StemRegenin 1 attenuates the RANKL-induced osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK-NFATc1 signaling pathway

Shengji Zhou; Jiarui Li; Tiantian Ying; Yuxin Wang; Quan Wang; Xiang Li; Fengchao Zhao

doi:10.1016/j.isci.2024.109682

. 2024 Apr 6;27(5):109682. doi: 10.1016/j.isci.2024.109682

StemRegenin 1 attenuates the RANKL-induced osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK-NFATc1 signaling pathway

Shengji Zhou ^1,², Jiarui Li ^1,², Tiantian Ying ^1,², Yuxin Wang ¹, Quan Wang ¹, Xiang Li ^1,^∗, Fengchao Zhao ^1,^3,^∗∗

PMCID: PMC11039397 PMID: 38660403

Summary

The aryl hydrocarbon receptor (AhR) pathway may play an important role in the regulation of osteoclasts, but there are still conflicting studies on this aspect, and the specific mechanism of action has not been fully elucidated. Therefore, we conducted this study to find a drug to treat osteoporosis that targets AhR. We found that StemRegenin 1 inhibited RANKL-induced osteoclastogenesis in a concentration-dependent and time-dependent manner. Through further experiments, we found that SR1 can inhibit nuclear transcription of AhR and inhibit c-src phosphorylation, and ultimately regulates the activation of the NF-κB and p-ERK/mitogen-activated protein kinase pathways. Therefore, for the first time, we discovered the way in which the AhR-c-src-NF-κB/p-ERK MAPK-NFATc1 signaling pathway regulates the expression of osteoclast differentiation-associated proteins. Finally, SR1 was shown to successfully reverse bone loss in OVX mice. These studies provide us with ideas for finding new way to treat osteoporosis.

Subject areas: Molecular biology, Immunology, Cell biology

Graphical abstract

Highlights

•
SR1 can inhibit RANKL-induced osteoclastogenesis
•
SR1 inhibits osteoclasts differentiation mainly by inhibiting AhR in the early stage
•
SR1 attenuates osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK
•
SR1 significantly improved bone loss in OVX mice in vivo

Molecular biology; Immunology; Cell biology

Introduction

Statistically, fractures due to osteoporosis are becoming more common in women after the age of 55, leading to a large number of bone-related diseases and increasing mortality and health care costs.¹ Bone metabolism is a dynamic process, and the dynamic activities of bone formation and reabsorption reaches a balance in normal physiological activities. However, in case of bone loss, the balance established by osteoclasts and osteoblasts is broken.² Due to the decrease of ovarian estrogen secretion after menopause, it will lead to increased bone resorption and a relative decrease in bone formation. As the only cell with bone resorption function in the body, the regulation of osteoclasts differentiation is extremely important for alleviating the development of postmenopausal bone shortening.³

Osteoclasts are derived from monocyte/macrophage hematopoietic progenitor cells.⁴ Osteoclasts are nonspecifically dependent on macrophage proliferation and survival cytokine macrophage colony stimulating factor (M-CSF) in the early stages of differentiation, followed by receptor activator of nuclear factor-kappa B ligand (RANKL) expressed by osteoblasts to activate RANK to differentiate into osteoclasts.⁵ After menopause, the deficiency of estrogen (E2) causes circulating monocytes to release interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), which are involved in bone resorbtion and lead to increased osteoclasts.⁶ Meanwhile, T cells secrete a large amount of RANKL, bind to its receptor rank, recruit TRAF, and activate the signaling pathway downstream of TNF receptor-associated factor (TRAF). And nuclear factor-kappa B (NF-κB) and mitogen activated protein kinases (MAPKs) activated the nuclear factor of T cell 1 (NFATc1), which is an important regulator of osteoclasts differentiation. NFATc1 can directly affect osteoclasts differentiation and expression of osteoclast-related genes, such as tartrate-resistant acid-base phosphatase (TRAP) and cathepsin K (CTSK) and dendritic cell-specific transmembrane protein (DC-STAMP).⁷^,⁸ Finally, NFATc1 can promote the formation of osteoclasts. Alternatively, RANKL may promote the expression of NFATc1 by inducing c-Fos and AP-1 activation, thus promoting the production of osteoclasts.⁹ In addition, experiments have shown that the formation of osteoclasts in mice knocked out by NFATc1 is severely hindered, and the expression of osteoclast differentiation-associated genes is also significantly inhibited.¹⁰

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the Pern-Arnt-Sim (PAS) superfamily. AhR is known to be involved in the progression of inflammatory and immune diseases by regulating Th17 and regulatory T cell differentiation.¹¹ Recent studies have also clarified that AhR plays an important role in bone remodeling.¹² In the unstimulated state, AhR interacts with helper p23, AhR interacting protein (XAP2), nonreceptor tyrosine kinase (c-src) and heat shock protein (Hsp90) in the cytoplasm and forms a stable protein complex. After binding AhR to its ligand, AhR is dissociated from the protein complex,¹³ transported to the nucleus, interacts with the aromatics receptor nuclear transporter (ARNT), and binds to the dioxin reaction element (DRE) sequence. Thus, the expression of cytochrome P450 subfamily B member 1 (CYP1B1), aryl hydrocarbon receptor repressor (AHRR) and other downstream genes were induced.¹⁴ As an inhibitor of the AhR signaling pathway, StemRegenin 1 (SR1) has been shown to improve the expansion of human primordial hematopoietic stem cells under certain conditions.¹⁵^,¹⁶^,¹⁷^,¹⁸ In addition, SR1 has the potential ability to promote human plasmacytoid and myeloid dendritic cell development from hematopoietic progenitor cells.¹⁹ It has also been reported that SR1 can promote the terminal differentiation of human erythroblasts and attenuates endothelial progenitor cell senescence by inhibiting AhR signaling.²⁰^,²¹ Recent studies have shown that the expression of the AhR pathway is associated with bone loss in mice, but various studies have contradicted each other and the specific mechanism of action has not been clarified.²²^,²³^,²⁴

In our study, we focused on exploring the role of SR1 in osteoclast formation and osteoblast formation. For the first time, we reveal the AhR-c-src-NF-κB/p-ERK MAPK-NFATc1 signaling axis in osteoclasts and confirm the important role of SR1 in ameliorating osteoporosis. This gives us new ideas for trying to find more effective drugs to treat osteoporosis.

