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Cell Biology International (2008) 32, 10641072 (Printed in Great Britain)
Licochalcone A inhibits the formation and bone resorptive activity of osteoclasts
Soon Nam Kimab, Myung Hee Kimac, Yong Ki Mina and Seong Hwan Kima*
aLaboratory of Chemical Genomics, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong-gu, Daejeon 305-600, Republic of Korea
bDepartment of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea
cDepartment of Biochemistry, Chungnam National University, Daejeon 305-764, Republic of Korea
Licochalcone A on the formation and bone resorptive activity of osteoclasts up to 5
Keywords: Licochalcone A, Osteoclast formation, Bone resorption, ERK, NF-κB, Fra-2.
*Corresponding author. Tel.: +82 42 860 7687; fax: +82 42 861 0307.
Bone homeostasis during remodeling is maintained by osteoclastic bone resorption and osteoblastic bone formation (Parfitt, 1987). Generally, an imbalance in bone remodeling due to increased bone resorption over bone formation leads to bone disorders such as osteoporosis (Boyle et al., 2003). Osteoclasts, multinucleated cells, are differentiated from hematopoietic cells of the monocyte/macrophage family in response to osteoclastogenic factors, especially RANKL, and mature osteoclasts have the ability to resorb mineralized bone (Teitelbaum, 2000).
Recently, beneficial effects of natural products and their derivatives on the skeleton were reported by their influencing the process of bone remodeling, in particular inhibiting bone resorption (Putnam et al., 2007). For example, soybean isoflavones, a subclass of flavonoids, mainly represented by genistein and daidzein, have received considerable attention for their potential role in preventing postmenopausal bone loss (Morabito et al., 2002), and their actions probably result in a decrease in osteoclast differentiation (Yamagishi et al., 2001; Rassi et al., 2002). During screening of natural compounds for their potential to inhibit osteoclast differentiation, we identified licochalcone A (Fig. 1) as a compound inhibiting RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity in mouse monocyte/macrophage RAW264.7 cells. Licochalcone A is a flavonoid derived from licorice, which is one of the most frequently used plants in traditional Oriental medicine (Shibata, 2000). Licochalcone A has anti-inflammatory activity (Shibata et al., 1991; Kolbe et al., 2006), anti-parasitic activity (Chen et al., 1993; Mi-Ichi et al., 2005), anti-cancer activity (Rafi et al., 2000; Fu et al., 2004), anti-bacterial activity (Tsukiyama et al., 2002) and anti-browning and depigmenting activity (Fu et al., 2005), but its effect on bone metabolism has not yet been studied.
Structure of licochalcone A.
Therefore, we investigated the effect of licochalcone A on the formation and bone resorptive activity of osteoclasts. To elucidate the action mechanism of licochalcone A in the processes of osteoclast differentiation and bone resorption, we also examined the effect of licochalcone A on the activation of mitogen-activated protein (MAP) kinases and transcription factors, such as NF-κB, activator protein (AP)-1 and nuclear factor of activated T cells (NFAT) c1, known to play a critical role in the induction of osteoclast-specific genes and the activation of mature osteoclasts to resorb mineralized bone (Boyle et al., 2003; Lee and Kim, 2003). The effect of licochalcone A on the expression levels of osteoclast-specific genes has also been examined.
