Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL- induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway

doi:10.21203/rs.3.rs-2730491/v1

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Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL- induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway

https://doi.org/10.21203/rs.3.rs-2730491/v1

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published 24 Jan, 2024

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Background: The overactivation of the osteoclasts is a key pathological factor in osteoporosis. Myeloid zinc finger 1 (MZF1) belongs to the scan-zinc finger family, which is involved in multiple processes of tumor malignant progression and is an important transcription factor regulating the expression of osteoblasts. However, the exact role of MZF1 in osteoclasts has not been determined. In this study, the purpose of our study was to elucidate the role of MZF1 in osteoclastogenesis.

Results: First, In vivo studies have shown that MZF1 defciency led to decreased bone mass and exhibited a low bone mass osteoporosis phenotype. Further, RANKL-induced osteoclast differentiation in vitro showed larger TRAP-positive osteoclasts and more complete sealing zone in the MZF1-knockout group. Mechanistically, the results showed that MZF1 deletion group promoted the expression of osteoclast-associated genes. MZF1 knockout can regulate RANKL-induced ferroptosis of osteoclasts by regulating Nrf2/GPX4 signaling pathway in the early stage of cell differentiation.

Conclusions: Our research revealed that MZF1 knockout promote osteoclast differentiation by regulating iron metabolism and exhibited osteolysis models. This may be a useful therapeutic target for the prevention and treatment of osteolytic diseases.

Myeloid zinc finger 1

knockout

iron metabolism

ferroptosis

osteoclastogenesis

The skeleton is a dynamic organ that supports the weight of the body, protects the organs, provides the primary site of hematopoiesis, while also serving as a mineral repository for humans[1]. The disorder of bone microenvironment in pathological conditions may break the delicate balance between osteogenesis and osteolysis leading to osteopetrosis or osteoporosis (OP), conditions of increased or decreased bone mass[2]. Osteoclasts (OCs) are mononuclear macrophages derived from bone marrow and their formation and abnormal functional activation are the main causes of related diseases[3, 4]. Therefore, it is of great significance to actively elucidate the key signaling molecules involved in the regulation of OCs under pathological conditions and elucidate their specific mechanism of action for the treatment of osteoclast-related diseases. OCs are the only cells that perform bone resorption functions in human body and are derived from hematopoietic stem cells (HSCs), which is a kind of multinucleated giant cell stimulated by multiple factors[5, 6]. Receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) are two key cytokines that induce osteoclast differentiation[7]. They bind to M-CSF receptor and RANK on the surface of OCs, respectively, to promote OCs proliferation, differentiation and other vital activities. RANK belongs to the tumor necrosis factor receptor family protein, which binds to RANKL to initiate the downstream classical signaling cascade, including NF-κB, MAPKs, PI3K/AKT, Ca2+/calmodulin-dependent protein kinase (CaMK)/ nuclear factor of activated T cells 1 (NFATc1) and activation of reactive oxygen species (ROS)[8, 9]. These classical osteoclastogenic pathways individually or collectively induce the expression and activation of NFATc1 and c-Fos, the major regulators of OCs formation, ultimately increasing the expression of osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metallopeptidase-9, integrin β3 and contributing to osteoclast differentiation and maturation[10-12]. Ferroptosis is a non-apoptotic cell death, which is significantly different from other programmed cell death including traditional apoptosis, autophagy, necrosis, pyrodeath, etc[13, 14]. Its main feature is the accumulation of iron dependent ROS in cells, which promotes cell death[15]. The dysregulation of ferroptosis is associated with many pathological processes, such as cancer, diseases of the nervous system, diseases of the cardiovascular system, and inflammation-related diseases[16-18]. Iron overload is an independent risk factor for OP[19]. Ni et al.[20] showed that ferroptosis was involved in the differentiation of OCs. The nuclear factor erythroid 2-related factor 2 (Nrf2), as a transcription factor that initiates endogenous antioxidant reaction elements, has nuclear transport under the stimulation of ROS, thus producing antioxidant stress effects[21]. As an important sensor of oxidative stress and cell death signals, glutathione peroxidase 4 (GPX4) in its downstream is considered to be one of the important targets that trigger the ferroptosis program[22]. Nrf2/GPX4 signaling pathway is a key target for regulating oxidative stress of osteoclasts, regulating iron metabolism, and preventing and treating osteoporosis.

