophage line with MTA1 gene knockout by technology to investigate the function of MTA1 in osteoclasts, and it is of great significance for the treatment of bone diseases to further understand its mechanism in osteoclast differentiation and bone catabolism. Our results indicate that MTA1 plays a pivotal role in RANKL-induced osteoclast differentiation and negatively regulates osteoclast differentiation via affecting ROS production and antioxidant production. It also provides a potential molecular target for the treatment of osteoclastmediated bone metabolic diseases.

| Tartrate-resistant acid phosphatase (TRAP) staining
The culture supernatants were inoculated into the 96-well plate. To

| Flow cytometric analysis
The effect of MTA1 knockout on the apoptosis of RAW264.

| Measurement of intracellular ROS production
ROS assay kit (Beyotime) was used to detect the intracellular production of ROS based on the cell permeant fluorogenic dye, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, the RAW264.7 cells were seeded into a 96-black well plate for 24 h and then pre-treated with 40 ng/mL RANKL. Cells were incubated with 10 μM DCFH-DA in the dark for 30 min at 37°C, rinsed in culture media three times and the fluorescence of DCF was measured using a fluorescence microscope (Leica). And the mean fluorescence intensity was quantitatively analysed by ImageJ software.

| Western blotting analysis
Total proteins from BMMs or RAW264.7 cells were isolated with RIPA buffer (Beyotime) containing PMSF and phosphatase inhibitors centrifuged at 13,000 × g for 5 min. The lysates were collected, and the protein concentration was quantitatively analysed using

| Real-time quantitative polymerase chain reaction (RT-qPCR)
RAW264.7 cells were inoculated into 6-well plates and stimulated with RANKL (40 ng/mL) for a period. BMMs were seeded in 6-well plates and stimulated with M-CSF (20 ng/mL) and RANKL (40 ng/ mL) for 5 days. Then, total RNA was extracted by TRIzol method according to the manufacturer's instructions. Prime Script RT kit was then used for reverse transcription into cDNA. The obtained cDNA was used as a template for real-time quantitative PCR on the Light Cycler® 96 system (Roche, Basel, Switzerland). The primers employed in this study are listed in Table 1. The housekeeping gene GAPDH was used as internal control.

| CRISPR/Cas9-mediated MTA1 knockout
Deletion of MTA1 in RAW264.7 cell lines was accomplished by Cyagen Biosciences Inc. US (China-branch). In brief, the MTA1 gene sequence was obtained from NCBI database, and the appropriate gRNA oligonucleotide was designed and constructed. Cas9 protein and gRNA oligonucleotide were transferred into RAW264.7 cell line via electroporation using CRISPR/Cas9 gene editing technique.
CRISPR/Cas9-mediated MTA1 deletion monoclonal cells were obtained by PCR amplification. Finally, Sanger sequencing was used for homozygous verification.

| Statistical analysis
Data are expressed as mean ± standard deviation (SD). Differences between two groups were evaluated using Student's t-test, and multiple group comparisons were tested using one-way anova, statistical analyses were performed using the GraphPad Prism software, *p < 0.05 was considered as a statistically significant difference.  Figure 3C). Subsequently, to explore the impact of MTA1 knockout on RAW264.7 apoptosis, flow cytometric analysis was conducted. The results suggested that MTA1 knockout reduced the RAW264.7 apoptosis rate ( Figure 3D).

| Deficiency of MTA1 accelerated osteoclast differentiation
After osteoclast differentiation in vitro was analysed using RAW264.7 KO and WT groups to evaluate the role of MTA1 in RANKL-induced

| Deletion of MTA1 promoted the expression of osteoclast-specific genes
To further compare the expression levels of osteoclast differentiation-related genes in KO group and WT group, we used western blotting and PCR to analyse the protein and mRNA expression levels of osteoclast differentiation-related genes. Total RNA was extracted at 0, 1, 3 and 5 days after RANKL induction, as shown in Figure 5C, the expression levels of osteoclast-associated genes (including CTSK, MMP9, c-Fos and NFATc1) were significantly increased in KO group. The difference between the two groups was more pronounced on day 3 and 5. In addition, compared with WT group, the protein levels of CTSK, MMP9, c-Fos and NFATc1 were significantly increased on day 3 in KO cells ( Figure 5A, B). These results suggested that MTA1 deficiency promoted osteoclast differentiation and functional expression.

| Lack of MTA1 facilitated RANKL-induced ROS production in osteoclastogenesis
Under physiological conditions, ROS can regulate intracellular environmental homeostasis, signal transduction, proliferation and differentiation, which are in a dynamic balance with antioxidants. 19 The inhibitory effect of ROS on osteoblasts and the promotion effect on osteoclasts will lead to greater bone resorption than bone formation, resulting in negative bone remodelling and ultimately reduced bone mass and bone strength. However, it is not clear whether MTA1 affects ROS production during osteoclastogenesis.
We assessed control and RANKL-induced intracellular ROS levels using DCFH-DA staining. In the absence of RANKL stimulation, our results showed that ROS levels were higher in the KO group than in the WT group ( Figure 6A NRF2, immunofluorescence was used to assess nuclear translocation of Nrf2, and western blot was used to assess Nrf2 protein levels in the nucleus and cytoplasm. As shown in Figure 7A, MTA1 knockdown significantly blocked nuclear translocation of Nrf2. In addition, western blot results also proved that the nuclear Nrf2 protein level was lower and the cytoplasmic Nrf2 level was higher in the KO group ( Figure 7B, C). These collective data suggested that MTA1 deficiency enhances osteoclastogenesis via inhibiting nuclear translocation of Nrf2 and downregulating the expression of NRF2 and its induced antioxidants.

| DISCUSS ION
In the process of bone metabolism, under the synergistic action of relevant regulatory hormones and various cytokines, osteoblasts generate new bone and osteoclasts absorb old bone. This cycle repeats itself, eventually leaving the human bone homeostasis in a relatively stable state. 21 Nevertheless, when bone resorption is increased or bone formation is reduced, the balance between bone resorption and bone formation is disrupted, leading to the destruction of the microenvironment and causing bone metabolism-related diseases such as OP. 22 The functional activation of osteoclasts is the key factor to induce osteoporosis pathology. At present, considering the limited of FDA-approved drugs for osteolysis treatments and their side effects cannot be ignored. 23

| CON CLUS IONS
In this study, we demonstrate that MTA1 may negatively regulate os- writing -original draft (equal).

FU N D I N G I N FO R M ATI O N
This study was supported by a grant from National Natural Science Foundation of China (82070909).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and materials were included in the article.