EX 527

Sirtuin 1 inhibits TNF-α-mediated osteoclastogenesis of bone marrow- derived macrophages through both ROS generation and TRPV1 activation

Shu Yan1 · Lujie Miao2 · Yahua Lu1 · Liangzhi Wang1

Abstract

Sirtuin 1 (SIRT1), also known as NAD-dependent deacetylase, has been reported to increase in vivo osteoclast-mediated bone resorption. However, its effects on osteoclastogenesis or bone loss in vitro have not been widely examined. There- fore, the effects and underlying mechanism of SIRT1 on osteoclast differentiation in mice in vitro were studied. During RANKL-induced osteoclastogenesis in differentiated bone marrow-derived macrophages (BMMs), SIRT1 downregulation was observed. The use of resveratrol (SIRT1 activator) and SIRT1 overexpression was found to inhibit osteoclastogenesis, which was confirmed by TRAP staining and activity loss, reduced expression of osteoclast markers and related genes, and a decrease in the number of multinuclear cells. In contrast, treatment with EX-527 (SIRT1 inhibitor) as well as SIRT1 silenc- ing promoted osteoclastogenesis. Furthermore, the tumor necrosis factor (TNF)-α level was reduced by resveratrol treatment and SIRT1 overexpression but increased following EX-527 incubation and SIRT1 depletion. TNF-α silencing blocked the osteoclastogenesis of BMMs promoted by SIRT1 depletion. Moreover, transient receptor potential vanilloid 1 (TRPV1) chan- nel activation and reactive oxygen species (ROS) production, which are associated with osteoclastogenesis, were impaired by TNF-α silencing. These data demonstrate that SIRT1 directly inhibits osteoclastogenesis by inhibiting ROS generation and TRPV1 channel activation under mediation of TNF-α.

Keywords Osteoclastogenesis · Bone marrow-derived macrophages · Sirtuin 1 · Tumor necrosis factor α · Reactive oxygen species

Introduction

Osteoclasts are multinucleated cells responsible for bone resorption throughout bone development. Numerous cytokines, hormones, and growth factors have been reported to regulate osteoclast generation and activation [1]. The pre- sent treatments for suppressing osteoclast-mediated bone resorption suppress the bone formation and anti-receptor activator of nuclear factor-κB ligand (RANKL), which is required for osteoclast differentiation, activation, and sur- vival, -antibody (such as AMG 162, also known as deno- sumab). The potential side effects of AMG162 are Frozen bone, Immunosuppression, and Osteonecrosis of the jaw [2]. Considering these side effects, long-term use of these is limited. RANKL and tumor necrosis factor-α (TNF-α), which have promoting effects on the recruitment, differentiation, and activation of osteoclasts, are abundant in inflamma- tion sites [3–5]. Osteoclast differentiation requires the RANK/RANKL pathway for activation [6, 7]. RANKL can be secreted by Th1 lymphocytes which also secrete the pro-inflammatory cytokines TNF-α and interleukin (IL)-1 [8]. TNF-α and many other osteoclastogenic factors boost the expression of RANKL via receptors on osteoblastic or stromal cells [9, 10]. However, the direct RANKL- independent-inducing effects of TNF-α on the differentia- tion of macrophages into osteoclasts were reported by a few recent studies [11]. Therefore, it is suggested there is orchestrating effect of TNF-α and RANKL on the dissolu- tion of tissues and bones in inflammatory bone diseases.

TNF-α induces various catabolic processes associated with chronic inflammatory osteolysis such as orthopedic implant loosening and pathogenesis rheumatoid arthritis [12–14]. Previous studies showed that in a mechanism involving activation of nuclear factor (NF)-κB sensors, TNF-α, via its p55 receptor, had a regulatory effect on lipopolysaccharide-stimulated osteoclastogenesis [15, 16]. As a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylase, sirtuin 1 (SIRT1) is critically important in metabolism, inflammation, and aging [17] and has been proposed to regulate bone mass. Additionally, targeted deficiency of SIRT1 in osteoclasts leads to enhanced bone resorption [18–22]. A potential method for bone resorption suppression is to increase SIRT1 activity, which may ameliorate other pathologies associated with age. Gurt et al. [23] detected inhibiting effects of Sirt1 activators (SRT2183 and SRT3025) on RANKL-induced osteoclast differentiation in a study of osteoclastogenesis in bone marrow-derived macrophages (BMMs) in vitro. The role of RSV in RANKL-induced osteoclastogenesis was evaluated by He et al. [24]. The present study proposed that RANKL-induced osteoclast differentiation was inhibited by RSV in a dose-dependent manner, which also augmented the apoptosis of differentiated cells.