Myeloid zinc finger 1 (MZF1) belongs to the scan-zinc finger protein family [23]. MZF1 was originally found to be a transcription factor involved in transcriptional regulation of hematopoietic development, which can regulate the differentiation and proliferation of hematopoietic cells, and upregulation of MZF1 expression could induce leukemia[24]. MZF1 regulates hematopoietic development, and its overexpression or underexpression may cause cancer[25]. In the past decade, many studies have shown that MZF1 is implicated in multiple processes of malignant progression of solid tumors, including invasion and metastasis, chemotherapy resistance and inflammatory response[26]. Although OCs originate from HSCs and MZF1, as a transcription factor, is expressed in totipotent hematopoietic cells as well as in myeloid progenitor cells, the effect of MZF1 on osteoclast differentiation has not been reported so far. Furthermore, document study has shown that MZF-1 acts as a tumor suppressor in prostate cancer, at least partially, through enhancing Ferroportin-conducted iron egress out of tumor cells[27]. MZF1 may affect osteoclastogenesis by regulating iron ion metabolism.

In this study, we verified for the first time that MZF1 plays a pivotal role in regulating osteoclast differentiation through in vitro and in vivo experiments. Further elucidate the molecular mechanism of MZF1 negatively regulating. It provides a new molecular target for the treatment of osteoclast - mediated bone metabolic diseases.

2.1 Cell culture and osteoclast differentiation

RAW 264.7 mouse monocyte/macrophage cell lineage was obtained from American Type Culture Collection (TIB-71; ATCC, Manassas, VA) and maintained in DMEM (Hyclone, Logan, UT, USA) medium containing 10% FBS (Gibco, Rockville, MD, USA) in a humidified atmosphere containing 5% CO₂ at 37˚C. For osteoclastic differentiation, we changed the induction from DMEM medium into α-MEM (Hyclone) containing 10% FBS at the first induction day and stimulated with 40 ng/mL RANKL (PeproTech EC, Ltd.) for 5 days into 96‐well‐plate. The medium was changed every 3 days and the cells used for osteoclastogenesis were from passage three.

2.2 Tartrate‑resistant acid phosphatase (TRAP) staining

Osteoclasts are usually characterized by the multiple nuclei (≥3) and TRAP staining positive activity. As a consequence, to identify osteoclasts, all media were removed and cells were washed with phosphate‐ buffered saline (PBS) three times. Next, fixed with 4% paraformaldehyde at room temperature for 10 min and then washed with PBS for 5 min. After, the cells were stained by a TRAP Kit (Sigma-Aldrich, USA) according to the manufacturer’s protocol for 60 min at 37°C in the dark room. The percentage of TRAP‐positive cells was measured using ImageJ software. Each experiment was repeated three times.

2.3 F-Actin ring formation assay

The bone resorption function of mature osteoclasts depends on the podosome patterns and sealing zone formation. We performed cytoskeletal fibrous actin (F-actin) staining. Briefly, After osteoclast differentiation and maturation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. To increase permeability, the 0.1% Triton X-100 was added for 10 minutes. Then, stained with Rhodamine-phalloidin (Yeasen, Shanghai, China) in the dark at room temperature for 40 min. For nuclear staining, DAPI was added to the osteoclasts nucleus. The sealing zone and nuclear were visualized by fluorescent microscope (Leica, Germany).

2.4 Targeted knockout of MZF1 using CRISPR-Cas9 in RAW264.7

Knockout of MZF1 in RAW264.7 cell line was accomplished by the Cyagen Company (China). In brief, the gene sequence of MZF1 was obtained from the 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 MZF1 deletion monoclonal cells were obtained by PCR amplification. Eventually, the sanger sequencing was employed for homozygous verification.