2 Materials and methods
2.1 Cell culture and induction of multinucleated osteoclasts
Osteoclast generation was achieved using either mouse monocyte/macrophage RAW264.7 cells or the primary culture of mouse bone marrow-derived macrophages (BMMs). RAW264.7 cells have been shown to retain the capacity to differentiate into osteoclast-like cells in the presence of RANKL (Hsu et al., 1999). RAW264.7 cells were purchased from the American Type Culture Collection and maintained in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, UT) supplemented with 10% fetal bovine serum (FBS, HyClone), 100
2.2 Cell viability assay
RAW264.7 cells were suspended in α-MEM with 10% FBS and 100
2.3 TRAP staining and activity assay
Multinucleated osteoclasts were fixed with 10% formalin for 10
2.4 Isolation of total RNA
Total RNA was isolated with TRIzol reagent (Life Technologies, MD, USA) according to the manufacturer's protocol. The concentration of total RNA was calculated from the absorbance at 260 and 280
2.5 Primer design and real-time quantitative PCR (QPCR)
Primers were chosen with an on-line primer design program (Rozen and Skaletsky, 2000; see Table 1). First-strand cDNA was synthesized with 2
Primer sequences used in this study
2.6 Western blotting analysis
Cells were homogenized in ice-cold protein extraction buffer consisting of 50
2.7 Pit formation assay
RAW264.7 cells were suspended in α-MEM with 10% FBS and 100
2.8 Lactate dehydrogenase (LDH) release assay
Cell cytotoxicity was evaluated by colorimetric assay based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant using a Cytotoxicity Detection Kit (Roche, Germany) according to the manufacturer's protocol. This experiment was performed in triplicate.
2.9 Actin rings and nucleus staining
Cells were washed with PBS twice, fixed with 10% formalin for 5
In a previous study, licochalcone A was one of the compounds found to inhibit RANKL-induced TRAP activity in RAW264.7 cells (data not shown). Since licochalcone A did not show any effect on cell viability up to 5
Effects of licochalcone A on cell viability in the presence of RANKL (A); RANKL-induced TRAP activity (B); and RANKL-induced formation of TRAP-positive multinucleated osteoclasts (C). The effect of licochalcone A on cell viability was evaluated as described in Section
To elucidate the action mechanism of licochalcone A in the process of osteoclast differentiation, the effect of licochalcone A on the activation of MAP kinases and NF-κB was evaluated by Western blotting analysis. RANKL treatment dramatically induced the phosphorylation of both the c-Jun N-terminal kinase and the extracellular signal-regulated kinase (ERK), but pretreatment with licochalcone A before RANKL treatment inhibited only RANKL-induced phosphorylation of ERK (Fig. 3A). In addition, translocation of the NF-κB p65 subunit into the nucleus was strongly induced by RANKL treatment, but pretreatment with licochalcone A before RANKL treatment also inhibited its translocation by RANKL. Phosphorylation of the inhibitor of κB-α (IκBα) was induced after RANKL treatment, but pretreatment with licochalcone A before RANKL treatment inhibited its phosphorylation by RANKL (Fig. 3B).
Effects of licochalcone A on activations of MAP kinases (A) and IκBα/NF-κB p65 subunit (B) in RAW 264.7 cells. Cells were plated in a 6-well plate at 2
We also looked at the effect of licochalcone A on the activations of osteoclastogenesis-related transcription factors AP-1 and NFATc1. When evaluated by real-time QPCR, all the genes studied were significantly induced by RANKL treatment, but only the induction of Fra-2 gene was significantly inhibited by pre-treatment with licochalcone A before RANKL treatment in a dose-dependent manner (Table 2).
The effect of licochalcone A on RANKL-induced mRNA expression levels of AP-1 family and NFATc1 in RAW264.7 cells
The effect of licochalcone A on bone resorptive activity of mature osteoclasts was also evaluated. RANKL-induced mature osteoclasts resorbed bone matrices, but the whole area of resorption pit excavations by mature osteoclasts was dramatically inhibited by treatment with licochalcone A in a dose-dependent manner (upper images in Fig. 4). To determine whether the inhibitory activity of licochalcone A in bone resorption could result from its potential to trigger the apoptosis (or cell death) of mature osteoclasts, we also evaluated the effect of licochalcone A on the appearance of apoptotic nuclear condensation, the disruption of actin rings and the release of LDH, which can be used as index of cell death. Interestingly, while licochalcone A inhibited bone resorptive activity of mature osteoclasts, there was neither apoptotic nuclear condensation nor disruption of the actin rings (bottom images in Fig. 4). Release of LDH by licochalcone A was not observed (data not shown). These results suggested the possibility that the inhibitory effect of licochalcone A on bone resorptive activity of mature osteoclasts results from its activity in suppressing the expression of genes required for functional osteoclasts, not triggering apoptosis (or cell death) of mature osteoclasts.