The crucial role of reactive oxygen species (ROS) pro- duction in RANKL-induced osteoclastogenesis has been reported. Following RANKL stimulation, pre-osteoclasts led to improved levels of intracellular ROS by activating NADPH oxidase homologs [25] or increased production of mitochondria ROS [26]. The transient receptor potential vanilloid 1 (TRPV1) channel facilitates the differentiation of osteoclasts and osteoblasts because of its high expres- sion in these two types of cells. The in vitro differentiation of osteoclasts and osteoblasts can be repressed by blocking TRPV1. Furthermore, TRPV1 activity suppression in vivo abates bone resorption and formation and protects mice against ovariectomy-induced bone loss [27]. These results suggest the direct effects of TRPV1 on the differentiation of osteogenic cells. However, the effects of ROS and TRPV1 on SIRT1-regulated osteoclast differentiation requires fur- ther analysis. In this study, the effects of SIRT1 on RANKL-induced osteoclastogenesis in BMMs were studied, as well as its role in TRAP activity, multinuclear cell generation, and oste- oclast-specific marker expression in differentiated BMMs. Moreover, we further investigated the effects of SIRT1 over- expression and silencing on osteoclast differentiation-related key mediators, such as ROS and TRPV1.

Materials and methods

Materials

Recombinant mouse RANKL (462-TEC) was commer- cially available from R&D Systems (Minneapolis, MN, USA); resveratrol (RSV) and EX-527 (EX) were commer- cially available from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, gentamicin, L-glutamine, and modified Eagle’s medium alpha (α-MEM) were commercially avail- able in Gibco (Carlsbad, CA, USA). Every procedure was approved by the Animal Care and Use Committee of The Third Affiliated Hospital of Soochow University and was in conformity with the guidelines of National Institute of Health (No81004).

Cell culture

The whole bone marrow was isolated from the tibiae and femora of C57/BL6 mice (8–10-weeks-old). The bone mar- row cells were plated in α-10 medium (α-MEM, 10% heat- inactivated fetal bovine serum, 1× penicillin-streptomycin- L-glutamine solution) containing 1/10 volume of CMG 14-12 (conditioned medium supernatant containing recom- binant M-CSF at 1 μg/mL) (30) in Petri dishes. The cells were incubated for 4–5 days in 5% CO2, 95% air, at 37 °C. Fresh media was replaced every other day, along with CMG 14-12 supernatant. After the BMMs were cultured with recombinant RANKL (100 ng/mL) and CMG 14-12 super- natant (1/100 volume) for 5 days, osteoclasts were generated.

Osteoclast differentiation

Osteoclast differentiation was performed by first seeding the macrophages into a 24-well plate (density: 1.25 × 104 cells/ well), followed by 4 days of cell culture with recombinant mouse RANKL (3 ng/mL). According to the manufacturer’s protocol, a Leukocyte Acid Phosphatase kit (Sigma-Aldrich) was used for TRACP staining. TRACP-positive multinucle- ated cells with more than two nuclei were classified as osteo- clasts, and their numbers were counted in randomly selected fields in different areas of each well.