Results

SR1 inhibits RANKL-induced osteoclastogenesis in a concentration-dependent manner

The chemical structure formula of SR1 is shown in the figure (Figure 1A), and we verified that the primary cells we used were bone marrow macrophages (BMMs) (Figure S1A). The cytotoxicity of SR1 on BMM cells was determined by 48 h and 96 h CCK8 cytotoxicity assay. The results showed that SR1 had no effect on the proliferative activity of BMMs when the concentration of SR1 was 10 μM or less (Figure 1B). Moreover, through qPCR results, we found that the expression of AhR was significantly increased in the early stage of osteoclast differentiation induced with RANKL (Figure 1C). Previous studies have shown that the stimulation of osteoclasts in the early stage had a more significant impact on their morphology and function.²⁵^,²⁶ Therefore, we suspect that AhR plays an important role in the early differentiation of osteoclasts. Therefore, we selected SR1, a specific inhibitor of AhR, and confirmed that SR1 can effectively reduce the expression of AhR in BMM cells when the BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL and stimulated with SR1 at indicated concentrations for four days (Figure 1D). It was found that when BMMs cells were cultured with the indicated concentrations SR1 for four days, the expression of osteoclast differentiation-associated genes (ACP5, NFATc1, CTSK, DC-STAMP, and ATP6v0d2) decreased with the increase of SR1 concentration (Figure 1E). In addition, at the protein expression level, we also verified that the expression of AhR and osteoclast differentiation-associated proteins (ACP5, CTSK, and NFTAc1) also decreased with the increase of SR1 concentration (Figure 1F). To understand the morphological role of AhR expression changes in osteoclasts formation, we selected an AhR agonist, Norisoboldine, we also first performed CCK8 cytotoxicity assay at 48 h and 96 h, respectively, to determine the toxicity of Norisoboldine on BMMs cells (Figure S2C). We used the indicated concentrations Norisoboldine and SR1 to stimulate BMMs, and observed the number and area of osteoclasts with TRAP staining for the indicated time. It was found that the number and area of osteoclasts could be significantly reduced after AhR was inhibited. In line with this, the number and area of osteoclasts increased after AhR was activated (Figures S2D, S2E, 1G, and 1H). To verify the necessary role of AhR in osteoclast differentiation, we used siAhR to knock down the AhR of BMMs, and found that the expression of osteoclast differentiation-associated proteins was decreased (Figures S1B, S2A, and S2B). At the same time, TRAP staining demonstrated that osteoclast formation was also inhibited when AhR expression was knocked down (Figures S1C and S1D). Moreover, we also confirmed by immunofluorescence experiments that the expression of NFATc1 and CTSK and the formation of F-actin rings in osteoclasts were significantly inhibited under the treatment of 10 μM SR1 (Figure 1I). In conclusion, during the normal differentiation of osteoclasts, inhibition of AhR expression will always result in the low expression of osteoclast differentiation-associated genes and proteins, and within the concentration range of cytotoxic tolerance, the expression levels of AhR and osteoclast-related genes and proteins decrease with the increase of SR1 concentration.

SR 1 inhibits RANKL-induced osteoclastogenesis in a time-dependent manner

We have demonstrated that the expression levels of osteoclast differentiation-associated genes and proteins are inhibited in a concentration-dependent manner by SR1 in the range of cytotoxicity tolerance, but we have not yet confirmed whether the duration of SR1 continuous action has any effect on the expression of osteoclast differentiation-associated gene and proteins. According to previous experiments, we selected 10 μM SR1 as the stimulation concentration to stimulate osteoclast differentiation of BMM cells for 0, 1, 3, and 5 days, respectively. We found that compared with the group stimulated by RANKL, the expression level of osteoclast differentiation-associated gene in the group stimulated by SR1 for the same time showed a downward trend, and this downward trend became more obvious with the extension of SR1 treatment time (Figure 2A). At the same time, we found that after treatment with SR1 for a specified time, the expression levels of AhR and osteoclast differentiation-associated proteins decreased compared with the normal group. With the extension of SR1 treatment time, this downward trend was more obvious. Here, we also detected the expression of AHRR protein during osteoclast differentiation, and found that the expression of AHRR protein increased with the extension of SR1 stimulation time (Figures 2B and 2C). Therefore, we speculated that the high concentrations of SR1 inhibited the expression of AhR by inducing the increase of AHRR expression level, and finally inhibited the osteoclast differentiation-associated gene and protein. In the previous experimental Figure 1C, we speculated that the change of AhR expression in the early stage of osteoclast differentiation would affect the differentiation of osteoclasts. To test this hypothesis, we stimulated the differentiation of BMMs with 10 μM SR1 at the early, middle, and late stages of osteoclasts differentiation (1–3, 3–5, 5–7 Day), and stained osteoclasts with TRAP staining on the 7th day (Figure 2D). We were surprised to find that the inhibition of AhR in the early stage of osteoclasts differentiation (1–3 Day) had the greatest effect on the inhibition of osteoclasts differentiation (Figures 2E, 2F, S1E, and S1F). In conclusion, the high concentrations of SR1 may inhibit the expression of AhR by inducing the expression of AHRR, thus presenting a time-dependent inhibitory effect on osteoclasts differentiation.

