The key royal jelly component 10-hydroxy-2-decenoic acid protects against bone loss by inhibiting NF-κB signaling downstream of FFAR4

Abstract

The supplementation of royal jelly (RJ) is known to provide a variety of health benefits, including anti-inflammatory and anti-obesity effects. RJ treatment also reportedly protects against bone loss, but no single factor in RJ has yet been identified as an anti-osteoporosis agent. Here we fractionated RJ and identified 10-hydroxy-2-decenoic acid (10H2DA) as a key component involved in inhibiting osteoclastogenesis based on mass spectrometric analysis. We further demonstrated free fatty acid receptor 4 (FFAR4) as directly interacting with 10H2DA; binding of 10H2DA to FFAR4 on osteoclasts inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced activation of NF-κB signaling, thereby attenuating the induction of nuclear factor of activated T cells (NFAT) c1, a key transcription factor for osteoclastogenesis. Oral administration of 10H2DA attenuated bone resorption in ovariectomized mice. These results suggest a potential therapeutic approach of targeting osteoclast differentiation by the supplementation of RJ, and specifically 10H2DA, in cases of pathological bone loss such as occur in postmenopausal osteoporosis. The supplementation of royal jelly (RJ) is known to provide a variety of health benefits, including anti-inflammatory and anti-obesity effects. RJ treatment also reportedly protects against bone loss, but no single factor in RJ has yet been identified as an anti-osteoporosis agent. Here we fractionated RJ and identified 10-hydroxy-2-decenoic acid (10H2DA) as a key component involved in inhibiting osteoclastogenesis based on mass spectrometric analysis. We further demonstrated free fatty acid receptor 4 (FFAR4) as directly interacting with 10H2DA; binding of 10H2DA to FFAR4 on osteoclasts inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced activation of NF-κB signaling, thereby attenuating the induction of nuclear factor of activated T cells (NFAT) c1, a key transcription factor for osteoclastogenesis. Oral administration of 10H2DA attenuated bone resorption in ovariectomized mice. These results suggest a potential therapeutic approach of targeting osteoclast differentiation by the supplementation of RJ, and specifically 10H2DA, in cases of pathological bone loss such as occur in postmenopausal osteoporosis. Royal jelly (RJ), an essential food required by the queen honeybee, is a product secreted from the hypopharyngeal and mandibular glands of worker honeybees. RJ has received considerable attention due to its pharmacological activities such as immunomodulatory, antitumor, antimicrobial, and vasoactive effects (1Cornara L. Biagi M. Xiao J. Burlando B. Therapeutic properties of bioactive compounds from different honeybee products.Front. Pharmacol. 2017; 8 (28701955): 41210.3389/fphar.2017.00412Crossref PubMed Scopus (219) Google Scholar, 2Pasupuleti V.R. Sammugam L. Ramesh N. Gan S.H. Honey, propolis, and royal jelly: a comprehensive review of their biological actions and health benefits.Oxid. Med. Cell Longev. 2017; 2017 (28814983): 125951010.1155/2017/1259510Crossref PubMed Scopus (362) Google Scholar). Raw RJ consists of water, lipids, proteins, carbohydrates, and other minor constituents. The lipid composition of RJ is comprised of 90–95% fatty acids (1Cornara L. Biagi M. Xiao J. Burlando B. Therapeutic properties of bioactive compounds from different honeybee products.Front. Pharmacol. 