Tartrate‑resistant acid phosphatase (TRAP) staining and TRAP activity assay

A 48-well tissue culture plate was used for BMM cul- ture (4–5 days; in α-10 medium containing RANKL and M-CSF). Cell fixation using paraformaldehyde/phosphate- buffered saline (4%) and TRAP staining with NaK tartrate and naphthol AS-BI phosphoric acid (Sigma-Aldrich) were performed as previously described [28]. A tissue culture plate (96-well) was used for BMM cul- ture (3–4 days; in α-10 medium containing RANKL and M-CSF). Next, 60 s cell fixation with 1:1 acetone and eth- anol was performed after washing twice with phosphate- buffered saline. The cells were dried at ambient tempera- ture in air. Each well was mixed with phosphatase substrate (80 μL; 20 mM) and tartaric acid (80 mM) in citrate buffer (0.09 M). Following 15 min of cell incubation at ambient temperature, the reaction was terminated after adding NaOH (20 μL; 0.1 N) to each well. The iMark microplate reader (Bio-Rad, Hercules, CA, USA) was employed to record the optical density at 405 nm [29].

Western blotting (WB)

The cell lysate was obtained after lysing the cells in RIPA buffer. After concentration determination, the protein was resolved by 10% SDS-PAGE and then delivered to the immo- bilon polyvinylidene difluoride (0.45 µm) membranes. This was followed by 60 min of membrane blocking using bovine serum albumin (5%) at 25 °C. With the indicated antibodies, the membranes were incubated at 4 °C for approximately 16 h. This was followed by further membrane incubation using secondary antibodies (4 °C; 60 min). Based on the C-DiGit® Blot Scanner, an enhanced chemiluminescence reaction (SuperSignal® West Femto Maximum Sensitivity Substrate Kit, Thermo Scientific, Waltham, MA, USA) was carried out for blot visualization.

qPCR

Extraction of RNA from the cells was carried out using Trizol reagent. Based a Light-Cycler® 480 RT-PCR sys- tem (Roche, Basel, Switzerland), mRNA levels for differ- ent genes were determined using SYBR Green master mix; GAPDH was used as an internal control. Next, the SYBR Green PCR master mix on a Light-Cycler® 480 RT-PCR system was further used for quantitative RT-PCR (reaction volume: 20 μL). PCR was carried out at 95 °C for 10 min, followed by cycling 40 times for 15 s (60 °C) and another 40 times for 30 s (72 °C). The copy numbers of target genes were determined by the comparative CT method (2− ΔΔCT) using an internal reference.

ELISA assay

According to the manufacturer’s protocol, the ELISA kit (R&D Systems, Minneapolis, MN, USA) was used to deter- mine the levels of CGRP in the cell supernatants. In contrast, a microplate reader (BMG Labtech, Offenburg, Germany) was used to read the absorbance at OD 543 nm.

RNA silencing

To reduce the expression of SIRT1 and TNF-α, siRNA against SIRT1 and TNF-α was used. SIRT1 (sequence: 5′-ACU UUG CUG UAA CCC UGU A-3′) and TNF-αα (5′-GAC AAC CAA CUA GUG GUG C-3′)-targeted siRNA was designed and manufactured by Genomics Co. (Beijing, China). Next, 5 μL RNase-free water was mixed with shRNA (100 μM), which was further mixed with 10 μL transfection reagent. This mixture was maintained at 25 °C for 15 min. The transfection of cells with siRNA was carried out 36 h before RANKL stimulation.

ROS measurement

ROS generation in differentiated BMMs was measured using a fluorescence probe, DCFH-DA. After incubation with P. acnes for the indicated times, these cells were further incu- bated using 10 μM of DCFH-DA in the dark for 30 min at 37 °C. An Olympus BX51 fluorescence microscope (Tokyo, Japan) was used to measure fluorescence intensity (at Ex 488/Em 525 nm).