SR 1 attenuates the RANKL-induced osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK signaling pathway

To explore the specific mechanism through which SR1 regulates the occurrence of osteoclasts, we demonstrated by immunofluorescence experiment that compared with the control group that 3 μM SR1 could inhibit the formation of F-actin ring in osteoclasts. 6 μM SR1 could not only inhibit the formation of F-actin ring in osteoclasts, but also prevent AhR from entering the nucleus to play a role. And 10 μM SR1 completely inhibited the formation of F-actin rings and AhR expression in osteoclasts (Figure 3A). We know that c-src is a component of the AhR complex that is stable in the cytoplasm. Moreover, p-c-src formed by c-src phosphorylation can play the role of protein kinase.²⁷ Therefore, we hypothesized that the low concentration of SR1 treatment of BMMs would affect c-src phosphorylation in BMMs. To test our hypothesis, we found that when BMMs were stimulated with SR1, AhR expression in the nucleus decreased significantly, and in contrast, AhR expression in the cytoplasm increased (Figure 3B). This suggests that at lower concentrations, SR1 inhibits nuclear translocation of AhR in a concentration-dependent manner. Moreover, we were surprised to find that SR1 can significantly inhibit c-src phosphorylation in BMMs (Figures 3C and 3D). Previous studies have found that p-c-src can activate the NF-κB pathway and p-ERK MAPK pathway.²⁸^,²⁹^,³⁰ We also know about the MAPKs family and NF-κB signaling pathway from previous reports in the literature play an important role in the process of osteoclasts differentiation.³¹ We hypothesize that SR1 blocks the activation of NF-κB and MAPK pathways by inhibiting c-src phosphorylation. To test our hypothesis, BMMs were treated with RANKL for 0, 15, 30, 45, and 60 min, with or without SR1. The results showed that the phosphorylation level of p-P65 had the most significant inhibitory effect by SR1 at the 15, 30 and 45 min, and the phosphorylation level of extracellular signal-regulated kinase (ERK) had the most significant inhibitory effect by SR1 at the 15 min (Figures 3E and 3F), while the phosphorylated protein expression levels of p-P38 and p-JNK did not change significantly (Figures S3A and S3B). Therefore, we found that the low concentration of SR1 inhibits the phosphorylation of c-src by inhibiting the nuclear translocation of AhR, and ultimately regulates the activation of the NF-κB and p-ERK/MAPK pathways.

SR 1 attenuates the RANKL-induced osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK-NFATc1 signaling pathway

(A) The BMMs were stimulated with the indicated concentrations of SR1. The detection of the AhR carried out immunofluorescence, Scale bar, 100 μm.

(B) BMMs were induced RANKL and stimulated with the indicated concentrations of SR1 for 4 days. The AhR protein levels in the cytoplasmic and nuclear were evaluated.

(C) BMMs were planted in a 6-well plate until the cells confluency reaches 90%, treated with serum-free medium starvation for 2 h, and then replaced with serum-containing medium and pretreated with or without 6 μM SR1 for 2 h. Finally, RANKL was stimulated the cells for 0, 15, 30, and 60 min. The cells were lysed with RIPA and the c-src and p-c-src protein levels were evaluated.

(D) BMMs were planted in a 6-well plate until the cells confluency reaches 90%, treated with serum-free medium starvation for 2 h, and then replaced with serum-containing medium and pretreated with or without 6 μM SR1 for 2 h. Finally, the cells were stimulated with or without RANKL for 15 min. The cells were lysed with RIPA and the c-src and p-c-src protein levels were evaluated.

(E) Treatment of BMMs was carried out with RANKL, with or without 6 μM SR1 for 0, 15, 30, 45, and 60 min. The p-P65, P65, p-ERK, and ERK protein levels were evaluated.

(F) Quantitative analysis results of p-P65, p-ERK relative to P65, ERK ratios are displayed. Data were presented as means ± SEM; n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

SR1 did not influence osteogenic differentiation of OB cells after inhibiting AhR expression

SR1 has a significant inhibitory effect on osteoclasts differentiation. To study the effect of SR1 on osteogenic differentiation, osteoblast (OB cells) were treated with different concentrations of SR1 for 48 h and 96 h to determine the toxic effect of SR1 on OB cells. The results showed that SR1 had no effect on the proliferation activity of OB cells when the concentration of SR1 was less than 4 μM (Figure 4A). Primary osteoblasts were induced to differentiate by vitamin C and β-glycerosodium phosphate salts, and different concentrations of SR1 were added to stimulate for 14 days and 21 days. The alkaline phosphatase (ALP) staining results and the ARS staining results showed that the effects of different concentrations of drugs on osteogenic differentiation were similar compared with the control group (Figures 4B–4E), While the expression levels of bone-specific genes ALP, OPN, RUNX2, and OCN were not affected after 7 days of SR1 stimulation (Figure 4F). Therefore, SR1 does not influence osteogenic differentiation of primary osteoblasts within the safe toxicity range of OB cells.

SR1 has no inhibitory effect on osteoblast differentiation

(A) Cell viability of primary osteoblasts with SR1 treated for 48 h and 96 h.

(B) After stimulating primary osteoblasts with osteogenesis induction solution (50 μg/mL vitamin C, 10 mM β-glycerol) and different concentrations of SR1 for 14 days. Representative image of ALP staining to show osteogenesis differentiation results, Scale bar, 200 μm.

(C) ALP activity was detected at different concentrations of SR1 after stimulating primary osteoblasts for 14 days.

(D) After stimulating primary osteoblasts with osteogenesis induction solution (50 μg/mL vitamin C, 10 mM β-glycerol) and different concentrations of SR1 for 21 days. Representative image of ARS staining to show osteogenesis differentiation results, Scale bar = 200 μm.

