Abstract
Increased inflammatory response is one of the major characteristics of osteonecrosis of the femoral head (ONFH). We aimed to investigate the function of bone morphogenetic protein 2 (BMP-2)/interleukin (IL)-34 axis in the inflammatory responses of ONFH. The systemic and local expression of BMPs in ONFH patients was detected by qRT-PCR and ELISA. In vitro osteoclast differentiation and ONFH mouse models, induced by 20 mg/kg methylprednisolone through i.m. injection, were established using WT and BMP-2−/− mice to explore the regulatory role of BMP-2 in pro-inflammatory responses and bone defects of ONFH. IL-34 expression and function were examined in vitro and in vivo through qRT-PCR, tartrate-resistant acid phosphatase (TRAP) staining, and gene knockout. The systemic and local expression of BMPs was elevated in ONFH patients. BMP-2 reduced the production of pro-inflammatory cytokines and inhibited the differentiation of osteoclasts. Mechanistically, BMP-2 inhibited osteoclasts formation through suppressing IL-34 expression and then promoted bone repair and alleviated ONFH. In conclusion, our study reveals that BMP-2 inhibits inflammatory responses and osteoclast formation through downregulating IL-34.
Introduction
Osteonecrosis of the femoral head (ONFH) is regarded as degenerative osteoarthritis of the hip caused by the interruption of blood supply and hypoxia of the femoral head, which then lead to the collapse of the femoral head (Moya-Angeler et al. 2015). There has been an increasing trend of ONFH cases in the past decades: about 20,000 people were diagnosed with ONFH in the USA, and more than 10% hip arthroplasties are carried out for ONFH treatment each year (Choi et al. 2015, Moya-Angeler et al. 2015). There are many causes of ONFH: genetic predisposition, radioactive factors, systemic steroid hormone application, metabolic factors, viral infection, and local factors that obstruct blood supply such as mechanical stress, intraosseous pressure, and vascular damage (Grose et al. 2008, Shah et al. 2015). So far, the pathophysiological mechanism of ONFH is not fully understood.
Cumulative evidence indicates that inflammation is involved in the occurrence and development of ONFH. The expression and secretion of pro-inflammatory cytokines are boosted in ONFH, including interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 (Samara et al. 2012, Li et al. 2015, Chen et al. 2018). Bone morphogenetic protein 2 (BMP-2) signaling pathway is composed of BMP-2, receptors and signal transducers, and a variety of cytokines, which promotes osteoblast differentiation and bone extracellular matrix synthesis and secretion and then affects bone metabolism, cartilage degeneration, destruction, and repair (Varas et al. 2015, Salazar et al. 2016, Hashimoto et al. 2020). Recent studies indicated that BMP-2 participates in inhibiting the expression of IL-34, a key mediator of inflammation and osteoclastogenesis in bone degenerative diseases, by affecting the activin receptor-like kinase 1 (ALK1) and ALK5 pathways in rheumatoid arthritis synovial fibroblasts (Bostrom & Lundberg 2013, Chemel et al. 2017). As a cytokine, IL-34 functionally overlaps with macrophage colony stimulating factor (M-CSF), which can regulate the survival, proliferation, and differentiation of monocytes and macrophages by binding to CSF-1R (Boulakirba et al. 2018, Easley-Neal et al. 2019). In addition, IL-34 is not only involved in regulating cell biological activity, but also is critical for the occurrence and development of various inflammatory diseases (Ge et al. 2019, Xu et al. 2019). However, the regulatory role of BMP-2 in inflammatory response and bone defects of ONFH is still unknown.