2.5 Immunofluorescence staining

WT and MZF1 KO preosteoclasts were seeded on cover slips in a 24-well plate for 12 h and then treated with RANKL for 24 h. For immunofluorescence staining, washed with PBS three times and then fixed with 4% paraformaldehyde for 15 min. Next, the cells permeabilized with 0.1% Triton X-100 for 15 min. Cells were incubated with anti-rabbit Nrf2 (Beyotime, Shanghai, China) at 4°C overnight and then probed with goat anti-rabbit Alexa Fluor 488 secondary antibody for 2 h in the dark at RT. After the cells were washed with PBS for three times. For nuclear staining, cells were counterstained with DAPI for 15 min and inspected using a confocal microscope (Leica).

2.6 Cell proliferation assays

A Cell Counting Kit-8 (Beyotime) was used for the cell viability assay. RAW264.7 cells were cultured in 96- well plates at a density of 4000 cells per well and stimulated with RANKL. At 0, 24, 48 and 72 hours of cell culture, the 10 μL CCK-8 reagent was added to each well. The plates were incubated for 3 hours in an incubator at 37 ℃, and optical density (OD) values were measured at 450 nm using a microplate reader.

2.7 Lipid reactive oxygen species, MDA and GSH assay

The intracellular production of ROS was investigated using 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye according to the manufacturer’s protocol (Beyotime). Briefly, RAW264.7 cells were inoculated and then stimulated with RANKL after 24 h. A Fluorescence probe (1:1000) containing DCFH-DA was used to detect and incubated in the dark for 30 min, and the fluorescence of DCF was measured using a fluorescence microscope. A minimum number of 10,000 cells were collected for further detection. MDA and GSH content were measured by commercial MDA and GSH assay kits (Beyotime) in accordance with manufacturers’ protocols.

2.8 Intracellular ferrous iron assay

The intracellular ferrous iron (Fe²⁺) was detected using a fluorescent probe FerroOrange (Dojindo, Japan) according to the manufacturer’s protocol. In brief, the cells were inoculated into 24-well plates and incubated overnight in incubators. After that, the supernatant was discarded and the cells were washed with PBS for 3 times. FerroOrange working liquid with a concentration of 1μmol/L was added and incubated at 37℃ and 5% CO₂ for 20-30 min. Directly removed and observed under a fluorescence microscope. The average fluorescence intensity was quantitatively analyzed using ImageJ software.

2.9 Western blot assay

RAW264.7 cells were seeded in 6-well plates. Total proteins were extracted from RAW264.7 cells using RIPA buffer (Beyotime) containing phenylmethylsulfonyl fluoride and phosphatase inhibitors. Then, the lysates were centrifuged at 13000 g for 15 min at 4 ° C and the supernatant was collected. Quantitative analysis of protein concentration was measured using a BCA assay kit (Mishu, Xi’an, China). Cellular proteins were separated by 6-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically onto polyvinylidene difluoride membranes. The membranes were blocked with 5% no-fat milk for 2 h at room temperature. The bands were probed at 4°C overnight with the following primary antibodies: c‐Fos, NFATc1 (Abcam, Cambridge, MA), MZF1 (Affinity, Jiangsu, China), TRAP, Nrf2, GPX4 (Cell Signaling Technology, Beverly, MA, USA), CTSK (Beyotime). Then washed 3 times with TBST and the membranes were incubated for 2 h at room temperature with horseradish-peroxidase conjugated secondary antibody (Beyotime). Finally, the signals of target proteins were detected using the Bio-Rad imaging system with the ECL imaging kit (Beyotime). The intensity of the bands was analysed using the ImageJ software.

2.10 Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR).

Total RNA was extracted from cultured RAW264.7 cells using TRIzol (Thermo Fisher Scientific), Then extracted RNA received reverse transcription utilizing a Prime Script RT kit. The mRNA levels were performed real-time quantitative PCR with SYBR green on Bio‐Rad PCR system (Bio‐Rad). The primers of selected target genes employed are listed in Table 1. Gene expression levels were measured by 2^−ΔΔCTmethod and the data were normalized by GAPDH.