(E) ARS activity was detected at different concentrations of SR1 after stimulating primary osteoblasts for 21 days.

(F) Primary osteoblasts were stimulated with the indicated different concentrations of SR1 for 7 days, using qPCR to show the mRNA level of the osteoblast-specific genes, including ALP, Runx2, OPN, and OCN, normalized to GAPDH. The Data were presented as means ± SEM; n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

SR1 inhibits OVX-induced bone loss in vivo

Previously, we studied the effect of SR1 on osteoclasts and osteoblast differentiation in vitro. Considering the complex physiological activities in animals, we selected 8-week-old female mice for this purpose and removed their bilateral ovaries to construct a mouse model of osteoporosis (OVX group), studied the effect of SR1 in mice, and recorded the weight changes of mice at 7 weeks after administration. It was found that OVX mice gained weight significantly faster than sham group, and SR1 treatment had no effect on the weight of mice (Figure 5A). Protein and RNA were extracted from the isolated mouse forelimbs. Western Blot (WB) and qPCR analysis showed that the expression of AhR protein and gene in OVX group were significantly increased compared with those in sham group. The expression of AhR protein and gene were inhibited after SR1 treatment in OVX group (Figures 5C–5E). Meanwhile, we found that after SR1 treatment, compared with the vehicle group, the expressions of osteoclasts specific genes CTSK, DC-stamp, ACP5, NFATc1, and ATP6v0d2 were significantly downregulated (Figure 5B). At the same time, micro CT showed obvious bone loss in tibia of OVX-induced osteoporotic animals. And SR1 treatment group significantly saved bone loss in mice (Figures 5F and 5G). We found that histological analysis of the tibia and femur using H&E staining showed that SR1 reduced bone loss in mice (Figures 6A and 6B). Similarly, TRAP staining presented that more TRAP-positive mature osteoclasts in OVX mice, while there were fewer stained osteoclasts in SR1-treatment group (Figures 6C and 6D). To further study the effect of SR1 on osteoblast formation, von Kossa staining and Calcein-alizarin red S labeling were occupied to examine femoral mineralization area and deposition rate, respectively. The von Kossa staining of femurs demonstrated that in the OVX group, the ratio of mineralized area (black part of the trabecular bone region) to bone area of distal femur decreased, while SR1 treatment can improve this ratio (Figures 6E and 6F). Calcein-alizarin red S labeling showed that the mineral apposition rate (MAR) of the femur was remarkably improved by SR1 when compared with the vehicle group (Figures 6G and 6H). And no toxic effects were identified in major organs (Figure S3C). Taken together, these results demonstrate that SR1 can inhibit osteoclasts activity and promote osteoblast activity in vivo, thereby reducing OVX-induced bone loss.

Evaluation and analysis of OVX-induced model via micro CT mainly

(A) Weight change diagram of C57BL/6J mice before and after OVX surgery.

(B and C) Tissue total RNA was extracted from Mice forelimb, and the mRNA expression levels of AhR, CTSK, NFATc1, DC-STAMP, ACP5, c-Fos, and ATP6v0d2 were displayed by qPCR, normalized to GAPDH.

(D and E) Mice forelimb bone tissue was isolated of from each experimental group. Adding tissue lysate to extract tissue protein, AhR specific primary antibody was used to show the results by western blot.

(F and G) The micro-CT was used to scan tibias of each mouse, and trabecular bone volume per total volume (BV/TV) was recorded for each specimen. And the relative data are compared. The Data were presented as means ± SEM; n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

SR1 inhibits bone loss in OVX mice

(A and B) H&E staining results detect bone mass loss (Femur and tibia).

(C and D) TRAP staining was used for sections of femurs. The number of osteoclasts and the area of osteoclasts per field of each specimen were measured.

(E and F) The von Kossa staining was used for the sections of femurs and the mineralized surface area/bone surface area (MS/BS (%)) was measured.

(G and H) Calcein-alizarin red S labeling to detect the femur mineral apposition rate (MAR) in mice. Scale bar, 100 μm. Data were presented as means ± SEM; n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Discussion

Human bone tissue is constantly remodeled throughout life, and the dynamic balance of osteoclasts and osteoblasts maintains the stability of bones. Excessive activation of osteoclasts will lead to the breakdown of this balance, which will lead to many bone diseases.⁷ Aging and a drop in estrogen levels during the menopause are common causes of osteoporosis.¹ For this reason, in recent years, a variety of drugs and targets for the treatment of osteoporosis have emerged, such as estrogen replacement therapy and drugs like dihydric phosphate. These drugs have some side effects such as vaginal bleeding and deep vein thrombosis,³² breast cancer,³³ osteonecrosis of the mandible³⁴ etc. Additional treatments are needed to alleviate these side effects, so the scope of use of these drugs is limited. Therefore, we are trying to find new therapeutic targets for osteoporosis and their corresponding drugs. Previous studies have known that the change of AhR expression is related to the occurrence of osteoclasts, but the specific relationship is still controversial in different studies, and the effect of the activation of AhR pathway on osteoporosis and osteoclasts is also different. And previous studies have not clearly explained how the AhR pathway affects osteoporosis.²³^,²⁷^,³⁵^,³⁶ In this study, we fully explored the important role of AhR in bone metabolism in vitro and in vivo. And we found SR1, a specific inhibitor of AhR can inhibit the phosphorylation of c-src by inhibiting the nuclear translocation of AhR, and ultimately regulates the activation of the NF-κB and p-ERK/MAPK pathways for the first time, while not inhibiting the occurrence of osteogenesis. At the same time, we successfully used SR1 to save bone loss in ovx mice in vivo.