Statistical analysis

The obtained data were denoted as the mean ± SD. One-way analysis of variance or two-tailed Student’s t test was used to calculate the statistical significance among groups, with p < 0.05 indicating a significant difference. Result SIRT1 expression was downregulated during RANKL‑induced osteoclastogenesis of BMMs The osteoclastogenesis of BMMs was induced by RANKL. During this period, the expression of SIRT1 in differenti- ated BMMs was investigated by WB and qPCR. It has been shown that SIRT1 expression was clearly decreased in the RANKL-induced group at 48 h post-induction compared to that in the non-induced group (Fig. 1a). Additionally, qPCR data also confirmed that the SIRT1 mRNA level was sig- nificantly reduced at 24 and 48 h post-induction (Fig. 1b). Fig. 1 SIRT1 expression decreased during RANKL- induced osteoclast differen- tiation. Total RNA and protein were isolated from non-induced and induced BMMs. SIRT1 expression during osteoclast differentiation was studied by WB analysis (a) and qPCR assay (b). *p < 0.05; **p < 0.01 compared to the non-induced group by Student’s t test SIRT1 activity and expression level is associated with development of osteoclastogenesis RSV and EX, known as a well-known SIRT1 activator and suppressor, respectively, were used to modulate SIRT1 activity during osteoclast differentiation to probe the effects of SIRT1 on RANKL-induced osteoclastogenesis. As suggested by the TRAP staining data shown in Fig. 2a, during the induction of BMMs to osteoclastogenesis, the RSV-treated group showed sharply abated yield of multi- nucleated osteoclasts compared to the EX group. TRAP activity was also significantly reduced after RSV treatment, but augmented following EX incubation (Fig. 2b). Although neither RSV nor EX treatment affected the total number of osteoclasts, RSV and EX significantly decreased and clearly increased the yield of large osteoclasts with a large nuclei number, respectively, as shown in Fig. 2c. We also detected osteoclast marker production via WB and qPCR. RSV administration resulted in significantly lower expression of TRAP, CALCR, NFATc1, and CTSK at the mRNA level compared to that in the control group; in contrast, EX treat- ment remarkably increased the levels of these four genes (Fig. 2d). The WB data also confirmed that the protein levels of NFATc1 and CTSK were modulated by RSV and EX administration (Fig. 2e). To further confirm the effects of SIRT1 on RANKL- induced osteoclastogenesis, BMMs were transfected with an SIRT1-expressing vector or SIRT1 siRNA to overexpress or silence SIRT1. The WB and qPCR data confirmed that SIRT1 expression was regulated by the different transfec- tions (Fig. 3a, b). We further found that TRAP activity was significantly decreased in the SIRT1 overexpression group but increased in the silencing group (Fig. 3c). The expres- sion of four osteoblast markers (TRAP, CALCR, NFATc1, and CTSK) was reduced by SIRT1 overexpression; in con- trast, their expression was promoted in the SIRT1 depletion group compared to in the control group (Fig. 3a, d). Col- lectively, these data strongly suggest the inhibitory effects of SIRT1 on RANKL-induced osteoclast differentiation of BMMs. SIRT1 is negative mediator of TNF‑α production during osteoclast differentiation It has been widely reported that TNF-α synergistically enhances RANKL-induced osteoclast differentiation both in vivo and in vitro as an important inflammatory cytokine [30]. Thus, TNF-α expression under RSV and EX treatments (or SIRT1 overexpression and silencing) were evaluated in this study. RSV administration significantly reduced TNF-α levels in cells at 48 h post-induction, while EX incubation led to increased levels of TNF-α, compared to in the con- trol group (Fig. 4a). The qPCR data also confirmed that the TNF-α level was negatively correlated with SIRT1 activity (Fig. 4b). In contrast, SIRT1 overexpression and silencing contributed to decreased and increased expression of TNF-α at both the protein and mRNA levels (Fig. 4c, d), indicating that SIRT1 inhibited TNF-α production during RANKL- induced osteoclastogenesis. TNF‑α enhances osteoclastogenesis caused by SIRT1 silencing To further evaluate the effects of SIRT1 and TNF-α on oste- oclastogenesis, BMMs were transfected with SIRT1 siRNA alone, TNF-α siRNA alone, and a combination of SIRT1 and TNF-α siRNA to knockdown the expression of SIRT1 or both SIRT1 and TNF-α. The WB and qPCR data confirmed