2017; 8 (28701955): 41210.3389/fphar.2017.00412Crossref PubMed Scopus (219) Google Scholar). RJ contains certain unique constituents, such as major royal jelly proteins. The requisite homeostasis of adult bone is maintained by a continuous physiological process, termed bone remodeling. This process requires a finely balanced activity of specialized groups of cells, including osteoclasts, which are multinucleated cells that resorb bone, and osteoblasts, which refill the resorption cavities created by osteoclasts. An imbalance between osteoclasts and osteoblasts can lead to metabolic bone diseases, such as osteoporosis. Osteoclasts are derived from monocyte/macrophage lineage precursor cells and their differentiation depends on receptor activator of NF-κB ligand (RANKL) (3Okamoto K. Nakashima T. Shinohara M. Negishi-Koga T. Komatsu N. Terashima A. Sawa S. Nitta T. Takayanagi H. Osteoimmunology: the conceptual framework unifying the immune and skeletal systems.Physiol. Rev. 2017; 97 (28814613): 1295-134910.1152/physrev.00036.2016Crossref PubMed Scopus (256) Google Scholar, 4Ono T. Hayashi M. Sasaki F. Nakashima T. RANKL biology: bone metabolism, the immune system, and beyond.Inflamm. Regen. 2020; 40 (32047573): 210.1186/s41232-019-0111-3Crossref PubMed Scopus (153) Google Scholar) RANKL binding to RANK, a transmembrane molecule expressed on osteoclast precursor cells and mature osteoclasts, activates NF-κB, mitogen-activated protein kinases, and AP-1, consequently leading to the activation of nuclear factor of activated T cell (NFAT) c1, a key transcription factor of osteoclast differentiation (3Okamoto K. Nakashima T. Shinohara M. Negishi-Koga T. Komatsu N. Terashima A. Sawa S. Nitta T. Takayanagi H. Osteoimmunology: the conceptual framework unifying the immune and skeletal systems.Physiol. Rev. 2017; 97 (28814613): 1295-134910.1152/physrev.00036.2016Crossref PubMed Scopus (256) Google Scholar, 4Ono T. Hayashi M. Sasaki F. Nakashima T. RANKL biology: bone metabolism, the immune system, and beyond.Inflamm. Regen. 2020; 40 (32047573): 210.1186/s41232-019-0111-3Crossref PubMed Scopus (153) Google Scholar). Although a previous study reported a significant effect of the administration of RJ on the mineral content of bone (5Hidaka S. Okamoto Y. Uchiyama S. Nakatsuma A. Hashimoto K. Ohnishi S.T. Yamaguchi M. Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model.Evid. Based Complement Alternat. Med. 2006; 3 (16951718): 339-34810.1093/ecam/nel019Crossref PubMed Scopus (72) Google Scholar), the molecular mechanisms by which RJ mediates the anti-osteoporotic effects were still unknown. In this study, we show that RJ partially prevents ovariectomy (OVX)-induced bone loss through the suppression of osteoclastogenesis in vivo. Furthermore, we show that 10-hydroxy-2-decenoic acid (10H2DA), one of a group of unique medium-chain fatty acids present in RJ, is a potent inhibitor of RANKL-induced osteoclastogenesis. These findings contribute to the understanding of the molecular basis for the beneficial effects of bone health elicited by RJ intake. A mouse model of postmenopausal osteoporosis due to estrogen deficiency was used to examine the effects of oral administration of RJ on bone remodeling. Ovariectomized 9-week–old mice were treated with RJ using oral gavage once a day for 4 weeks. Microcomputed tomography (µCT) analysis revealed that ovariectomy-induced significant decreases in bone volume/tissue volume (BV/TV) and trabecular number (Tb.N) in mice, but RJ administration protected against bone loss as measured by BV/TV and Tb.N (Fig. 1, A and B). Similarly, OVX-induced decrease in bone mineral content per tissue volume, increases in trabecular separation, and marrow space star volume were blunted by RJ administration (Fig. 1A and Fig. S1A). A significantly lower osteoclast number was found in RJ-treated ovariectomized mice when compared with vehicle-treated controls using histological analysis (Fig. 1, C and D). Eroded surface and osteoclast surface also showed a tendency to decrease compared with vehicle-treated control (Fig. S1B). In contrast, RJ did not significantly induce the modulation of osteoblast surface and osteoblastic bone formation (Fig. 1, C and D, and Fig. S1B). These results suggest that RJ administration prevented OVX-induced bone loss by inhibiting osteoclastic bone resorption. These results prompted us to investigate the cellular and molecular mechanisms