2.11 Experimental animals

C57BL/6 female mice were bought from the Animal Center of Xi’an Jiao Tong University. All mice were housed in the animal facility of the translation center of Xi’an Honghui Hospital under a 12-hour light and 12-hour dark cycle at room temperature (22± 2 °C) with 50% humidity and provided with water and standard diet ad libitum. All animal care and experimental procedures were approved by Animal Care Committee of Hong-Hui Hospital, Xi’an Jiaotong University College of Medicine and conducted strictly followed by “the institutional guidelines for the care and use of laboratory animals at the Jiaotong University College of Medicine”. Short interfering RNAs (siRNA) targeting MZF1 were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). The sequence of siMZF1 in vivo is GCCCAGAAGUACAUUCCAATT. For transfection, we injected siMZF1 into the tail vein of C57BL/6 female mice for 8 weeks.

2.12 Ovariectomy (OVX)-induced osteoporosis mouse model

For the OVX-induced osteoporosis model, Eight‐week‐old C57BL/6 female mice were anesthetized with chloral hydrate administered intraperitoneally and bilateral ovaries were removed. Mice in the sham group underwent the same procedure without bilateral ovaries resected. After eight weeks, all the mice were sacrificed and the femurs were harvested for micro-CT analysis and histological staining.

2.13 Micro-computed tomography (CT) and analysis

The micro-CT device (SKYSCAN 1172, Belgium) was applied to evaluate the distal femur samples. The scanning parameters were set as 90 kV of source voltage; 120 μA of source current; 0.5 mm of AI filter; 8 μm of pixel size; 180 degrees rotation step. After scanning, relevant software was used to select appropriate regions of interest for quantitative analysis. The relative bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) parameters were obtained for evaluation.

2.14 TRAP and Histological staining

The femur bones were fixed in 4% paraformaldehyde overnight and decalcified in 14% ethylenediaminetetraacetic (EDTA) acid at room temperature for 3 weeks. Thereafter, the femur samples were embedded into the paraffin cassettes and cut into 4-5 µm thick sections by using a microtome. Histological sections were prepared for hematoxylin and eosin (H&E) and TRAP staining and histological analysis. TRAP staining was used to observe multinucleated osteoclasts, and H&E staining were performed to observe trabecular bones. The images were captured using a light microscope (Nikon, Tokyo, Japan).

2.15 Statistical analysises

Data are expressed as means ± standard deviation (SD). GraphPad Prism was utilized for the statistical analysis and visualization (9.5.0). The two-tailed Student’s t-test was applied for comparisons of two groups, and one-way analysis of variance (ANOVA) was implemented for three or more groups comparisons. The value of p < 0.05 was considered indicative of statistical significance.

3.1 MZF1-deficient female mice exhibit an osteoporotic phenotype.

To determine the potential role of MZF1 in osteoporosis, we generated siMZF1 mice and then performed morphological three-dimensional (3D) micro-CT to evaluate the phenotype of isolated distal femur bones (Fig. 1A). Based on reconstructed 3D micro-CT images, quantitative bone morphological parameter evaluation showed that compared with wild-type (WT) group, trabecular number (Tb. N), trabecular thickness (Tb. Th,) and volume of bone to tissue (BV/TV) were decreased and the trabecular separation (Tb. sp) was increased in siMZF1 group (Fig. 1B). Interestingly, these skeletal changes were consistent with the osteoporotic phenotype. Meanwhile, the microstructure of bone tissue was further evaluated by histological staining with H&E. The osteoporosis phenotype of siMZF1 mice was also evident in H&E stained tissue samples (Fig. 1C). Increased osteoclast formation and bone resorption are important causes of the low bone mass osteoporosis phenotype. The number and activity of osteoclasts were evaluated by TRAP staining of femoral bone sections. As shown in the figure (Fig. 1C), femur bone sections of siMZF1 mice exhibited a higher number of osteoclasts and greater bone resorption activity compared with WT mice. In addition, OVX mouse models also demonstrated reduced MZF1 expression in osteoporosis. OVX mice had a smaller femoral bone volume than control mice (Fig. 1D). Similarly, compared with the control group, the expression of MZF1 gene was downregulated in the OVX group (Fig. 1E). Taken together, these results suggested that MZF1 expression is downregulated in osteoporosis.