Effect of licochalcone A on bone resorptive activity of mature osteoclasts derived in RAW264.7 cells. The bone resorptive activity was measured by using BioCoat Osteologic multitest slides. After the formation of multinucleated osteoclasts, cells were incubated with fresh medium containing RANKL and licochalcone A for 3
Therefore, the effect of licochalcone A on the mRNA expression levels of genes required for functional osteoclasts such as TRAP, matrix metalloproteinase-9 (MMP-9), c-Src, v-ATPase V0 subunit d2 (ATP6v0d2) and cathepsin K was further evaluated by real-time QPCR. When normalized with GAPDH expression levels as described in Section
The effect of licochalcone A on RANKL-induced mRNA expression levels of osteoclast-specific genes in RAW264.7 cells
The effect of licochalcone A on the osteoclastogenesis of BMMs was also evaluated. Licochalcone A did not decrease the viability of BMMs (data not shown), but consistent with results in RAW264.7 cells, it inhibited the formation of osteoclast (Fig. 5A) and the TRAP activity (Fig. 5B) that were induced by both stimulators, RANKL and M-CSF. The treatment of both stimulators into BMMs induced phosphorylation of ERK, but pre-treatment of licochalcone A inhibited the induction of ERK phosphorylation (Fig. 5C). In addition, both stimulators also induced the translocation of NF-κB p65, but this was inhibited by pre-treatment of licochalcone A. The bone resorptive activity of BMMs-derived mature osteoclasts was dose-dependently inhibited by licochalcone A (Fig. 5D).
Effects of licochalcone A on RANKL/M-CSF-induced formation of multinucleated osteoclasts (A); TRAP activity (B); activations of ERK and translocation of NF-κB p65 subunit (C); and bone resorptive activity of mature osteoclasts (D) derived in BMMs. The generation of multinucleated osteoclasts in BMMs and the bone resorptive activity assay were achieved as described in Section
Osteoclastogenesis takes place through multiple steps such as differentiation, fusion and activation of mature osteoclasts by the factors such as RANKL. The binding of RANKL to its receptor RANK, which is a member of tumor necrosis factor receptor (TNFR) superfamily, recruits adaptor molecules such as TNFR-associated factor 6 (TRAF6), induces its trimerization and subsequently leads to the activation of MAP kinase families and transcription factors such as NF-κB (Lee et al., 2002; Lee and Kim, 2003). In differentiated osteoclasts, ERK and NF-κB play an especially important role in the survival and resorption activity of osteoclast (Miyazaki et al., 2000). However, considering that several natural compounds, including tanshinone IIA, coumestrol and curcumin, inhibit osteoclastogenesis by preventing the RANKL-induced activations of ERK and/or NF-κB (Bharti et al., 2004; Kanno et al., 2004; Kim et al., 2004), both signaling molecules may play an important role in the process of osteoclast differentiation. In this study, when licochalcone A significantly inhibited the RANKL-induced formation of osteoclasts, it inhibited the RANKL-induced activations of ERK and NF-κB in both RAW264.7 cells and BMMs.