In our experiments, SR1, previously a purine derivative, was first identified in an unbiased screening of a compound that promotes the expansion of CD34⁺ hematopoietic progenitors. In vitro expansion of CD34⁺ cells was promoted by inhibiting AhR expression,¹⁸ and Phase I/II trials of expanded cord blood hematopoietic stem cells with SR1 support testing as standalone grafts.³⁷ Therefore, we tried to test the effect of SR1 on osteoclasts genesis and further study the relationship between SR1 and osteoporosis and its specific mechanism. It was found that during normal osteoclasts development, AhR would break away from the stable complex structure in the cytoplasm and enter the nucleus to play a role, which was also confirmed in the previous literature.³⁸ In our current study, we found that treating BMMs with 6 μM SR1 can inhibit cytoplasmic AhR from entering the nucleus. However, how this process occurs remains to be further investigated in the future.

In addition, when we used Norisoboldine to stimulate the differentiation of BMM cells into osteoclasts, we found that Norisoboldine significantly promoted the formation of osteoclasts in a concentration-dependent manner, which was quite contrary to the results of previous studies.³⁵ We suspect that this may be due to differences in the type of cells treated and the dosage of Norisoboldine. In addition, we also found that early suppression of AhR had the greatest effect on inhibiting osteoclasts formation, indicating that AhR was mainly involved in the early stage of osteoclasts differentiation. According to our study and previous literature reports,²⁵^,²⁶ we guessed that the early stage of osteoclast differentiation is the period most susceptible to external factors. The signal pathways of osteoclasts formation have formed a complex network with the diversities of explorations and researches. The NF-κB, MAPK protein family is two important signaling pathways for osteoclasts formation. The MAPK family, including ERKs, JNKs, and P38, is phosphorylated under RANKL stimulation and is involved in osteoclasts differentiation. In addition, blocking the NF-κB signaling pathway can inhibit osteoclastogenesis and promote osteogenesis.³⁹^,⁴⁰ The results of WB in the experiment proved that SR1 inhibits the phosphorylation of c-src by inhibiting the nuclear translocation of AhR, and ultimately regulates the activation of the NF-κB and p-ERK/MAPK pathways. Numerous studies have demonstrated that NFATc1 is a key transcription factor for osteoclasts differentiation and is capable of continuous self-amplification.⁴¹^,⁴² However, SR1 did not promote osteogenesis in vitro, which we speculated was due to the co-regulation of multiple factors. This needs to be further explored.⁸

In summary, we found that in the natural environment, there are ligands of AhR in environmental pollutants, which can activate AhR, and AhR in the activated state can promote the expression of osteoclast differentiation-associated genes and the formation of osteoclasts after entering the nucleus. Our study demonstrates for the first time that SR1 can inhibit c-src phosphorylation by inhibiting nuclear translocation of AhR. P-c-src is a protein kinase, and inhibition of p-c-src prevents the activation of NF-κB and pERK/MAPK pathways, ultimately inhibiting osteoclast formation and bone resorption (Figure 7). Therefore, SR1 can inhibit bone loss by inhibiting nuclear translocation of AhR, which has a therapeutic effect on osteoporosis and discovers a new therapeutic target for the study of bone metabolic diseases.

Mechanism diagram of SR1 alleviating bone loss

In the natural environment, air pollutants contain different AhR ligands. When these ligands enter the cell, they bind specifically to the stable inactive AhR complex in the cytoplasm. AhR is released from the stable complex structure, and the activated AhR is transferred to the nucleus to promote the expression of the downstream osteoclast differentiation-associated gene NFATc1. At the same time, c-src is also released from the stable complex structure, and c-src is phosphorylated to form p-c-src, which promotes the expression of NFATc1 by activating the NF-κB and MAPKs pathways. This promotes the formation of osteoclasts, which ultimately leads to bone loss. Low concentrations of SR1 can prevent the dissociation of c-src in the AhR complex, resulting in a decrease in free c-src and p-c-src. The decrease in p-c-src expression can inhibit the activation of the NF-κB and MAPKs pathways, while high concentrations of SR1 can directly inhibit the transcription of NFATc1 by suppressing the expression of AhR, and ultimately alleviate bone loss.

Limitations of the study

Our study also has some problems worth further research. AhR can regulate the direction of T cell differentiation, and the direction of T cell differentiation has two completely different directions.⁴³ This leads to changes in inflammatory factors in the organism. Activation of AhR also leads to the production of ROS,³⁸ ROS can activate the downstream MAPK signaling pathway and promote osteoclasts formation.⁴⁴ Therefore, the activation of AhR pathway leading to the increase of ROS and inflammatory factors may also have some influence on bone loss in mice.⁴⁵^,⁴⁶

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

anti-GAPDH antibody	Cell Signaling Technology	Cat# 51332
anti-β-actin antibody	Cell Signaling Technology	Cat# 3700
anti-Histone H3 antibody	Cell Signaling Technology	Cat# 9715
anti-AhR antibody	Proteintech	Cat# 67785-1-Ig
anti-NFATc1 antibody	Proteintech	Cat# 66963-1-lg
anti-CTSK antibody	Cell Signaling Technology	Cat# 57056
anti-ACP5 antibody	Proteintech	Cat# 11594-1-AP
anti-P65 antibody	Cell Signaling Technology	Cat# 4764
anti-p-P65 antibody	Cell Signaling Technology	Cat# 3033
anti-ERK antibody	Cell Signaling Technology	Cat# 4695
anti-p-ERK antibody	Cell Signaling Technology	Cat# 4370
anti-JNK antibody	Cell Signaling Technology	Cat# 9252
anti-p-JNK antibody	Cell Signaling Technology	Cat# 4668
anti-P38 antibody	Cell Signaling Technology	Cat# 9212
anti-p-P38 antibody	Cell Signaling Technology	Cat# 4511
anti-c-src antibody	Proteintech	Cat# 60315-1-Ig
anti-p-c-src antibody	Cell Signaling Technology	Cat# 2101
anti-AHRR antibody	Proteintech	Cat# 13241-1-AP
anti-c-Fos antibody	Cell Signaling Technology	Cat# 2250