3.2 MZF1 expression is negatively correlated with osteoclast differentiation independently induced by RANKL.

Murine RAW264.7 macrophage cells, as a classical cell model for osteoclast differentiation, is a mature preosteoclast cell line[28]. Document studies, including our previous study, have demonstrated that RANKL can independently induce differentiation of RAW264.7 into mature multinuclear osteoclasts in α-MEM[29, 30]. In the whole process of our experiment, as shown in (Fig. 2A, B and C ), TRAP and F-actin staining results showed that, morphologically, 40 ng/mL RANKL could independently induce RAW264.7 to differentiate into osteoclasts, and with the increase of induction time, especially on the 5th day of differentiation, there were a large number of TRAP positive multi-nucleated macrophages and the osteoclastic cytoskeletal sealing zone with bone erosion function. In addition, ROS has been reported as key regulators of osteoclast differentiation and a major therapeutic target for bone loss diseases. Therefore, we evaluated the effect of intracellular ROS production during RANKL-mediated RAW264.7 cell differentiation by using a DCFH-DA fluorescence-based ROS assay. The results showed that the fluorescence intensity of control group was significantly decreased than the RANKL treatment group (Fig. 2D and E). Furthermore, to study whether MZF1 is involved in osteoclast differentiation and formation, to test whether MZF1 deficiency–induced bone loss is associated with osteoclast differentiation and activity, we used RANKL-induced RAW264.7 cells to detect the expression levels of mRNA and protein. In osteoclast differentiation, the expression levels of specific proteins and genes such as TRAP, CTSK and c-Fos, NFATc1, increase significantly as cell differentiation progresses. (Fig. 2F and G). However, mRNA and protein expression levels of MZF1 decreased gradually in a time-dependent manner and decreased to the minimum at day 7 (Fig. 2F and G). These data indicate that the expression of MZF1 is negatively correlated with the osteoclastogenesis.

3.3 CRISPR/Cas9 mediated knockout of MZF1 in RANKL-stimulated RAW264.7 cells.

To confirm the role of MZF1 in osteoclast differentiation by knockout of MZF1 using the CRISPR/Cas9 gene editing technique (Fig. 3A). Compared to the original sequence, the sequencing results demonstrate a single base deficiency of the MZF1 gene accompanied by a frame shift, resulting in MZF1 gene mutation (Fig. 3C). Furthermore, PCR results showed that the expression level of MZF1 mRNA in RAW264.7 cells stably transfected with MZF1 was significantly higher than that in normal RAW264.7 cells, and the homozygous cells with mouse gene MZF1^-/- were successfully constructed (Fig. 3B). At the same time, the expression of MZF1 was detected at the protein level in MZF1^+/+(WT) and MZF1^-/- cell lines. Western blotting also verified the expression levels of the MZF1 protein were significantly reduced in the MZF1^-/- group compared to normal RAW264.7 cells (Fig. 3D). These data confirmed that knockout of MZF1 in RAW264.7 cells were successfully established.