Materials and methods
Human studies
In a cross-sectional study, we examined the BMP genes (BMP2, BMP4, and BMP7) expression in proximal femur tissue samples from 24 patients (10 male and 14 female) with ONFH and 24 subjects (12 male and 12 female) with osteoarthritis (OA). From 2018 to 2021, subjects (20–60 years old) with newly diagnosed ONFH were selected based on International Classification of Disease, 9th Revision of Clinical Modification (ICD-9). The diagnosis date of ONFH was defined as the index date. OA patients were diagnosed by clinical and radiological criteria and fulfilled the American College of Rheumatology (ACR) criteria for the classification of OA. The study has been approved by the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Subjects gave written informed consent before taking part in the study. Proximal femur tissue was immediately frozen in liquid nitrogen after surgery.
Animals
The 8-week-old C57BL/6 WT mice were ordered from Cyagen Biosciences Inc, Suzhou, China. Il34tm1e(EUCOMM)Wtsi (2010004A03Rik) with C57BL/6 background were purchased from Shanghai Yunhao Biotech Company (Shanghai, China). To generate Bmp2 knockout mice, Bmp2 floxed (bmp2 flox/flox) homozygous mice with C57BL/6 background (B6; 129S4-Bmp2tm1Jfm/J) were obtained from Shanghai Yunhao Biotech Company and crossed with ERT2-cre mice. Tamoxifen (#10540-29-1, Sigma) was injected to generate the Bmp2 knockout mice (BMP-2−/−). Animal studies were approved by Shanghai Jiao Tong University Affiliated Sixth People’s Hospital.
Osteoclastogenesis and osteoclast differentiation
The femurs and tibia were used to isolate whole bone marrow cells (BMCs) as described previously (Suzuki et al. 2002). Briefly, flushed BMCs were placed in 100 mm plates with minimum essential media (MEM) plus 10% fetal bovine serum (FBS, Hyclone). After overnight incubation, non-adherent cells were collected and transferred to a new dish with normal culture medium. The differentiation of osteoclasts was induced by culturing bone marrow-derived monocytes in 50 ng/mL M-CSF (M6518, Sigma) or 50 ng/mL IL-34 (SRP3287, Sigma) for 3 days and then with the stimulation of 25–100 ng/mL RANKL (# PHP0034, Thermo Fisher Scientific) for additional 3 days.
Tartrate-resistant acid phosphatase (TRAP) analysis
The differentiation of IL-34-induced osteoclasts from BMCs was analyzed by using leukocyte acid phosphatase kit (MilliporeSigma, Burlington, USA), following the manufacturer’s instruction.
ONFH model
Sixty 8-week-old mice were used in this study to establish the steroid-induced ONFH model. The mice of the experimental group received 20 mg/kg methylprednisolone (MPS; Pfizer) through i.m. injection. The mice of the control group received normal saline three times per week, for 3 weeks. Six weeks later, the mice were euthanized; serum, proximal femur tissue, and the femoral heads were collected for the subsequent analyses.
Micro-CT analysis
Femoral heads dissected from mice of the above control and experimental groups were fixed in formalin for 16 h and then scanned and analyzed by using the SkyScan1178 scanner (Bruker, Kontich, Belgium). Changes in the bone trabeculae and excised femoral head sample were detected using a Micro-CT. The scanning parameters were set as follows: 500 μA source current, 0.5 mm aluminum filter, 50 kV, 9 μm isotopic resolution, a voxel resolution of 20 μm, exposure time 700 ms, and three projection images per 0.3° rotation step. NRecon software was used for data reconstruction, with 40% beam hardening correction, ring artefact correction, and Gaussian smoothing applied. Using DataViewer software, each dataset was normalized regarding its orientation and saved in transaxial (X-Y) projections and then exported to CTAn software. Microarchitecture parameters included trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), trabecular number (Tb.N), and bone volume/tissue volume (BV/TV). DataViewer was used to image the sagittal section of samples.