that contribute to the RJ-induced protection of bone loss induced by estrogen deficiency. The addition of RJ significantly inhibited the osteoclast differentiation induced by stimulation with macrophage-colony stimulating factor (M-CSF)/RANKL (Fig. 2A) and co-culture of bone marrow cells with calvarial osteoblastic cells (Fig. 2B) in a dose-dependent manner. Interestingly, RJ treatment had no effect on the expression of Tnfsf11 (encodes RANKL) or Tnfrsf11b (encodes OPG) in calvarial cells, both of which are involved in the supporting activity of osteoclastogenesis (Fig. 2C), indicating that RJ inhibited osteoclastogenesis directly. To gain insight into the RJ constituents that suppress osteoclastogenesis, we first examined whether any of the putative anti-osteoclastogenic molecules in RJ are resistant to heating. The inhibitory effect of RJ on osteoclastogenesis was not affected by heating (Fig. 3A). Next, RJ was divided into a methanol-soluble portion and a methanol-insoluble residue (Fig. 3B). We confirmed that the methanol-soluble fraction hardly contained any protein (Table S1) and this deproteinized fraction had similar inhibitory activity toward osteoclastogenesis as RJ itself (Fig. 3C). The lipid-soluble constituents in the deproteinized fraction, which were extracted in ethyl acetate exhibited anti-osteoclastogenic activity (Fig. 3C), were fractionated using silica gel column chromatography (Fig. 3B). An anti-osteoclastogenic factor was eluted in chloroform-methanol (9:1, v/v). Among 30 fractions, we found that the pooled fractions of 16–20 had a similar inhibitory activity on osteoclastogenesis as before purification (Fig. 3D). To isolate the anti-osteoclastogenic molecule, the pooled fraction was then separated using reversed phase HPLC (Fig. 3E). Among the peaks we detected, the purified peak C still exhibited a similar inhibitory effect on osteoclastogenesis (Fig. 3E). To obtain information on the molecules contained in peak C, we utilized liquid chromatography–tandem MS (LC–MS/MS) (Fig. 3F). Mass analysis at the 10.7-min retention time of peak C (Fig. S2A) indicated the presence of a molecule that showed a strong molecular ion peak at m/z 185.1, which represented 10H2DA (Fig. S2B). The MS/MS spectra of these fractions were consistent with that of 10H2DA (Fig. 3F). Indeed, the LC–MS chromatograms of peak C exhibited a retention time that was similar to that of 10H2DA (Fig. S2A). Consistent with this, the treatment with 10H2DA remarkably decreased the formation of osteoclasts in a dose-dependent manner (Fig. 3G). We analyzed the expression of the genes implicated in osteoclastogenesis and bone resorption to elucidate the molecular mechanism of the 10H2DA-mediated inhibitory effect. Neither 10H2DA nor RJ had any influence on the expression of Tnfrsf11a (encodes RANK), Csf1r (encodes M-CSFR), Fos, and Mitf (Fig. 4, A and B). In contrast, the expression of the key transcription factor of osteoclastogenesis Nfatc1 and its downstream elements was significantly decreased in osteoclasts treated with 10H2DA or RJ, without affecting the expression of Irf8 and Mafb, factors that are known to be involved in the inhibition of Nfatc1 expression (Fig. 4, A and B). Protein levels of cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), V-type proton ATPase subunit D2 (V-ATPase D2), and matrix metalloproteinase 9 (MMP9), all of which are downstream molecules of the RANK signaling pathway and involved in bone resorption, were decreased in osteoclasts treated with 10H2DA (Fig. 4, C and D). Indeed, the areas of hydroxyapatite resorption in 10H2DA-treated osteoclasts were significantly decreased (Fig. 4E). To confirm 10H2DA as an inhibitor of bone resorption in vivo, OVX mice were treated with 10H2DA using oral gavage once a day for 4 weeks. Serum levels of the N-terminal propeptide of type I collagen (PINP), which is used to measure bone formation activity, was not changed by 10H2DA treatment. However, serum