3.4 MZF1 downregulation stimulates osteoclastogenesis of RAW264.7 cells.

To detect the effect of MZF1 KO on RAW264.7 cells, we evaluated cell viability by CCK-8 assay at 0, 24, 48 and 72 h during the early stages of osteoclast formation in RANKL stimulation. As shown in (Fig. 4E), the CCK-8 absorbance showed that MZF1^-/- group cells promoted cell viability compared with the WT group. Next, to explore the impact of MZF1 on osteoclast differentiation and bone resorption, we performed in vitro cellular and biochemical analyses. We first examined the differentiation potential of osteoclast precursor cells in WT and MZF1^-/- groups. RAW264.7 cells were treated with RANKL for 5 days to investigate whether MZF1 is involved in osteoclastogenesis. Surprisingly, the results were contrary to our expectations, with light microscopy and TRAP staining results showed that the MZF1^-/- group produced more TRAP-staining positive multinucleated macrophages in number and size than the WT group (Fig. 4A and B). Upon contact with the bone matrix, mature osteoclasts secrete H⁺ to the bone surface, and the cytoskeleton is reorganized, and then sealing zone and ruffled border are formed, making osteoclasts tightly bind to the bone surface. The sealing zone, consisting of actin rings, is an important functional domain separating the osteoclast adhesion zone from the microenvironment between bone tissues[31]. Therefore, we performed F-actin assay to evaluate the osteoclastic cytoskeletal sealing zone structure formation in WT and MZF1^-/- groups after stimulation by RANKL, and to evaluate the effect on bone resorption function of osteoclasts. After 5 days of RANKL stimulation, phalloidin-FITC staining revealed that MZF1^-/- group formed more and larger actin rings compared with WT group (Fig. 4C and D). These outcomes provide the functional evidence that, morphologically, MZF1 deficiency promotes osteoclast precursors toward their differentiation into mature and more active osteoclasts.

3.5 MZF1 deficiency changed the gene expressions during osteoclast differentiation.

As mentioned above, MZF1 negatively regulates osteoclast differentiation morphologically. In order to further investigate the molecular mechanism of MZF1^-/- promoting osteoclast differentiation, we then investigated the regulatory effect of MZF1^-/- group on key genes of osteoclast differentiation. TRAP and CTSK are specific genes for osteoclast differentiation, and c-Fos and NFATc1 are master regulator of osteoclast differentiation. Total RNA was extracted at 1, 3 and 5 days after RANKL stimulation, and the expression levels of key genes in osteoclast differentiation were detected by RT-qPCR. The results showed that the expression levels of genes related to osteoclast differentiation, such as TRAP, CTSK, c-Fos and NFATc1 were significantly increased in MZF1^-/- cells (Fig. 5A). In addition, Western blot analysis and protein quantitation revealed that the expression level of CTSK, TRAP, c-Fos and NFATc1 proteins in MZF1^-/- cells were also higher than in control cells at day 1, 3 and 5(Fig. 5B). These results suggest that MZF1 can negatively regulate osteoclast differentiation through the transcription of target genes.

3.6 MZF1 may regulate RANKL-induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway during the early stages of cell differentiation.

MZF1^-/- group cells significantly promoted the morphological differentiation of osteoclasts and the expression of key genes for osteoclastogenesis. In addition, the above results showed the MZF1^-/- group was significantly enhanced cell viability in the early stages of cell differentiation. To further identify the specific molecular mechanisms of this phenotype. We wanted to know whether MZF1 regulation of osteoclastogenesis affects the process of cell death. Ferroptosis is a regulatory cell death characterized by iron and lipid peroxidation[13]. We examined some prevalent ferroptosis markers, including malondialdehyde (MDA), a byproduct of lipid peroxidation, glutathione (GSH), a cofactor of GPX4, and intracellular Fe, the active form of iron in the cytoplasm during the early stages of osteoclastogenesis[14, 32, 33]. The results showed that MDA and ferrous iron were significantly down-regulated in the MZF1^-/- group under RANKL stimulated early stage compared with the WT group. GSH level was increased in response to RANKL stimulated early stage in the MZF1^-/- group (Fig. 6A and B). We also tested total ROS level with DCFH-DA, since total ROS level always increase significantly in ferroptosis. Expectedly, we observed that ROS levels in the MZF1^-/- group were significantly down-regulated in the early stages of RANKL-mediated osteoclastogenesis (Fig. 6C). Nrf2/GPX4 is a key signaling pathway that regulates ferroptosis in cells. Nrf2 signaling pathway is the direct downstream pathway of ROS, which can trigger downstream antioxidants to remove excess ROS in cells[34, 35]. Immunofluorescence staining was used to evaluate the expression and localization of Nrf2 protein in WT and MZF1^-/- cells. The results showed that the expression and nuclear localization of Nrf2 were increased in the MZF1^-/- group (Fig. 6D). Furthermore, western blotting and PCR were used to detect the expression levels of GPX4 and Nrf2 protein and mRNA. The results showed that the expression levels of GPX4 and Nrf2 protein and gene were increased in the MZF1^-/- group (Fig. 6E-G). In conclusion, MZF1 may regulate RANKL-induced ferroptosis of osteoclasts in the early stage of cell differentiation through Nrf2/GPX4 signaling pathway.