Quantitative real-time PCR (qRT-PCR)
Total RNAs of the osteoclasts were isolated by using TRIzol RNA isolation reagents (Thermo Fisher Scientific). Human and mice proximal femur tissue samples were homogenized using LabGEN 125 Homogenizer (Cole-Parmer, Vernon Hills, USA), and total RNA was extracted as described above. The cDNA was synthesized using random primers and M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, USA). The qRT-PCR was performed using iQTM SYBR green supermix on the iCycler Sequence Detection System (Bio-Rad). Gene expression was calculated by 2−ΔΔCT method and normalized to the expression of Actin. Primers used in this study are listed below: Cstk forward 5’-cca gtgg gag cta tgg aaga-3’; Cstk reverse 5’-aag tggt tca tgg cca gtt c-3’; Acp5 forward 5’-cgt ctct gca cag att gca t-3’; Acp5 reverse 5’-gta gtcc tcc ttg gct gct g-3’; Calcr forward 5’-cgg act tga cac agc aga a-3’; Calcr reverse 5’-gtc accc tct ggc agc taa g-3’; Il6 forward 5’-ctg atgc tgg tga caa cca c-3’; Il6 reverse 5’-cag act gcc att gca caa c-3’; Tnf forward 5’-cat cttc tca aaa ttc gag tga caa-3’; Tnf reverse 5’- cca gctg ctc ctc cac ttg -3’; Il1b forward 5’-aag cctc gtg ctg tcg gac c-3’; and Il1b reverse 5’-tga ggcc caa ggc cac agg t-3’.
ELISA
Serum of patients with ONFHH or OA and serum of WT and BMP-2−/− mice with steroid-induced ONFH were collected for the measurement of BMPs and cytokines using commercial ELISA kits (Thermo Fisher Scientific Inc.). The concentration of BMP2 and BMP4 in patient serum was determined by using BMP-2 Human ELISA Kit (# EHBMP2) and BMP-4 Human ELISA Kit (# EHBMP4), respectively. Pro-inflammatory cytokines, IL-6, TNF-α, and IL-1β, in mice serum were measured by using IL-6 Mouse ELISA Kit (# BMS603-2), TNF alpha Mouse ELISA Kit (# BMS607-3TWO), and IL-1 beta Mouse ELISA Kit (# BMS6002), respectively, following the protocols provided by the manufacturer.
Statistical analysis
Statistical analyses were conducted by using the Prism software (V8.0, GraphPad Software). One-way ANOVA with a post hoc test, or Student’s t-test was used to analyze the differences between groups. Data were represented as mean ± s.e.m. Experiments were repeated at least three times unless indicated.
Results
BMPs are upregulated in the patients with ONFH
To evaluate the correlation between ONFH pathogenesis and factors that regulate bone formation and transformation in patients, we analyzed the mRNA level of BMP2, BMP4, and BMP7 in the proximal femur tissues of ONFH patients and OA patients using qRT-PCR. In comparison with OA patients, the expression level of the three BMPs was significantly increased in ONFH patients (Fig. 1A). Moreover, ELISA results showed that both BMP2 and BMP4 protein levels in ONFH patients were higher than those in OA patients (Fig. 1B). These data suggested that bone formation and transformation factors might contribute to the pathogenesis of ONFH in patients.
