Hydrogen sulfide donor GYY4137 attenuates RANKL-induced osteoclast differentiation and multi-nucleation

Abstract

Hydrogen sulfide (H₂S) is a novel gasotransmitter produced in mammalian cells and is known to various regulate physiological functions. Previous study reported that an imbalance in H₂S metabolism is associated with defective bone homeostasis. However, the detailed mechanism of how H₂S affect osteoclast differentiation remains unclear. In the present study, we demonstrated that the effect of H₂S donor GYY4137 on osteoclast differentiation and multi-nucleation. Treatment of GYY4137 significantly decreased the number of receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive cells and inhibited the expression of osteoclast-related genes, nuclear factor of activated T-cells 1 (NFATc1) and Cathepsin K(Ctsk). Additionally, the increased gene expression of dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), and v-ATPase V0 subunit d2 (Atp6v0d2), which are cell-cell fusion-related molecules by RANKL treatment, was attenuated by GYY4137. Furthermore, GYY4137 suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK1/2, and p38MAPK, compared to RANKL-treated cells. Thus, our data suggested that H₂S donor GYY4137 as a novel osteoclast genesis inhibitor, significantly decreases osteoclast differentiation and multi-nucleation by inhibiting the expression of the cell-cell fusion molecules.

INTRODUCTION

Bone metabolism is regulated by a balance between bone resorption by osteoclasts and bone formation by osteoblasts (Hadjidakis and Androulakis, 2006). Inhibition of osteoclast differentiation is effective in preventing osteoporosis. Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential factor for osteoclast differentiation and is expressed in osteoblasts. RANKL specifically binds to its receptor RANK and activates osteoclast differentiation (Teitelbaum, 2000). Interaction between RANK and RANKL induces the expression of nuclear factor of activated T cells c1 (NFATc1), a master regulator for osteoclast differentiation (Boyle et al., 2003). Subsequently, NFATc1 regulates a number of osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Ctsk) (Suda et al., 1997; Wong et al., 1998). Additionally, activation of RANK/RANKL signaling also triggers distinct signaling cascades mediated by c-Jun N-terminal Kinase 1/2 (JNK1/2), p38MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2), which are critical for RANKL-induced osteoclast differentiation (Wei et al., 2002; Mansky et al., 2002; Lee et al., 2018). In particular, activation of p38MAPK results in the downstream expressional activation of several osteoclast marker genes, such as c-Fos, TRAP and Ctsk during osteoclast differentiation (Matsumoto et al., 2004; Li et al., 2002).

A recent study showed that osteoclast differentiation and maturation are necessary for cell-cell fusion (Oursler, 2010). Osteoclast cell fusion through cell-cell fusion molecules, such as dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), and v-ATPase V0 subunit d2 (Atp6v0d2), is one of the main factors involved in the regulation of bone resorption (Hartgers et al., 2000; Yang et al., 2008; Lee et al., 2006). In fact, DC-STAMP or OC-STAMP knock-out cells are unable to develop into multinucleated osteoclasts (Yagi et al., 2005; Ishii et al., 2018). Therefore, the agents that suppress RANK/RANKL-mediated pathways can be a potential therapeutic target.

Hydrogen sulfide (H₂S) has been investigated as a newly discovered third gasotransmitter following nitric oxide (NO) and carbon monoxide (CO) (Kajimura et al., 2010). The generation of H₂S is mainly catalyzed by pyridoxal-5′-phosphate-dependent enzymes from sulfur-containing amino acids, including cystathionine-γ-synthase (CBS) and cystathionine γ-lyase (Kajimura et al., 2010; Kamoun, 2004). Several studies reported that multiple physiological functions of H₂S improved atherosclerosis and cardiovascular (Xie et al., 2016; Meng et al., 2018). On the other hand, bone loss and fracture were caused in CBS^+/− deficient mice via accumulation of homocysteine, which is a detrimental factor (Behera et al., 2018). In fact, mutation of CBS leads to decreased bioavailability of H₂S and to increased homocystinuria (Jhee and Kruger, 2005). Kruger et al., reported that patients with CBS deficiency (G307S, I278T, V320A, T353M, and L101P, A226T, N228S, A231L, D376N, Q526K) exhibit elevated homocysteine plasma levels at excess of 200 µM compared to 5-15 µM in healthy adults (Kruger et al., 2003). Mutation of CBS or homocysteine enhances bone resorption by stimulation of osteoclast formation and activity (Koh et al., 2006). In addition, CBS^–/– mice showed elevated numbers of TRAP-positive osteoclasts in their femurs and elevated serum RANKL levels compared with wild-type mice (Zheng et al., 2017). In contrast, endogenous CSE promoted osteoblast biologic function such as increasing ALP activity, calcium nodules, and matrix protein expression (Lan et al., 2011). Thus, H₂S levels may affect the osteopenia phenotype. Recently, morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) is a new synthetic H₂S donor. Interestingly, GYY4137 generates a more stable and slower production rate of H₂S (Lee et al., 2011; Li et al., 2008). Importantly, H₂S protects against chemical stress-induced cytotoxicity in various cells through inhibition of reactive oxygen species (ROS)-mediated signaling pathways (Yang et al., 2011; Lan et al., 2011; Luo et al., 2012). Additionally, 100 µM GYY4137 treatment promoted osteoblast differentiation by decreasing the ROS level (Lv et al., 2017). On the other hand, GYY4137 (200 µM) treatment in CSE knockout mice induced osteoclastogenesis and alveolar bone resorption (Mo and Hua, 2018). Although GYY4137 treatment might serve as a regulator of osteoclast-osteoblast balance during bone remodeling, inhibition of osteoclast differentiation and maturation mechanisms by low doses of GYY4137 that induce osteoblast differentiation has not been reported.