Bone homeostasis disorder is the main mechanism of osteoporosis. When the homeostasis between bone resorption and bone formation is broken, osteoclast differentiation and function are active, forming bone lacunae on the bone surface and then destroying bone[36, 37]. Abnormal activation of osteoclasts is the main pathological factor of OP[38]. Therefore, it is still a key strategy to delay the development of OP to further study the signal pathways related to osteoclast fusion, screen the key molecular targets that regulate osteoclast differentiation and function, improve the cognition level of bone metabolism, and then maintain the dynamic balance between osteoclast differentiation and osteoblast differentiation. MZF1 is a member of the SCAN zinc finger transcription factor family. MZF1 protein plays an important role in the occurrence and development of tumors. Moreover, MZF1 is an important transcription factor that regulates N-cadherin promoter activity and osteoblast expression[39]. However, the role of MZF1 in osteoclast differentiation and osteoclast-mediated bone resorption has not been demonstrated. Here, we demonstrated in vitro and in vivo that MZF1 deficiency promotes osteoclast differentiation and that MZF1 plays a negative regulatory role in RANKL-induced osteoclast differentiation and activation.

In this study, the in vivo anabolism of MZF1 and its regulatory effect on skeletal phenotype was investigated by injecting siMZF1 intermittently into mice for 8 weeks. Microstructure of bone tissue was assessed by 3D micro-CT scans and histological staining. Our results showed that the Tb. N, Tb. Th and BV/TV of distal femur of mice were significantly reduced in siMZF1 group, and Tb. Sp was increased. Interestingly, we found a novel anabolic effect of MZF1 in bone. Deficiency of MZF1 results in low bone volume in young adult mice, this change in bone structure is consistent with the osteoporotic skeletal phenotype. Further, we established the OVX-induced postmenopausal osteoporosis model, and RT-PCR detection showed that MZF1 expression decreased in OVX group compared with WT group. Our in vivo study demonstrated that the expression of MZF1 was decreased in osteoporosis models. As previously reported, RANKL independently stimulated RAW264.7 cells to establish osteoclast models in vitro[30]. To test whether MZF1 deficiency–induced bone loss is associated with osteoclastic differentiation and activity. Our results demonstrated that the expression level of MZF1 decreased gradually during the differentiation of osteoclasts, suggesting MZF1 could affect the differentiation and function of osteoclasts. To further understand the cellular effects of MZF1 on osteoclast differentiation and bone resorption function, in vitro cellular and biochemical analyses were performed.

Firstly, we used CRISPR/Cas 9 gene editing to establish a MZF1 knockout RAW264.7 cell model. Using the sequencing results and western blotting to identify the expression of MZF1, the precursor osteoclast model with stable low expression of MZF1 was successfully constructed. The MZF1^-/- group cells promoted cell proliferation or viability, suggesting that MZF1 may be involved in cell division in a macrophage-like cell lineage. Further, morphological analysis of the differences in cell differentiation and function between the two groups showed that the RAW264.7 cells with MZF1^-/- group cells differentiated into more TRAP-positive multi-nucleated osteoclasts, and the knockout group also showed more complete sealing zone with bone resorption function. MZF1-deficient RAW264.7 cells promoted the expression of osteoclast marker genes (TRAP, CTSK, c-Fos, NFATc1), increased mature osteoclast formation, and enhanced bone resorption capacity. Finally, the results confirmed that MZF1 negatively regulated osteoclast differentiation, formation and function.