The expression levels of BMPs were upregulated in ONFH patients. (A) The mRNA level of Bmp2, Bmp4, and Bmp7 in the proximal femur of ONFH or OA patients was assessed by qRT-PCR (n = 24). (B) BMP-2 and BMP-4 levels in the serum of ONFH or OA patients were detected by ELISA (n = 24). The data were expressed as means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
The expression levels of BMPs were upregulated in ONFH patients. (A) The mRNA level of Bmp2, Bmp4, and Bmp7 in the proximal femur of ONFH or OA patients was assessed by qRT-PCR (n = 24). (B) BMP-2 and BMP-4 levels in the serum of ONFH or OA patients were detected by ELISA (n = 24). The data were expressed as means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
The expression levels of BMPs were upregulated in ONFH patients. (A) The mRNA level of Bmp2, Bmp4, and Bmp7 in the proximal femur of ONFH or OA patients was assessed by qRT-PCR (n = 24). (B) BMP-2 and BMP-4 levels in the serum of ONFH or OA patients were detected by ELISA (n = 24). The data were expressed as means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 deficiency promoted inflammatory response and bone defects in ONFH mice
In order to investigate the regulatory role of Bmp2 in ONFH progression, we established the steroid-induced ONFH model using WT and tamoxifen-mediated BMP-2−/− mice and detected the pro-inflammatory cytokines levels in proximal femur tissues and serum. In WT mice, both the mRNA and protein levels of pro-inflammatory cytokines, IL-6, TNF-α, and IL-1β, were significantly elevated in ONFH mice compared with those in control mice (Fig. 2A and B). Interestingly, the mRNA level of pro-inflammatory cytokines was further increased in BMP-2−/− ONFH mice compared to that in WT ONFH mice (Fig. 2A). Similarly, protein level of IL-6, TNF-α, and IL-1β was significantly upregulated in BMP-2−/− ONFH mice (Fig. 2B). Next, we evaluated the microstructural bone defect of WT and BMP-2−/− with or without ONFH through Micro-CT analysis. As shown in Fig. 3, multiple microstructural parameters of bone, BV/TV (%), Tb.Th (μm), and Tn.N, were decreased in ONFH mice compared to those in non-ONFH mice. In contrast, bone defect marker Tb.Sp (μm) was increased in ONFH mice relative to non-ONFH mice. Importantly, the extent of ONFH symptoms was significantly worse in BMP-2−/− ONFH mice compared to that in WT ONFH mice. The above data indicated that BMP-2 deficiency could further exacerbate inflammatory response and bone defects, which might be an important regulator in ONFH pathogenesis.
BMP-2 deficiency promoted inflammatory response in the mice with steroid-induced ONFH. (A) The expressions of IL-6, TNF-α, and IL-1β in the proximal femur tissues of mice in the indicated groups were measured by qRT-PCR (n = 4). (B) ELISA of the above cytokines in the serum of WT and BMP-2−/− mice with steroid-induced ONFH (n = 4). Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. **P < 0.01.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 deficiency promoted inflammatory response in the mice with steroid-induced ONFH. (A) The expressions of IL-6, TNF-α, and IL-1β in the proximal femur tissues of mice in the indicated groups were measured by qRT-PCR (n = 4). (B) ELISA of the above cytokines in the serum of WT and BMP-2−/− mice with steroid-induced ONFH (n = 4). Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. **P < 0.01.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 deficiency promoted inflammatory response in the mice with steroid-induced ONFH. (A) The expressions of IL-6, TNF-α, and IL-1β in the proximal femur tissues of mice in the indicated groups were measured by qRT-PCR (n = 4). (B) ELISA of the above cytokines in the serum of WT and BMP-2−/− mice with steroid-induced ONFH (n = 4). Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. **P < 0.01.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 deficiency promoted the pathogenic symptoms in the mice with steroid-induced ONFH. Statistical analysis was performed on BV/TV, Tb. N, Tb.sp., and Tb.Th (n = 5) of WT and BMP-2−/− mice with ONFH. The data were presented as bar graph. The data were expressed as means ± s.e.m. *P < 0.05; **P < 0.01.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 deficiency promoted the pathogenic symptoms in the mice with steroid-induced ONFH. Statistical analysis was performed on BV/TV, Tb. N, Tb.sp., and Tb.Th (n = 5) of WT and BMP-2−/− mice with ONFH. The data were presented as bar graph. The data were expressed as means ± s.e.m. *P < 0.05; **P < 0.01.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 deficiency promoted the pathogenic symptoms in the mice with steroid-induced ONFH. Statistical analysis was performed on BV/TV, Tb. N, Tb.sp., and Tb.Th (n = 5) of WT and BMP-2−/− mice with ONFH. The data were presented as bar graph. The data were expressed as means ± s.e.m. *P < 0.05; **P < 0.01.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 is dispensable for pro-inflammatory cytokines production
To explore the underlying mechanism of BMP-2-regulated pro-inflammatory response in ONFH progression, we induced the in vitro differentiated osteoclasts derived from BMCs of WT and BMP-2−/− mice. Upon the stimulation of LPS, the mRNA level of pro-inflammatory cytokines was significantly increased in differentiated osteoclasts compared to non-LPS control; however, there was no difference between WT and BMP-2−/− (Fig. 4A). In line with the mRNA data, ELISA results showed that the production of pro-inflammatory cytokines was comparable in differentiated osteoclasts derived from WT and BMP-2−/− mice (Fig. 4B). These results suggested that BMP-2 might not regulate the inflammatory response of osteoclasts directly.