Chemicals, peptides, and recombinant proteins

PBS	Beyotime Biotechnology	Cat# P0010S
2×SYBR Green qPCR master mix	EZBioscience	Cat# A0001-R2
StemRegenin 1	Selleck	Cat# S2858
FBS	Thermo Fisher Scientific	Cat# 10099141
1% penicillin-streptomycin	Biological Industries	Cat# 03-031-1B
α-MEM	Thermo Fisher Scientific	Cat# 12561056
ECL Luminous Liquid	FD Biotech	Cat# FD8000
RIPA	Sigma-Aldrich	Cat# V900854
RANKL	R&D	Cat# 462-TEC
M-CSF	R&D	Cat# 416-ML
0.25% Trypsin	Beyotime Biotechnology	Cat# P0013B
Alizarin red S	Sigma-Aldrich	Cat# a5533-25g
EDTA	solarbio	Cat# E1171
Norisoboldine	Selleck	Cat# S9092
DMEM	Thermo Fisher Scientific	Cat# 11995065
DAPI	Beyotime Biotechnology	Cat# C1002
calcein	Sigma-Aldrich	Cat# C0875-5g

Critical commercial assays

TRAP dyeing kit	Sigma-Aldrich	Cat# 387A-1KT
CCK-8 assay solution	solarbio	Cat# CA1210
SDS-PAGE gel preparation kit	Beyotime Biotechnology	Cat# P0012A
RNAiso Plus Kit	Takara Bio	Cat# 9108
HiFiScipt cDNA Synthesis kit	CWBIO	Cat# CW0741M
bicinchoninic acid protein (BCA) assay kit	Millipore	Cat# BCA1

Experimental models: Organisms/strains

Mouse: C57BL/6 mice	Animal Experiment Center, the First Affiliated Hospital of Zhejiang University School of Medicine	N/A

Oligonucleotides

See Table S1	Tsingke Biotechnology	N/A

Software and algorithms

GraphPad Prism version 9.0	GraphPad Software	N/A
FlowJo version 10.0	FlowJo Software	N/A
ImageJ software	NIH	N/A

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Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfifilled by the lead contact, Dr. Fengchao Zhao (zhaofengchao@zju.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

•
This paper does not report original code.
•
Data reported in this paper will be shared by the lead contact upon request.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Cell isolation and culture

Eight-week-old C57BL/6 male mice was prepared for the extraction of bone marrow macrophages (BMMs). The mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 10 min, then the lower limbs were separated with sterile scissors, the muscle tissue on the lower extremity surface was removed, and the femur and tibia were separated. The bone marrow was rinsed into a 10 cm Petri dish using a 1 mL syringe. The BMMs cells will be extracted to cultivated in an α-MEM medium (Gibco-BRL, Gaithersburg, MD, USA), FBS (10%) (Gibco-BRL, Gaithersburg, MD, USA), 1% penicillin/streptomycin (1%, Gibco-BRL, Gaithersburg, MD, USA), and 25 ng/mL M-CSF (R&D, Minneapolis, MN, USA) in 37°C and 5% CO₂ incubator. After 48 h, the medium was changed to remove the unattached cells and attached cells were used for subsequent experiments.

The mouse osteoblast cells were extracted from the skull of 8-day-old lactating mice. The lactating mice were first killed, soaked in 75% alcohol for 10 min and sterilized. The skulls of lactating mice were separated with sterile scissors, then the excess intracranial tissue was cut off and the skull was separated. Then the medium was changed into type 1 collagenase and digested in the incubator (37°C, 5% CO₂) for 12 h. Finally, the medium was replaced by DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. After 48 h, the medium was changed to remove the unattached cells and attached cells were used for subsequent experiments.

Ovariectomized (OVX)-induced bone loss mouse model

8-week-old C57BL/6 female mice (n = 15) weighing 20 to 25 g were anesthetized with 3 mg/mL pentobarbital for bilateral ovariectomy (OVX). The mice were randomly divided into three groups (each; n = 5). The sham and vehicle group were injected with corn oil (MedChemExpress, NJ, USA) containing 10% DMSO (MedChemExpress), and the SR1 group was injected with SR1 (2.5 mg/kg) every 3 days for 42 days. Next, the isolated femur and tibia were fixed in 4% paraformaldehyde for 48 h and the micro-CT and histological experiments were carried out. The study protocol was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University. All efforts were made to minimize the number and suffering of animals.

Method details

Flow cytometry

Bone marrow macrophages (BMMs) were extracted and cultured with 25 ng/mL M-CSF. After 96 h, BMMs were stained for 30 min at 4°C in the dark using the following antibodies: APC-Cy7 CD11b (101226), APC F4/80 (123116), all purchased from BioLegend (USA). 7-AAD (559925, B.D., USA) staining was used to identify living cells. Flow cytometry was performed on the Gallios Flow Cytometer (Beckman, USA). FlowJo Software Version 10 (FlowJo LLC, USA) was used for subsequent analyses.⁴⁷

Cell counting Kit-8 assay

The cytotoxicity of SR1 to BMMs and OBs was determined by CCK-8 method. Simply put, 6×10³ cells were added to each of the 96-well plates, With different concentrations of SR1 (Selleck, Houston, Texas, United States) of different concentrations for 48 and 96 h. Then, 10 μl CCK8 reagent (Solarbio, Beijing, China) was added to each well and cultured for 1 h. The absorbance value at 450 nm was detected by microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

In vitro osteoclastogenesis assay and osteoclast count

In 96 well plates, 6×10³ BMMs were added to each well and cultured for 12 h to adhere to the wall. BMMs were then incubated with 25 ng/mL M-CSF and 50 ng/mL RANKL (R&D, Minneapolis, MN, USA), and different concentrations of SR1 (0, 3, 6, 8, 10 μM) were added. The medium was changed every two days for 7 days, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, washed twice with PBS, and finally stained with tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min. Osteoclasts were observed under a fluorescence microscope (NIKON TE2000, Nikon Corporation, Minato, Tokyo, Japan). The cell body of osteoclasts is large, and osteoclasts are multinucleated cells. We first photographed each group of osteoclasts under a fluorescence microscope, and then counted the number of osteoclasts with more than 3 nuclei by ImageJ software (NIH, Bethesda, MD, USA).