In the present study, we hypothesized that a low dose of H₂S donor GYY4137 inhibits osteoclastogenesis. We provide the first demonstration that GYY4137 inhibits osteoclast differentiation and cell-cell fusion by down-regulation of RANKL-NFATc1 signal and cell-cell fusion signal.

MATERIALS AND METHODS

Reagents

GYY4137 was purchased from DOJINDO (Kumamoto, Japan). Anti-JNK1/2, anti-phospho-JNK1/2, anti-ERK1/2, anti- phospho-ERK1/2, anti-p38MAPK, anti-phospho-p38MAPK, and antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A primary antibody against β-actin and secondary antibodies (anti-rabbit or mouse IgG goat antibody) conjugated to HRP were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Cell Culture

RAW264.7 mouse macrophage/monocytes were purchased from American Type Culture Collection (Manassas, VA, USA). In the culture system, RAW264.7 cells were cultured in α-MEM (phenol red free) (Gibco BRL/Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Inc., Kerrville, TX, USA), 100U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL/Invitrogen, Carlsbad, CA, USA) at 37°C in 5% CO₂.

Cell viability assay

RAW264.7 cells (2×10³ cells/well) were cultured in a 96-well plate and then cells were treated with various concentrations of GYY4137 for 24 hr in α-MEM containing 10% FBS. After treated cells were washed twice with phosphate-buffered saline (PBS) at room temperature, the cell viability was assessed using Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan). The effect of GYY4137 on cell viability was expressed as percent cell viability, with vehicle-treated control cells set at 100%.

Osteoclast differentiation and TRAP staining assay

RAW264.7 cells (1×10⁴ cells/well) were seeded in a 24-well plate for 24 hr, and the medium was replaced, followed by an additional incubation for 4 days in RANKL (50 ng/mL) (R&D Systems, Minneapolis, MN, USA) and the indicated GYY4137. After incubation, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), a marker enzyme for osteoclast differentiation. TRAP-positive cells with ≥3 nuclei were counted as osteoclasts per well. The effect of GYY4137 on osteoclast differentiation was expressed as rate of osteoclast formation, with vehicle-treated control cells set at 100%.

Real-time PCR analysis

Total RNA was isolated from RAW264.7 cells by using the Sepasol-RNA I Super G (Nacalai Tesque, Tokyo, Japan). cDNA was synthesized from total RNA using PrimeScript RT reagent Kit (TAKARA Biotechnology, Otsu, Japan). Real-time PCR was performed on the ABI Step one Plus System (Applied Biosystems, Foster City, CA, USA) using THUNDERBIRD qPCR Mix (Toyobo, Osaka, Japan). The sequences of primers used have been reported previously (Takagi et al., 2017). All reactions were normalized to the housekeeping gene β-Actin.

Statistical Analysis

Results are presented as the mean ± S.E. All data are the means of three independent experiments. Multiple comparisons were performed with Tukey’s test. P < 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSIONS

In the present study, H₂S inhibited osteoclast-differentiation and multi-nucleation. Firstly, we examined the effect of GYY4137 as a H₂S donor on osteoclast differentiation. RAW264.7 cells were treated with various concentrations of GYY4137, and the cell viability was evaluated using CCK-8 assay. GYY4137 showed no cytotoxic effects in RAW264.7 cells (Fig. 1A). Additionally, many researchers use a GYY4137 concentration of up to 100 µM (Cui et al., 2023; Karaman et al., 2021; Yurinskaya et al., 2020; Liu et al., 2020). Therefore, this assay indicated that the maximum concentration used in our subsequent studies (50 μM). Next, to analyze the direct effects of GYY4137 on osteoclast differentiation, RAW264.7 cells were cultured with RANKL. As a results, mature TRAP-positive multinucleated osteoclasts were seen in the RANKL-treated cells, whereas GYY4137 treatment significantly decreased the osteoclast differentiation compared to RANKL-treated cells (Fig. 1B). To further explore the role of GYY4137 in osteoclast differentiation, we analyzed the mRNA expression of osteoclast-associated genes in RANKL-treated RAW264.7 cells. Total RNA from osteoclast cultures was collected and real-time PCR for mRNA expression of TRAP, Ctsk, NFATc1, and c-Fos was executed in the presence of RANKL were treated with GYY4137. Our present study revealed that those mRNA expression levels were significantly suppressed by GYY4137 during osteoclast differentiation (Fig. 2A-D). Importantly, osteoclasts are highly developed multinucleated cells that are involved in cell-matrix interactions, cell spreading and membrane-membrane fusion (Iwasaki et al., 2008). This phenomenon is a necessary step for the formation of multinucleated osteoclasts. Monomer osteoclasts promote cell-cell fusion by increasing the expression levels of DC-STAMP or OC-STAMP, which are major factors for activating osteoclasts and inducing bone resorption (Hartgers et al., 2000; Yang et al., 2008). According to a previous study, Atp6v0d2-deficient mice exhibit impaired osteoclast cell fusion (Lee et al., 2006). Interestingly, the cooperation of osteoclast-associated receptor (OSCAR) and RANK is indispensable for osteoclast differentiation (Kim et al., 2010). Therefore, we focused on DC-STAMP, OC-STAMP, OSCAR and Atp6v0d2, which are fusogenic molecules in RANKL-induced osteoclast. As shown in Fig. 3A-D, GYY4137 decreased the mRNA expression levels of DC-STAMP, OC-STAMP, OSCAR and Atp6v0d2 compared to RANKL-induced differentiated osteoclasts. To date, DC-STAMP and Atp6v0d2 are originally identified in osteoclasts and are essential for the multi-nucleation of osteoclasts in the presence of RANKL (Kim et al., 2008). Importantly, the promoter regions of DC-STAMP are bind to NFATc1 in osteoclasts, resulting in induction of the expression of DC-STAMP (Chiu et al., 2017). Another fusogenic molecule OC-STAMP is also induced by RANKL treatment (Yang et al., 2008). Both DC-STAMP and OC-STAMP are cooperatively regulated by RANKL-NFATc1 signaling during osteoclast maturation (Miyamoto et al., 2012). In an in vivo model, DC-STAMP transgenic mice exhibited accelerated bone resorption and a concomitant bone loss (Liu et al., 2014). Thereafter, NFATc1-DC-STAMP signaling plays a key role in the osteoclast multi-nucleation process. Additionally, we observed that GYY4137 strongly inhibited the gene expression of OSCAR, which may be a component of an alternative signaling pathway for osteoclast differentiation. We revealed for the first time that GYY4137 exerts an inhibiting effect on osteoclast differentiation and multi-nucleation by inactivating RANKL-NFATc1 signals.

Fig. 1

Effects of GYY4137 as a hydrogen sulfide donor on osteoclast formation in RAW264.7 cell. (A) RAW264.7 cells (2×10³ cells/well) were cultured in a 96-well plate and then treated with various concentrations of GYY4137 for 24 hr in α-MEM containing 10% FBS. Cell viability was assessed using the CCK-8 assay. Cell viability was expressed as a percentage of the values obtained for hydrogen sulfide donors-untreated cells. Cell viability of GYY4137 treated RAW264.7 cells. (B) RAW264.7 cells (1×10⁴ cells/well) were seeded in a 24-well plate for 24 hr. Then, the cells were cultured with various concentrations of GYY4137 (30 or 50 µM) in the presence of RANKL (50 ng/mL) for 4 days. After incubation, the cells were fixed and stained for TRAP, a marker enzyme for osteoclast differentiation. TRAP-positive multinuclear cells (≥3 nuclei) were counted. The effect of GYY4137 on osteoclast differentiation was expressed as rate of osteoclast formation, with GYY4137-untreated cells set at 100%. The data are representative of n = 3 biological replicates, each measured in triplicate and are expressed as means ± SE. A single asterisk (*P<0.05) indicates a significant difference compared with the RANKL group, and a double asterisk (**P<0.05) indicates a significant difference compared with the GYY4137 (30 µM) group.

Fig. 2

Effects of GYY4137 on expression of osteoclast-differentiation associated genes. RAW264.7 cells were treated with indicated doses of GYY4137 for 4 days in the presence of RANKL (50 ng/mL). mRNA expression levels of (A) TRAP (B) Ctsk, (C) NFATc1, and (D) c-Fos were analyzed by real-time PCR and the results were normalized to the expression of β-actin. The data are representative of n = 3 biological replicates and are expressed as means ± SE. A single asterisk (*P<0.05) indicates a significant difference compared with the CTL group, and a double asterisk (**P<0.05) indicates a significant difference compared with the RANKL group.