BMP-2 was dispensable for the expression of proinflammatory cytokines in osteoclast. (A) The expression of indicated cytokines in primary M-CSF-induced osteoclast stimulated with LPS (100 ng/mL) was measured by qRT-PCR. Relative mRNA expression for each gene was expressed as fold induction based on the Actin. (B) ELISA of the indicated cytokines in the supernatants of osteoclast stimulated with LPS for 24 h. Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. ns, no significance.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 was dispensable for the expression of proinflammatory cytokines in osteoclast. (A) The expression of indicated cytokines in primary M-CSF-induced osteoclast stimulated with LPS (100 ng/mL) was measured by qRT-PCR. Relative mRNA expression for each gene was expressed as fold induction based on the Actin. (B) ELISA of the indicated cytokines in the supernatants of osteoclast stimulated with LPS for 24 h. Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. ns, no significance.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 was dispensable for the expression of proinflammatory cytokines in osteoclast. (A) The expression of indicated cytokines in primary M-CSF-induced osteoclast stimulated with LPS (100 ng/mL) was measured by qRT-PCR. Relative mRNA expression for each gene was expressed as fold induction based on the Actin. (B) ELISA of the indicated cytokines in the supernatants of osteoclast stimulated with LPS for 24 h. Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. ns, no significance.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP2 does not inhibit IL-34-induced osteoclastogenesis
The previous studies showed that IL-34 mediated the regulatory effect of BMP-2 in rheumatoid arthritis synovial fibroblasts and played an important role in osteoclastogenesis (Chen et al. 2011, Chemel et al. 2017). To investigate whether IL-34 was also involved in BMP-2-regulated inflammatory response and ONFH progression, we examined the expression of osteoclast markers and the ratio of osteoclast formation with the induction of IL-34 and RANKL using qRT-PCR and TRAP staining. As shown in Fig. 5A, IL-34 treatment significantly upregulated the expression of Acp5, Calcr, and Cstk in control osteoclasts; however, there was no difference in the osteoclast marker expression levels with the co-treatment of BMP-2 recombinant protein. TRAP results also showed that ratios of osteoclast formation and osteoclastogenesis were comparable in IL-34+PBS and IL-34+BMP-2 groups (Fig. 5B). The above data indicated that BMP-2 did not inhibit IL-34-induced osteoclastogenesis and the expression of osteoclast-related genes.