Osteoblast differentiation

Osteogenesis induction of OB cells was performed in DMEM medium containing 50 μg/mL ascorbic acid and 10 μM β-glycerophosphatester (Sigma-Aldrich, St. Louis, MO, USA). After 7 days of osteogenic induction, the expression levels of bone-specific genes were detected. After 14 days of osteogenic induction, alkaline phosphatase (ALP) staining was performed with a BCIP/NBT kit (CWBIO, Beijing, China), and ALP activity was quantified with an alkaline phosphatase assay kit (Beyotime, Nanjing, China). Alizarin red S (ARS) staining was performed with 1% alizarin red staining solution (Sigma-Aldrich, Shanghai, China) 21 days after osteogenesis induction to observe the mineralization deposition in the extracellular matrix. To quantify the mineralization, the absorbance at 405 nm was measured with a microplate reader (Bio-Tek Instruments) by elution staining with 10% glacial acetic acid.

Cell transfection

The BMMs were laid into 6-well plates, 12-well plates or 96-well plates, and the medium was changed once every two days. When the cell density reached 50–60%, they were stimulated with RANKL for 4 h, and then transfected with AhR siRNA (50 nM) or negative controls (control siRNA) for another 6 h, which were purchased from Tsingke Biotechnology Co (Beijing, China). Transfections were performed using TSnanofect transfection reagent (Tsingke Biotechnology Co, Beijing, China) according to the manufacturer’s instructions. After that, the previous medium was replaced with the medium containing 25 ng/mL M-CSF and 50 ng/mL RANKL and continued for the specified time (The protein and RNA extraction experiments were cultured for 48 h, and the TRAP staining experiments were cultured for 5 days). AhR siRNA: sense (5′-3′) GAGGUUAAAGUAUCUUCAUTT, antisense (5′-3') AUGAAGAUACUUUAACCUCTT.

RNA extraction and quantitative real-time PCR (qPCR)

The cultured cells were lysed with RNAiso Plus Kit (Takara Bio, Otsu, Japan) to extract total RNA, according to the manufacturer’s protocol. Total RNA was reverse transcribed into cDNA using the HiFiScipt cDNA Synthesis kit (CWBIO). cDNA was amplified via quantitative real-time PCR. qPCR was performed using 2× SYBR qPCR Master Mix (EZBioscience, USA) with ABI Prism 7500 fast system (Applied Biosystems, Foster City, CA, USA). The conditions for PCR cycling were as follows: 95°C for 5 min, then the next step consists of 40 cycles at 95°C for 10 s and 60°C for 30 s, and a final step at 4°C for 10 min the expression level of each gene was calculated by the method of 2^−ΔΔCt. The primer sequences were listed in Table 1.

Protein extraction and western blotting

Total protein was extracted from BMMs and OB cells and bones of mice used in the OVX experiment using radio immune precipitation assay lysis buffer (RIPA) (Sigma-Aldrich) and the protein concentration was measured using a bicinchoninic acid protein (BCA) assay kit (Millipore, Bedford, MA, USA). Equivalent amounts of protein via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 at room temperature for 1 h. Following the overnight incubation of primary antibodies at 4°C, the membranes were nurtured with corresponding HRP-conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA). The bands were visualized employing ECL Luminous Liquid (FDbio, Hangzhou, China). The gray level of proteins was calculated by ImageJ software (NIH, Bethesda, MD, USA). The gray level of the target protein is compared with the gray level of the internal reference protein to obtain a value, and then the comparison value is compared with the comparison value of the CTL group, the result is the relative gray level. Anti-AhR (Cat No: 67785-1-Ig), AHRR (Cat No: 13241-1-AP), ACP5 (Cat No: 11594-1-AP), NFATc1 (Cat No: 66963-1-Ig), c-src (Cat No: 60315-1-Ig) were procured from Proteintech (Wuhan, China). Anti-GAPDH (Cat No: 51332), β-actin (Cat No: 3700), Histone H3 (Cat No: 9715), CTSK (Cat No: 57056), c-Fos (Cat No: 2250), p-P65 (Cat No: 3033), P65 (Cat No: 4764), p-P38 (Cat No: 4511), P38 (Cat No: 9212), p-JNK (Cat No: 4668), JNK (Cat No: 9252), p-ERK (Cat No: 4370), ERK (Cat No: 4695), p-c-src (Cat No: 2101) were all purchased from Cell Signaling Technology (Danvers, MA, USA).

Fluorescent staining and actin ring formation of osteoclasts

The BMMs were cultivated in the presence of SR1, using a 96-well plate. The cells washed with PBS three times, fixed in 4% paraformaldehyde for 15 min at room temperature. Triton X-100 (0.1%, v/v) was carried out for 20 min to permeabilize the cells. After blocking with goat serum, anti-AhR, NFATc1, CTSK antibody were added to cells overnight at 4°C followed by a goat anti-mouse Alexa Fluor-488-conjugated secondary antibody (Abcam). To visualize F-actin ring formation, rhodamine phalloidin was used for F-actin staining at room temperature away from light for an hour. After washed with PBS for three times, cells were then stained with DAPI (Beyotime Institute of Biotechnology, Shanghai, China) for 15 min at room temperature and observed under a fluorescence microscope (NIKON TE2000, Nikon Corporation, Minato, Tokyo, Japan).