that TNF-α was first upregulated after SIRT1 silencing, but significantly depleted at the protein and mRNA levels after transfection with TNF-α siRNA (Fig. 5a–c). As clearly shown by the TRAP staining results for SIRT1 silencing in BMMs, the yield of large multi-nucleated osteoclasts was abated by TNF-α silencing, which was similar to the effect observed in SIRT-overexpressing BMMs (Fig. 5d). Addi- tionally, TNF-α depletion also impaired the increased TRAP activity caused by SIRT1 silencing (Fig. 5e) and reduced the number of large osteoclasts with a high nuclei number, rather than by affecting the total osteoclast number (Fig. 5f). Com- bined with the significantly reduced level of four osteoclast markers (TRAP, CALCR, NFATc1, and CTSK) because of Excessive production of ROS and TRPV1 activation was associated with TNF-α and promoted RANKL-induced osteoclastogenesis. Therefore, we further examined whether ROS generation and TRPV1 channel was regulated by SIRT1, and how these factors functioned during osteoclas- togenesis. First, we found that ROS production was sig- nificantly enhanced after SIRT1 silencing, but significantly reduced by TNF-α depletion, suggesting that SIRT1 inhib- ited ROS generation through mediation by TNF-α (Fig. 6a). NAC, an ROS suppressor, was applied in SIRT1-silenced BMMs to probe the effect of ROS on osteoclastogenesis. NAC administration significantly abated the augmented expression of osteoclast markers (CTSK, NFATc1, and TRAP) caused by SIRT1 knockdown (Fig. 6b), suggesting that ROS was, at least partially, responsible for SIRT1-medi- ated osteoclast differentiation. The expression of TRPV1 was examined in the presence of SIRT1 and/or TNF-α silencing, and TRPV1 and its ligand CGRP were found to be highly produced following SIRT1 silencing. However, their production was significantly decreased after TNF-α was knocked down (Fig. 6c–e), demonstrating that SIRT1 suppressed TRPV1 channel activation through TNF-α. Capsaicin, a TRPV1 channel agonist, was used to increase the activity of the TRPV1 channel in BMMs with SIRT1 and TNF-α depletion. Its efficacy was confirmed by the observation that capsaicin treatment remarkably increased the amount of released CGRP without increasing TRPV1 expression (Fig. 6d, e). Moreover, administration of the agonist capsaicin significantly restored the transcription levels of the three osteoclast markers (CTSK, NFATc1, and TRAP), which was first increased by SIRT1 silencing and then decreased by TNF-α silencing (Fig. 6f). These data indicate that SIRT1 regulated osteoclast differentiation par- tially via TNF-α-controlled TRPV1 channel activation. Discussion For differentiated BMMs, RANKL-stimulated osteoclas- togenesis was found to be responsible for reducing SIRT1 expression. Additionally, overexpression or silencing of SIRT1 may attenuate or promote RANKL-stimulated osteoclast differentiation to some extent. Moreover, TNF-α was obviously reduced by overexpression of SIRT1. Addi- tionally, ROS generation and the levels of released CGRP through the TRPV1 channel were decreased or increased by SIRT1 overexpression and silencing by regulating intracel- lular TNF-α levels, respectively. In conclusion, the present study strongly indicated that SIRT1 suppressed RANKL- stimulated osteoclast differentiation in BMMs through TNF-α abrogation, which was partially regulated by ROS generation and CGRP release. RSV is the first SIRT1 agonist to be investigated. A previ- ous study demonstrated that RSV inhibits the generation and functions of osteoclasts [31], which may function through their cellular targets beyond SIRT1 (e.g., estrogen recep- tor α, an important regulator of osteoclast generation) [32, 33]. RSV is thought to conformationally alter SIRT1, which showed lower Km values for both the acetylated substrate and NAD+ cofactor and resulted in apparently increased enzymatic activity [11]. EX-527 has been widely used in the physiological field as an effective SIRT1 inhibitor. The mined. c TRAP activity results indicated that SIRT1i and SIRT1o led to significantly altered formation of osteoclasts compared to that in the control group. d Gene expression of TRAP, CTSK, CALCR, and NFATc1 (osteoclast marker genes) were measured by qPCR. *p < 0.05; **p < 0.01; ***p < 0.001, compared to in the control group by Student’s t test crystal structures of some SIRT inhibitor complexes have been determined, and EX-527 was found to stabilize the closed enzyme conformation