BMP-2 did not inhibit IL-34-induced osteoclastogenesis and the expression of osteoclast-related genes. (A) Bone marrow cells were cultured in IL-34 (50 ng/mL) with or without BMP-2 (100 ng/mL) for 3 days and added RANKL (50 ng/mL) for other 3 days. The expressions of osteoclast genes Calcr, Acp5, and Cstk were determined by qRT-PCR. (B) The differentiation of IL-34-induced osteoclast from bone marrow cells was analyzed by TRAP staining under BMP-2 treatment. Scale bar, 10 μm. TRAP activity was measured using an ELISA reader. Relative mRNA amounts for each gene were expressed as fold induction based on the Actin. Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. ns, no significance.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 did not inhibit IL-34-induced osteoclastogenesis and the expression of osteoclast-related genes. (A) Bone marrow cells were cultured in IL-34 (50 ng/mL) with or without BMP-2 (100 ng/mL) for 3 days and added RANKL (50 ng/mL) for other 3 days. The expressions of osteoclast genes Calcr, Acp5, and Cstk were determined by qRT-PCR. (B) The differentiation of IL-34-induced osteoclast from bone marrow cells was analyzed by TRAP staining under BMP-2 treatment. Scale bar, 10 μm. TRAP activity was measured using an ELISA reader. Relative mRNA amounts for each gene were expressed as fold induction based on the Actin. Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. ns, no significance.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 did not inhibit IL-34-induced osteoclastogenesis and the expression of osteoclast-related genes. (A) Bone marrow cells were cultured in IL-34 (50 ng/mL) with or without BMP-2 (100 ng/mL) for 3 days and added RANKL (50 ng/mL) for other 3 days. The expressions of osteoclast genes Calcr, Acp5, and Cstk were determined by qRT-PCR. (B) The differentiation of IL-34-induced osteoclast from bone marrow cells was analyzed by TRAP staining under BMP-2 treatment. Scale bar, 10 μm. TRAP activity was measured using an ELISA reader. Relative mRNA amounts for each gene were expressed as fold induction based on the Actin. Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. ns, no significance.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 inhibits ONFH via downregulating the induction of IL-34
To further explore the in vivo relationship between BMP-2 and IL-34 in ONFH mice, we examined the IL-34 expression in proximal femur tissues and serum of WT and BMP-2−/− mice. In WT mice, IL-34 mRNA level was significantly upregulated in ONFH compared to non-ONFH mice; interestingly, the highest IL-34 expression was observed in proximal femur tissues of BMP-2−/− ONFH mice (Fig. 6A). Moreover, IL-34 protein showed the similar pattern that BMP-2 deficiency significantly increased the production of IL-34, which plays an important role in osteoclastogenesis (Fig. 6B). Next, we established BMP-2 and IL-34 double knockout mice and profiled the expression of pro-inflammatory cytokines in proximal femur tissues. Strikingly, IL-34 knockout significantly suppressed the BMP-2 deficiency-induced cytokines expression in ONFH mouse model (Fig. 6C). All these results suggested that BMP-2 inhibited ONFH through downregulating the induction of IL-34 in proximal femur tissues.
BMP-2 inhibited ONFH via downregulating the induction of IL-34. The mRNA expression (A) and the protein production (B) of IL-34 in the proximal femur tissues of WT and BMP-2−/− mice with steroid-induced ONFH were measured by qRT-PCR (n = 4). (C) The mRNA expression of IL-6, TNF-α, and IL-1β in the proximal femur tissues of WT and BMP-2−/− mice with steroid-induced ONFH under IL-34-deficient background were measured by qRT-PCR (n = 4). Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. *P < 0.05.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 inhibited ONFH via downregulating the induction of IL-34. The mRNA expression (A) and the protein production (B) of IL-34 in the proximal femur tissues of WT and BMP-2−/− mice with steroid-induced ONFH were measured by qRT-PCR (n = 4). (C) The mRNA expression of IL-6, TNF-α, and IL-1β in the proximal femur tissues of WT and BMP-2−/− mice with steroid-induced ONFH under IL-34-deficient background were measured by qRT-PCR (n = 4). Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. *P < 0.05.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
BMP-2 inhibited ONFH via downregulating the induction of IL-34. The mRNA expression (A) and the protein production (B) of IL-34 in the proximal femur tissues of WT and BMP-2−/− mice with steroid-induced ONFH were measured by qRT-PCR (n = 4). (C) The mRNA expression of IL-6, TNF-α, and IL-1β in the proximal femur tissues of WT and BMP-2−/− mice with steroid-induced ONFH under IL-34-deficient background were measured by qRT-PCR (n = 4). Data are presented as mean ± s.e.m. values and are representative of at least three independent experiments. *P < 0.05.