Micro-CT scanning

Micro-CT scanner (1072 μCT System; Sky Scan, Bruker micro CT, Kontich, Belgium) was used to examine the fixed tibia according to a previously reported procedure.⁴⁸ The parameters are set as follows: The X-ray energy was 70 kV and 300 μA, the aluminum filter was 0.5 mm, the rotation step was 0.4°C and the isometric resolution was 9 μm. For reconstruction, the NRecon program (Bruker micro CT, kontich, Belgium) was put into use. For rebuilding, the settings are as follows: 1. Smoothing; 2. Ring artifact correction; 3. correction of beam hardening, 40%. Utilizing the software CTAn (Bruker micro CT, Kontich, Belgium), each sample was examined BV/TV (trabecular bone volume per total volume).²⁵^,²⁶

Histological analysis (TRAP and H&E staining)

The fixed femur and tibia were immersed in 10% ethylene diamine tetraacetic acid (EDTA) for 4 weeks for decalcification and then embedded in paraffin to perform histological sections. The segments were examined by TRAP and H&E staining, using premium microscope known as Aperio Scanscope (Mt Waverley, VIC, Australia) to observe and photographed. The number of TRAP positive cells and osteoclasts surface or bone surface area (OCs/BS) of each field were calculated by ImageJ software (NIH, Bethesda, MD, USA).

Calcein-alizarin red S labeling and von Kossa staining

20 mg/kg calcein (Sigma-Aldrich, C0875-5g, 1 mg/mL in 2% NaHCO3 solution) and 40 mg/kg alizarin red S (Sigma-Aldrich, a5533-25g, 2 mg/mL in H₂O) were injected intraperitoneally on days 0 and 4, respectively. On day 7, the mice were sacrificed, the bones were fixed, dehydrated and embedded with embedded-812 (Electron Microscope Science). The samples were cut into 5 μm with a hard tissue cutter (RM2265, Leica, Wetzlar, Germany). Fluorescent labeling images were obtained with a microscope (BX51, Olympus).

For von Kossa staining, the femur samples were sliced into 6 μm dense pieces. Subsequently, the sections were irradiated with ultraviolet light in 2% silver nitrate solution for 1 h and then incubated with 5% sodium thiosulfate for 2 min. Finally, the staining results were observed by a Diplan light microscope (Leitz).

Quantification and statistical analysis

Utilizing Graphpad Prism 9 (Graphpad Software Inc., San Diego, CA, USA), the data were examined from triplicate experiments expressed as mean ± SEM. One-way or two-way ANOVA and Student’s t test were used for comparison between groups and p < 0.05 was considered to indicate a statistically significant difference.

Acknowledgments

This work was supported by Zhejiang Provincial Natural Science Foundation of China (LGF22H060003).

Author contributions

F.Z. and X.L. contributed to the study conception and design. Material preparation, experiments conduction and data collection were performed by S.Z., J.L., and T.Y. Statistical analysis was performed by Y.W. and Q.W. S.Z. wrote the first draft of the manuscript and X.L. revised the manuscript. All authors read and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.

Published: April 6, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.109682.

Contributor Information

Xiang Li, Email: idea_lixiang@zju.edu.cn.

Fengchao Zhao, Email: zhaofengchao@zju.edu.cn.

Supplemental information

Document S1. Figures S1‒S3, Table S1, and Data S1

mmc1.pdf^{(7.4MB, pdf)}

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48.Boerckel J.D., Mason D.E., McDermott A.M., Alsberg E. Microcomputed tomography: approaches and applications in bioengineering. Stem Cell Res. Ther. 2014;5:144. doi: 10.1186/scrt534. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1‒S3, Table S1, and Data S1

mmc1.pdf^{(7.4MB, pdf)}

Data Availability Statement

•
This paper does not report original code.
•
Data reported in this paper will be shared by the lead contact upon request.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

StemRegenin 1 attenuates the RANKL-induced osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK-NFATc1 signaling pathway

Shengji Zhou

Jiarui Li

Tiantian Ying

Yuxin Wang

Quan Wang

Xiang Li

Fengchao Zhao

Summary

Graphical abstract

Highlights

Introduction

Results

SR1 inhibits RANKL-induced osteoclastogenesis in a concentration-dependent manner

Figure 1.

SR 1 inhibits RANKL-induced osteoclastogenesis in a time-dependent manner

Figure 2.

SR 1 attenuates the RANKL-induced osteoclastogenesis via inhibiting AhR-c-src-NF-κB/p-ERK MAPK signaling pathway

Figure 3.

SR1 did not influence osteogenic differentiation of OB cells after inhibiting AhR expression

Figure 4.

SR1 inhibits OVX-induced bone loss in vivo

Figure 5.

Figure 6.

Discussion

Figure 7.

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Data and code availability

Experimental model and study participant details

Cell isolation and culture

Ovariectomized (OVX)-induced bone loss mouse model

Method details

Flow cytometry

Cell counting Kit-8 assay

In vitro osteoclastogenesis assay and osteoclast count

Osteoblast differentiation

Cell transfection

RNA extraction and quantitative real-time PCR (qPCR)

Protein extraction and western blotting

Fluorescent staining and actin ring formation of osteoclasts

Micro-CT scanning

Histological analysis (TRAP and H&E staining)

Calcein-alizarin red S labeling and von Kossa staining

Quantification and statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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