C-terminal telopeptides of type I collagen (CTX-I) level, which reflects osteoclastic activity, was significantly inhibited by 10H2DA treatment (Fig. 4F). These results showed that treatment with 10H2DA significantly inhibited OVX-induced osteoclastic bone resorption without affecting bone formation. Thus, 10H2DA is the molecule responsible for the inhibitory activity of RJ on OVX-induced bone loss. We next searched for a putative receptor expressed in osteoclasts to determine the molecular mechanisms underlying 10H2DA-induced inhibition of osteoclastogenesis. Because 10H2DA is classified as a medium-chain fatty acid, we examined the expression of free fatty acid receptors (6Ichimura A. Hasegawa S. Kasubuchi M. Kimura I. Free fatty acid receptors as therapeutic targets for the treatment of diabetes.Front. Pharmacol. 2014; 5 (25414667): 23610.3389/fphar.2014.00236Crossref PubMed Scopus (117) Google Scholar, 7Ulven T. Christiansen E. Dietary fatty acids and their potential for controlling metabolic diseases through activation of FFA4/GPR120.Annu. Rev. Nutr. 2015; 35 (26185978): 239-26310.1146/annurev-nutr-071714-034410Crossref PubMed Scopus (77) Google Scholar). Although the Ffar1, Ffar2, Ffar3, and Gpr84 expression patterns were greatly reduced, Ffar4 expression was strikingly increased after RANKL stimulation (Fig. 5A). Therefore, we tested whether FFAR4 serves as a receptor for 10H2DA. In a reporter assay, 10H2DA dose-dependently activated FFAR4 with EC50 of 1.025 mm (Fig. 5B), indicating that 10H2DA had a moderate activity for FFAR4 activation. It has been demonstrated that the FFAR4 expressed in macrophages mediates the anti-inflammatory effects of fatty acids via inhibiting the NF-κB signaling pathway (8Oh D.Y. Talukdar S. Bae E.J. Imamura T. Morinaga H. Fan W. Li P. Lu W.J. Watkins S.M. Olefsky J.M. GPR120 is an ω-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.Cell. 2010; 142 (20813258): 687-69810.1016/j.cell.2010.07.041Abstract Full Text Full Text PDF PubMed Scopus (1770) Google Scholar). Because NF-κB signaling is crucial for RANKL-induced osteoclastogenesis, we examined the effect of 10H2DA treatment on NF-κB signaling. RANKL-induced phosphorylation and the degradation of IκBα were both diminished in 10H2DA-treated osteoclasts (Fig. 5, C and D). When Ffar4 expression was knocked down with a lentiviral vector expressing a short hairpin RNA (shRNA) targeting Ffar4 (Fig. 5E), the inhibitory effect of 10H2DA on osteoclastogenesis was abrogated (Fig. 5F). In addition, 10H2DA-induced inhibition of IκBα phosphorylation and degradation was abrogated in Ffar4-silenced osteoclasts (Fig. 5, G and H). These results collectively indicate that a single factor contained in RJ, 10H2DA, suppressed the NF-κB signaling pathway through the FFAR4 receptor so as to inhibit osteoclastogenesis. RJ is increasingly used as a dietary supplement for its health-promoting effects (1Cornara L. Biagi M. Xiao J. Burlando B. Therapeutic properties of bioactive compounds from different honeybee products.Front. Pharmacol. 2017; 8 (28701955): 41210.3389/fphar.2017.00412Crossref PubMed Scopus (219) Google Scholar). Thus, the characterization of the molecular mechanisms underlying these activities in normal and pathological contexts will help not only to better understand the molecular basis of these health-promoting effects of RJ, but also highlight potential pathways that can be targeted for therapy. In this study, we found that RJ administration to ovariectomized mice ameliorated bone loss through the suppression of OVX-induced activation of osteoclastogenesis. The anti-osteoclastogenic factor 10H2DA, which we identified in this study, is a medium-chain fatty acid specifically found in RJ. Although it is known to be one of the molecules responsible for the various pharmacological effects of RJ (9Sugiyama T. Takahashi K. Mori H. Royal jelly acid, 10-hydroxy-trans-2-decenoic acid, as a modulator of the innate immune responses.Endocr. Metab. Immune Disord. Drug Targets. 