Citation: Journal of Molecular Endocrinology 68, 1; 10.1530/JME-21-0163
Discussion
The current study investigated the function of BMP-2-IL-34 axis in osteoclast differentiation and mouse ONFH models. We found that BMP-2 not only affects the differentiation of osteoclasts but also affects the expression of pro-inflammatory cytokines. Mechanistically, BMP-2 inhibits the formation of osteoclasts through suppressing the production of IL-34 and then promotes bone repair and alleviates ONFH. Our data suggested that BMP-2 might be a potential drug target for the treatment of ONFH.
ONFH is characterized by increasing inflammatory response, cartilage collapse, loss of joint function, and persistent pain (Qu et al. 2019). Recent studies reported that non-steroidal anti-inflammatory medicines, stem cell therapy, cytokines treatment, and viral vector gene therapy could suppress the inflammatory responses and alleviate the symptoms of ONFH (Tanaka et al. 2003, Rackwitz et al. 2012, Arbab & Konig 2016). The concentration of multiple inflammatory factors in the serum of patients is elevated significantly and involved in the pathological process of ONFH. As the major systemic pro-inflammatory factors, TNF-α and IL-1β have been reported to increase the production of IL-3, IL-12, and MMP-13 and to promote the progression of ONFH (Liacini et al. 2003, Dai et al. 2006). In addition, the overexpression of IL-6 is closely associated with the activation of osteoclasts, and blocking IL-6 could decrease the inflammatory response and promote bone reconstruction (Yamaguchi et al. 2016, Kuroyanagi et al. 2018). Fukui et al. demonstrated that pro-inflammatory cytokines could stimulate BMP-2 expression in osteoarthritic chondrocytes (Fukui et al. 2003). In line with the above studies, our data showed that BMP-2 mRNA and protein levels were elevated significantly in the proximal femur tissues and serum of ONFH patients. Interestingly, the production of pro-inflammatory cytokines was further increased in BMP-2-deficient mice compared to WT mice, which indicated there might be negative feedback regulation between BMP-2 and pro-inflammatory cytokine in the ONFH condition.
IL-34 is a newly identified cytokine that is able to bind to CSF-1R and promote the proliferation, differentiation, and survival of macrophages, monocytes, and osteoclasts (Lin et al. 2008, Baud’huin et al. 2010, Guillonneau et al. 2017). Baudhuin et al. reported that IL-34 could stimulate RANKL-induced osteoclastogenesis through promoting the adhesion and proliferation of osteoclast progenitors in giant cell tumors (Baud’huin et al. 2010). IL-34 facilitates the differentiation of peripheral blood mono-nucleated cells; moreover, IL-34 administration could increase the ratio of CD11b+ cells and promote bone loss of trabecular bone (Chen et al. 2011). In this study, we found that IL-34 was the downstream effector of BMP-2, which was significantly upregulated in the BMP-2-deficient mice, and it then promoted cytokines production and bone defects. Interestingly, knockout of IL-34 in BMP-2-deficient mice was able to restore the expression of cytokines to the normal level, indicating the inhibitory effect of BMP-2 on ONFH depending on the downregulating of IL-34 in vivo. However, BMP-2 administration could not inhibit IL-34-induced osteoclastogenesis and suppress osteoclast differentiation in vitro, which might be due to the difference in microenvironments between the in vitro and in vivo systems. In addition, other BMPs (BMP-4 and BMP-7) were also elevated in ONFH; their regulatory role needs to be addressed in the future studies.
Conclusion
The current study demonstrates the expression and function of BMP-2-IL-34 axis in ONFH. It is revealed that BMP-2 inhibits the formation of osteoclasts through suppressing the production of IL-34 and then promotes bone repair and alleviates ONFH.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was funded by National Natural Science Foundation of China (81873993), the Clinical Science and Technology Innovation Project of Shanghai Hospital Development Center (SHDC12019X24), and the Shanghai Pudong New Area Science and Technology Development Fund (PKJ2019-Y02).
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