The binding of RANKL to RANK can also trigger activation of other transcription factors, such as AP-1 and NFATc1. RANKL activates AP-1 partly through an induction of its critical component, c-Fos (Takayanagi et al., 2002; Wagner and Eferl, 2005). In mice lacking the c-Fos gene, severe osteopetrosis due to a complete block of osteoclast differentiation has been observed (Johnson et al., 1992; Wang et al., 1992). Another member of the Fos family, Fra-1, is a transcriptional target of c-Fos during osteoclast differentiation (Matsuo et al., 2000) and can compensate for the loss of c-Fos (Eferl et al., 2004). Although the compensatory ability of Fra-2 was relatively weaker than that of Fra-1, Fra-2 also rescued the blockade of differentiation in c-Fos-deficient osteoclast precursor cells (Matsuo et al., 2000). In addition, considering that NF-κB and c-Fos were recruited to the NFATc1 gene promoter immediately and 24
Mature osteoclasts are characterized by multinuclearity, an actin ring structure and acidic cell condition; a major function of these cells is to resorb mineralized bone surface. The bone resorptive activity of mature osteoclasts can be inhibited by disrupting the actin ring, triggering the apoptosis of mature osteoclasts and/or inhibiting expression/activity of molecules required for functional osteoclasts. We have shown that licochalcone A inhibits the bone resorptive activity of mature osteoclasts by attenuating the RANKL-induced osteoclast-specific genes (TRAP, MMP-9, c-Src, ATP6v0d2 and cathepsin K) required for osteoclastic differentiation, osteoclast fusion and/or bone resorption (Soriano et al., 1991; Lowe et al., 1993; Halleen et al., 1999; Ishikawa et al., 2001; Ishibashi et al., 2006; Lee et al., 2006), but not with the appearance of apoptotic nuclear condensation, the disruption of actin rings and the release of LDH. Licochalcone A significantly attenuated the induction of all genes by RANKL in the process of osteoclast formation and also significantly inhibited the induction of mRNA expression of both MMP-9 and ATP6v0d2 in mature osteoclasts. TRAP is highly expressed in osteoclasts and widely used as a phenotypic marker of osteoclasts. A binuclear iron atom in the active center of TRAP allows the formation of reactive oxygen species, especially highly destructive hydroxyl radicals; in the presence of H
In this study, licochalcone A has been shown to have dual activity: it inhibited the formation of osteoclasts and the bone resorptive activity of mature osteoclasts. Momordin I has been shown to suppress osteoclastogenesis through inhibition of NF-κB and AP-1; it also reduces osteoclast activity and survival (Hwang et al., 2005). The similarity between licochalcone A and momordin I is that they inhibit osteoclastogenesis via inhibition of NF-κB and AP-1 and reduce the bone resorptive activity of mature osteoclasts. However, there are two differences between them: (1) licochalcone A, but not momordin I, inhibits RANKL-induced activation of MAP kinase; and (2) momordin I, but not licochalcone A, stimulates actin ring disruption. This suggests that the inhibitory effect of momordin I on the bone resorptive activity of mature osteoclasts may result from its potential to stimulate the apoptosis of osteoclasts.
In conclusion, we first demonstrated that licochalcone A has the potential to inhibit the formation of osteoclasts via preventing activation of the ERK and NF-κB signaling pathways that might consequently affect activation of AP-1 components such as Fra-2; and second, that it may also suppress the bone resorptive activity of mature osteoclasts by regulating the expression of bone resorption-related genes in part. Further studies are needed to determine its precise mechanism of action and biological efficacy in both ex vivo and in vivo models.
This work was supported by grants to S.N.K., Y.K.M. and S.H.K. from the Korea Science and Engineering Foundation (KOSEF), funded by the Korea government (MOST) No. M10526020001-07N2602-00110.
Asagiri M, Sato, K, Usami, T, Ochi, S, Nishina, H, Yoshida, H. Auto amplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med 2005:202:1261-9
Bharti AC, Takada, Y, Aggarwal, BB. Curcumin (diferuloylmethane) inhibits receptor activator of NF-κB ligand-induced NF-κB activation in osteoclast precursors and suppresses osteoclastogenesis. J Immunol 2004:172:5940-7