2012; 12 (23061418): 368-37610.2174/187153012803832530Crossref PubMed Scopus (41) Google Scholar, 10Li X. Huang C. Xue Y. Contribution of lipids in honeybee (Apis mellifera) royal jelly to health.J. Med. Food. 2013; 16 (23351082): 96-10210.1089/jmf.2012.2425Crossref PubMed Scopus (47) Google Scholar), there is no report of a 10H2DA effect on osteoclastogenesis. Moreover, we speculate that RJ contains other anti-osteoclastogenic molecule(s) in addition to 10H2DA, because at concentrations of 10H2DA equivalent to those found in RJ (1.54%) (9Sugiyama T. Takahashi K. Mori H. Royal jelly acid, 10-hydroxy-trans-2-decenoic acid, as a modulator of the innate immune responses.Endocr. Metab. Immune Disord. Drug Targets. 2012; 12 (23061418): 368-37610.2174/187153012803832530Crossref PubMed Scopus (41) Google Scholar) osteoclastogenesis was inhibited, but to a lesser extent. FFAR4 is a member of the rhodopsin-like G protein–coupled receptor family, also known as G protein–coupled receptor 120 (GPR120). FFAR4, along with FFAR1, functions as a receptor for saturated and unsaturated fatty acids of medium- to long-chain E. and therapeutic Pharmacol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, E. acid signaling mechanisms in fatty acid fatty acid signaling mechanisms in fatty acid Cell PubMed Scopus Google Scholar), including acid, and are receptors for fatty of acid to FFAR4 on the of to FFAR4, thereby it from and activation (8Oh D.Y. Talukdar S. Bae E.J. Imamura T. Morinaga H. Fan W. Li P. Lu W.J. Watkins S.M. Olefsky J.M. GPR120 is an ω-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.Cell. 2010; 142 (20813258): 687-69810.1016/j.cell.2010.07.041Abstract Full Text Full Text PDF PubMed Scopus (1770) Google Scholar). In addition to FFAR4 been and with the ω-3 fatty acids acid and acid mediates phosphorylation of both the short and of the GPR120 2010; PubMed Scopus Google Scholar). In it has been reported that a of FFAR4 and FFAR1, inhibited osteoclast differentiation G receptor 120 signaling osteoclast and PubMed Scopus Google Scholar, S.H. H. S.H. J.M. Free fatty acid receptor 4 bone formation and bone resorption in the presence of fatty acid PubMed Scopus Google Scholar). Consistent with these we found that 10H2DA has an inhibitory effect on osteoclast differentiation and by the NF-κB signaling pathway and its downstream molecules including and via FFAR4 (Fig. Moreover, it has been that FFAR4 is implicated in physiological and pathological including Y. C. A. Huang C. ω-3 free fatty acids suppress activation by inhibiting NF-κB activation and 2014; 9 PubMed Scopus Google Scholar), of A. K. T. S. T. M. Y. S. Free fatty acids through Med. PubMed Scopus Google Scholar), differentiation T. Y. J. Y. T. J. J. H. GPR120 through and PubMed Scopus Google Scholar), of N. C. stimulation of fatty acid in the with and J. 2017; Scopus Google Scholar), and K. K. Y. K. S. K. N. K. T. of and in the of cellular functions in Cell PubMed Scopus Google Scholar). of the beneficial effects of RJ are to it an FFAR4 Therefore, FFAR4 a for the treatment of metabolic such as and in addition to due to its in the of and bone resorption. In we demonstrated that 10H2DA, a key RJ osteoclastogenesis by inhibiting NF-κB signaling through its Because RJ is one of the used health-promoting RJ as as 10H2DA a potential therapeutic approach against various metabolic bone diseases, including osteoporosis. RJ was a from or by 10H2DA was from the mice were from were in groups of per a and were and food for a OVX or was as M. Nakashima T. M. T. A. Takayanagi H. by 2012; PubMed Scopus Google Scholar). were oral of of of RJ in or 40 of of 10H2DA in in with a for 4 weeks. did not occur were to confirm the of were with 4 and day to day after the of RJ, all mice were and were and with for to and bone were by the and of and was using the were using the a of with no were and the using a analysis M. Nakashima T. N. Okamoto K. S. Takayanagi H. of estrogen and bone Metab. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Hayashi M. T. K. M. T. A. J.M. Takayanagi H. for of bone homeostasis through RANKL Med. PubMed Scopus Google Scholar, A. F. volume of marrow space and of the first and biological PubMed Scopus Google Scholar). were in and with and for osteoblast and osteoclast were in from per mice by the in along the and were using a and all histological were using M. Nakashima T. N. Okamoto K. S. Takayanagi H. of estrogen and bone Metab. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Hayashi M. T. K. M. T. A. J.M. Takayanagi H. for of bone homeostasis through RANKL Med. PubMed Scopus Google Scholar). Serum and levels were measured with the and In osteoclast differentiation in a was as with certain minor M. Nakashima T. N. Okamoto K. S. Takayanagi H. of estrogen and bone Metab. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Nakashima T. Hayashi M. N. T. H. S. Takayanagi H. analysis activates osteoclastogenesis by 2014; PubMed Scopus Google Scholar). bone marrow cells were maintained in a culture essential and with of for to obtain were in with of of RANKL and RJ or RJ for 3 The culture was changed the of osteoclasts in in the system, bone marrow cells and calvarial cells were in the presence of and for was by 3 were Raw RJ was in and were with a and used for in analysis. treatment of RJ was at for The of RJ were extracted with 4 of at were This was once and all were with and for protein content using the Protein The pooled was and in or ethyl acetate the effect of and lipid-soluble fractions on osteoclastogenesis was the lipid-soluble fraction was to a silica gel column mm and with 30 fractions were after with (9:1, and the column was with the anti-osteoclastogenic effects of fraction, fractions 16–20 were and in The was analyzed by HPLC system, the mm of and of column 40 16–20 were further to a column 30 and fractionated with and the fractions in which anti-osteoclastogenic activity was confirmed were The pooled fractions were with and then purified by chromatography mm analysis was on a of MS with an was on an column mm at 40 with a of phase A acetate in and The was at increased to at and maintained for 5 at at a of Mass spectra were in the ion with a at and at the mass m/z To confirm the of the 10H2DA the was analyzed RNA was extracted by the M. Nakashima T. N. Okamoto K. S. Takayanagi H. of estrogen and bone Metab. Full Text Full Text PDF PubMed Scopus Google Scholar). were from of RNA using analysis was with using of the are to The of expression was with osteoclasts were on These cells were by treatment with and cells were on and in the presence of of and of RANKL with or without 10H2DA for 3 areas were with and using were with and then with inhibitor and on for were by at for were to and to The were with or and with at for by The were using were from Cell and and (TRAP), and (V-ATPase D2 and FFAR4 reporter was using the FFAR4 per the was with an The targeting FFAR4 and control were from G receptor 120 signaling osteoclast and PubMed Scopus Google Scholar, Y. Hayashi M. T. T. Takayanagi H. Nakashima T. of cell the bone with 30 Scopus Google Scholar). The lentiviral were by the into cells with a lentiviral using the were with for before RANKL analysis was using the or analysis of with or the are expressed as the are of analysis was using are in the or from the and We all the for and their with to this We also for and RJ. with royal jelly receptor activator of NF-κB ligand nuclear factor of activated T cell ovariectomy 10-hydroxy-2-decenoic acid tomography bone volume/tissue volume macrophage-colony stimulating factor cathepsin K tartrate-resistant acid phosphatase V-type proton ATPase subunit D2 matrix metalloproteinase 9 N-terminal propeptide of type I collagen C-terminal telopeptides of type I collagen short hairpin RNA bone monocyte/macrophage precursor

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