Fig. 3

Effects of GYY4137 on expression of osteoclast cell fusion molecules. RAW264.7 cells were treated with RANKL (50 ng/mL) for 4 days in the absence or presence of GYY4137. mRNA expression levels of (A) DC-STAMP, (B) OC-STAMP, (C) OSCAR and (D) Atp6v0d2 were analyzed by real-time PCR and the results were normalized to the expression of β-actin. The data are representative of n = 3 biological replicates and are expressed as means ± SE. A single asterisk (*P<0.05) indicates a significant difference compared with the CTL group, and a double asterisk (**P<0.05) indicates a significant difference compared with the RANKL group.

In general, RANKL-activated MAPK signaling, such as JNK1/2, ERK1/2 and p38MAPK, has been known to be involved in osteoclast differentiation (Wei et al., 2002; Mansky et al., 2002; Lee et al., 2018; Huang et al., 2016). To gain insight into how GYY4137 regulates proliferation of osteoclast precursors, we examined whether GYY4137 regulates the activation of MAPKs by RANKL treatment. The phosphorylation levels of signal transduction molecules, such as JNK1/2, ERK1/2, and p38MAPK, were analyzed by western blotting. The addition of RANKL induced JNK1/2, ERK1/2, and p38MAPK phosphorylation observed 15 min after stimulation, whereas treatment with GYY4137 decreased the phosphorylation of JNK1/2, ERK1/2, and p38MAPK (Fig. 4A-D). In fact, several studies showed that H₂S inhibited MAPK phosphorylation under various stress conditions (Guo et al., 2013; Lan et al., 2013; Liu et al., 2018; Cong et al., 2017). In particular, suppressing of p38MAPK phosphorylation down-regulated several osteoclast marker genes, such as c-Fos, TRAP and Ctsk (Matsumoto et al., 2004; Li et al., 2002; Böhm et al., 2009). These results indicate that GYY4137 mainly inhibits osteoclastogenesis via suppressing the phosphorylation of RANKL-mediated p38MAPK signaling.These results suggest that GYY4137 inhibits RANKL-induced cell-cell fusion via the down-regulation of osteoclast fusogenic molecules and suppresses osteoclast-multi-nucleation.

Fig. 4

Effects of GYY4137 on the phosphorylation of MAPK signaling. After pretreatment with GYY4137 for 3 hr, the RAW264.7 cells were stimulated with RANKL (50 ng/mL) for 15 min in the absence or presence of GYY4137(30 μM). (A) The phosphorylation levels of JNK1/2, p-JNK1/2, ERK1/2, p-ERK1/2, p38MAPK, and p-p38MAPK were analyzed by western blotting. Densitometry quantification of (B) p-ERK1/2, (C) p-JNK1/2 and (D) p-p38MAPK were performed using ImageJ. Phosphorylation levels were normalized to total-MAPKs, respectively. The data are representative of n = 3 biological replicates and are expressed as means ± SE. A single asterisk (*P<0.05) indicates a significant difference compared with the CTL group, and a double asterisk (**P<0.05) indicates a significant difference compared with the RANKL group.

In conclusion, the present study demonstrated that GYY4137 inhibits the osteoclast differentiation and cell-cell fusion. Interestingly, it was previously reported that GYY4137 (200 µM) treatment induced osteoclastogenesis (Mo and Hua, 2018), whereas a low dose of GYY4137 (up to 50 µM) inhibits osteoclast differentiation by decreasing RANKL/NFATc1 gene expression. Therefore, GYY4137 concentration may act as a regulator of osteoclast-osteoblast balance during bone remodeling. Particularly, the present study is the first to demonstrate that GYY4137 strongly inhibits the multi-nucleation of mononuclear pre-osteoclasts by downregulating DC-STAMP, OC-STAMP and Atp6v0d2. Further studies on the mechanisms by which H₂S donor GYY4137 improves bone resorption and osteoporosis in vivo are still required.

ACKNOWLEDGMENTS

The authors thank Dr. S. Fujii, Mr. N. Hanawa, Mr. D. Yamauchi and Mr. Y. Wakui (Tokyo University of Agriculture) for their technical assistance. We would like to thank Editage (www.editage.com) for English language editing.

Funding

No funding was provided for the work.

Conflict of interest

The authors declare that there is no conflict of interest.

Data availability

The data in this study are included in the article/materials. Contact the corresponding author directly to request the underlying data.

Author contributions

H.I. designed the experiments. T.T. and H.I. performed the experiments and analyzed the data. T.T. and H.I. wrote the manuscript. H.M., N.T., and M.U. contributed to the development of the manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

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