Chen M, Christensen, SB, Blom, J, Lemmich, E, Nadelmann, L, Fich, K. Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania. Antimicrob Agents Chemother 1993:37:2550-6
Fu B, Li, H, Wang, X, Lee, FS, Cui, S. Isolation and identification of flavonoids in licorice and a study of their inhibitory effects on tyrosinase. J Agric Food Chem 2005:53:7408-14
Fu Y, Hsieh, TC, Guo, J, Kunicki, J, Lee, MY, Darzynkiewicz, Z. Licochalcone-A, a novel flavonoid isolated from licorice root (Glycyrrhiza glabra), causes G2 and late-G1 arrests in androgen-independent PC-3 prostate cancer cells. Biochem Biophys Res Commun 2004:322:263-70
Halleen JM, Raisanen, S, Salo, JJ, Reddy, SV, Roodman, GD, Hentunen, TA. Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase. J Biol Chem 1999:274:22907-10
Hsu H, Lacey, DL, Dunstan, CR, Solovyev, I, Colombero, A, Timms, E. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci USA 1999:96:3540-5
Hwang YH, Lee, JW, Hahm, ER, Jung, KC, Lee, JH, Park, CH. Momordin I, an inhibitor of AP-1, suppressed osteoclastogenesis through inhibition of NF-κB and AP-1 and also reduced osteoclast activity and survival. Biochem Biophys Res Commun 2005:337:815-23
Ishibashi O, Niwa, S, Kadoyama, K, Inui, T. MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration. Life Sci 2006:79:1657-60
Ishikawa T, Kamiyama, M, Tani-Ishii, N, Suzuki, H, Ichikawa, Y, Hamaguchi, Y. Inhibition of osteoclast differentiation and bone resorption by cathepsin K antisense oligonucleotides. Mol Carcinog 2001:32:84-91
Kim HH, Kim, JH, Kwak, HB, Huang, H, Han, SH, Ha, H. Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge. Biochem Pharmacol 2004:67:1647-56
Kolbe L, Immeyer, J, Batzer, J, Wensorra, U, Dieck, KT, Mundt, C. Anti-inflammatory efficacy of Licochalcone A: correlation of clinical potency and in vitro effects. Arch Dermatol Res 2006:298:23-30
Lee SE, Woo, KM, Kim, SY, Kim, HM, Kwack, K, Lee, ZH. The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. Bone 2002:30:71-7
Lee SH, Rho, J, Jeong, D, Sul, JY, Kim, T, Kim, N. v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation. Nat Med 2006:12:1403-9
Lowe C, Yoneda, T, Boyce, BF, Chen, H, Mundy, GR, Soriano, P. Osteopetrosis in src-deficient mice is due to an autonomous defect of osteoclasts. Proc Natl Acad Sci USA 1993:90:4485-9
Matsuo K, Owens, JM, Tonko, M, Elliott, C, Chambers, TJ, Wagner, EF. Fosl1 is a transcriptional target of c-Fos during osteoclast differentiation. Nat Genet 2000:24:184-7
Mi-Ichi F, Miyadera, H, Kobayashi, T, Takamiya, S, Waki, S, Iwata, S. Parasite mitochondria as a target of chemotherapy: inhibitory effect of licochalcone A on the Plasmodium falciparum respiratory chain. Ann NY Acad Sci 2005:1056:46-54
Miyazaki T, Katagiri, H, Kanegae, Y, Takayanagi, H, Sawada, Y, Yamamoto, A. Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts. J Cell Biol 2000:148:333-42
Morabito N, Crisafulli, A, Vergara, C, Gaudio, A, Lasco, A, Frisina, N. Effects of genistein and hormone-replacement therapy on bone loss in early postmenopausal women: a randomized double-blind placebo-controlled study. J Bone Miner Res 2002:17:1904-12
Okada Y, Naka, K, Kawamura, K, Matsumoto, T, Nakanishi, I, Fujimoto, N. Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase=gelatinase B) in osteoclasts: implications for bone resorption. Lab Invest 1995:72:311-22
Putnam SE, Scutt, AM, Bicknell, K, Priestley, CM, Williamson, EM. Natural products as alternative treatments for metabolic bone disorders and for maintenance of bone health. Phytother Res 2007:21:99-112
Shibata S, Inoue, H, Iwata, S, Ma, RD, Yu, LJ, Ueyama, H. Inhibitory effects of licochalcone A isolated from Glycyrrhiza inflata root on inflammatory ear edema and tumour promotion in mice. Planta Med 1991:57:221-4
Takayanagi H, Kim, S, Koga, T, Nishina, H, Isshiki, M, Yoshida, H. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002:3:889-901
Yamagishi T, Otsuka, E, Hagiwara, H. Reciprocal control of expression of mRNAs for osteoclast differentiation factor and OPG in osteogenic stromal cells by genistein: evidence for the involvement of topoisomerase II in osteoclastogenesis. Endocrinology 2001:142:3632-7
Received 26 November 2007/10 March 2008; accepted 25 April 2008doi:10.1016/j.cellbi.2008.04.017