and prevent the release of prod- ucts by occupying the nicotinamide site and a neighboring pocket as well as by contacting the ribose of NAD+ or of the coproduct 2′-O-acetyl-ADP ribose [34]. In the present study, these two tools were utilized to regulate SIRT1 dea- cetylase activity and subsequently to probe the effects of SIRT1 activity on osteoclastogenesis. We found that RSV administration inhibited RANKL-stimulated osteoclastogen- esis and the expression of several osteoclast markers as well as TNF-α. In contrast, EX-527 treatment had a stimulatory effect on osteoclast differentiation by increasing TNF-α lev- els in the cells. These data support the SIRT1 expression data, which also suggested that overexpression or silencing of SIRT1 upregulated or downregulated RANKL-induced osteoclast differentiation in BMMs, as evidenced by the results of osteoclast marker expression, as well as TRAP staining and activity determination. Previous studies demonstrated that ROS was deleterious to cells because of the oxidative stress caused by their inter- actions with lipids, proteins, and nucleic acids [35]. ROS production increased by RANKL induction is considered essential for osteoclastogenesis [36]. Indeed, ROS had cru- cial effects on bone resorption, in addition to its various points and multiple pathway functions in TNF-α signaling. Using an osteoclast differentiation model based on bone marrow precursor cells, Lee and co-workers suggested that ROS production is essential for RANKL-induced osteoclas- togenesis [25]. Notably, He et al. [24] demonstrated that the suppression of ROS production mediated the direct inhibi- tory effect of RSV treatment on osteoclastogenesis. In our study, RSV treatment inhibited osteoclast differentiation by inhibiting ROS production; in contrast, EX-527 administra- tion led to decreased levels of ROS in differentiated BMMs and osteoclastogenesis. Moreover, treatment with NAC, an ROS suppressor, contributed to attenuated expression of osteoclast-specific proteins, suggesting that the loss of SIRT1 activity was essential for osteoclastogenesis by pro- moting ROS generation. Initially found to be localized on the peripheral and cen- tral endings [37], TRPV1 receptors function as molecu- lar integrators of nociceptive stimuli. The crucial role of TRPV1 in regulating pain and inflammation has been reported [38, 39]. Additionally, the differentiation of either osteoclasts or osteoblasts can be promoted by TRPV1. Idris et al. [39] reported that the capsazepine-induced pharmaco- logical blockade of TRPV1 ion channels suppressed bone formation and osteoblast activity, which could also protect against ovariectomy-induced bone loss in mice and inhibit osteoclastic bone resorption. This work reported the loss of CGRP release and inactivation of the TRPV1 channel during osteoclastogenesis in SIRT1 and TNF-α-silencing BMMs, corresponding to the reduced expression of osteo- clast markers. Administration of capsaicin in this SIRT1 and TNF-α-silencing BMMs resulted in increased CGRP release, in which osteoclast-specific gene expression was also increased. Therefore, TNF-α-mediated TRPV1 chan- nel activation may explain the SIRT1 silencing involved in osteoclastogenesis promotion.Therefore, these results strongly suggest that decreased TNF-α expression and SIRT1 overexpression in BMMs attenuated the differentiation of osteoclasts. Whether SIRT1 also affects in vivo bone homeostasis through TNF- α-mediated ROS production and TRPV1 channel activation requires further investigation. Remarkably, knockdown/ knockout of SIRT1 in mice caused adipogenesis suppres- sion [40] and glucose regulation [41]. Additional studies of this type of mouse model and their skeletal phenotypes must be further analyzed. SIRT1 alone, co-transfected with siRNA-SIRT1 and TNF-α, or sub- sequently incubated with capsaicin, followed by 48 h of stimulation with RANKL (200 ng/mL). WB analysis was carried out to detect the protein expression of TRPV1 and CGRP. d qPCR assay was con- ducted to detect the mRNA expression of TRPV1 channel. e ELISA was carried out to detect released CGRP levels in cells with differ- ent treatments. f qPCR assay was performed to detect the mRNA expression of TRAP, CTSK, and NFATc1 (osteoclast marker genes). *p < 0.05; **p < 0.01; ***p < 0.001, compared to the control group by Student’s t test Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. 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