Iron is an essential trace element in the body that plays a key role in physiological processes such as oxygen transport, DNA synthesis, production of ATP as an energy source, and involvement in the immune system's defense against bacterial infection[40, 41]. Iron homeostasis is also closely related to bone health. Abnormal iron metabolism can destroy bone balance and lead to osteoporosis[42]. In some iron overload models, enhanced osteoclast differentiation and osteoblast apoptosis were induced, showing a phenotype of reduced bone density and trabecular number, leading to increased osteolysis[43, 44]. Iron overload is widely regarded as an independent risk factor for osteoporosis[40]. Ferroptosis is a modulated cell death pattern characterized by the accumulation of ROS and lipid peroxidation products caused by the disturbance of iron metabolism[13, 45]. Several studies reported that Ferroptosis is involved in RANKL-mediated osteoclastogenesis[20, 46, 47]. Chen et al. showed that MZF1 acts as a tumor suppressor in prostate cancer, partly through enforcing ferroportin-conducted iron egress[27].

In the present study, we found that MZF1^-/- group cells increased cell viability in the early stages of cell differentiation, and then we wondered whether knockout cells attenuated cell aging and affected RANKL-induced ferroptosis. We detected the cell ferroptosis markers including MDA, GSH and intracellular Fe. In MZF1 knockout group, MDA and ferrous iron decreased, while GSH level increased. The accumulation of lipid ROS was also the most significant feature of ferroptosis. As we expected, our results demonstrated that ROS expression was also decreased in the knockout group. In addition, we examined the expression of some key genes involved in ferroptosis, such as Nrf2 and GPX4. Nrf2, as a transcription factor that initiates endogenous antioxidant reaction elements, occurs nuclear transport under the stimulation of ROS, thus producing antioxidant stress effects[48]. GPX4, as an important sensor of oxidative stress and cell death signals, has been considered as one of the important targets triggering iron death[49]. In the early stage of osteoclast differentiation stimulated by RANKL, MZF1^-/- group causes up-regulation of Nrf2 and GPX4 genes and transcription proteins. The results of fluorescence staining showed that MZF1^-/- group reduced the nuclear translocation of Nrf2 at the early stage of RANKL stimulation. In conclusion, in the early stage of osteoclast differentiation, MZF1 may interfere with iron metabolism of RANKL-induced osteoclasts and regulate osteoclasts by influencing the expression of Nrf2 and GPX4.

In conclusion, we demonstrated for the first time the important role and mechanism of MZF1 in osteoclast differentiation. Morphologically, MZF1 knockout significantly increased osteoclast differentiation, and further mechanism analysis indicated that MZF1 may affect ferroptosis during RANKL-induced osteoclast differentiation. However, other downstream molecular mechanisms involved in MZF1 regulation of osteoclast differentiation remain elusive and need to be further confirmed by clinical and animal experiments. The regulatory effect of MZF1 on osteoclasts may be a useful therapeutic target for the prevention and treatment of osteolytic diseases.

Authors’ contributions

ZQ, LK, and LY. conceptualized the work; ZQ. design the study and draft the manuscript; ZQ, BZ YZ and YZ. analysis and interpretation of data and carried out the experiments; LK and YG. intellectual input and supervision; XG, MF and JZ. collected the literature; LY. funding acquisition.

All authors have reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (82260181), National Natural Science Foundation of China (82070909), Key project of Natural Science Basic Research Plan of Shaanxi Province (2022JZ-43) and General Project of Natural Science Basic Research Plan of Shaanxi Province (2021JQ-069).

Availability of data and materials

The data supporting the conclusions of this article are included within the article or are available from the corresponding author on reasonable request.

Acknowledgements

The authors thank the staff of the translation center of Xi’an Honghui Hospital for their support during this study.

Ethics approval and consent to participate

All animal care and experimental procedures were approved by Animal Care Committee of Hong-Hui Hospital, Xi’an Jiaotong University College of Medicine and conducted strictly followed by "the institutional guidelines for the care and use of laboratory animals at the Jiaotong University College of Medicine ".

Competing Interests

The authors declare that they have no competing interests.

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Myeloid zinc finger 1 knockdown promotes osteoclastogenesis and bone loss in part by regulating RANKL- induced ferroptosis of osteoclasts through Nrf2/GPX4 signaling pathway

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