Punicalagin Attenuates Osteoclast Differentiation by Impairing Nfatc1 Expression and Blocking Akt- and JNK-Dependent Pathways
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Mol Cell Biochem DOI 10.1007/s11010-015-2466-3 Punicalagin attenuates osteoclast differentiation by impairing NFATc1 expression and blocking Akt- and JNK-dependent pathways 1 1 2 1 Mayumi Iwatake • Kuniaki Okamoto • Takashi Tanaka • Takayuki Tsukuba Received: 13 March 2015 / Accepted: 29 May 2015 Ó Springer Science+Business Media New York 2015 Abstract Punicalagin is a bioactive polyphenol that is kappa-B alpha compared with untreated OCLs. Thus, classified as an ellagitannin. Although punicalagin has been punicalagin may affect bone metabolism by inhibiting shown to have various pharmacological effects, such as OCL differentiation. anti-oxidative, anti-inflammatory, and anti-tumor effects, no studies have reported the effects of punicalagin on Keywords Osteoclasts Á Punicalagin Á NFATc1 osteoclasts (OCLs). In this study, we investigated the effects of punicalagin on OCL differentiation by receptor Abbreviations activator of nuclear factor kappa-B ligand in the murine OCLs Osteoclasts monocytic RAW-D cell line and bone marrow-derived RANKL Receptor activator of nuclear factor kappa-B macrophages (BMMs). Treatment with punicalagin sig- ligand nificantly inhibited OCL formation from RAW-D cells and BMMs Bone marrow-derived macrophages BMMs and prevented bone resorption of BMM-derived NFATc1 Nuclear factor of activated T cells cytoplasmic- OCLs. Moreover, punicalagin impaired multinucleation 1 and actin-ring formation in OCLs, and decreased the pro- IjBa Nuclear factor kappa-B alpha tein levels of nuclear factor of activated T cells cytoplas- TRAP Tartrate-resistant acid phosphatase mic-1 (NFATc1), which is a master regulator of OCL M-CSF Macrophage colony-stimulating factor differentiation, and concomitantly reduced the expression NF-jB Nuclear factor kappa-B levels of Src and cathepsin K, which are transcriptionally PI3 K Phosphatidylinositol 3-kinase regulated by NFATc1. The effects of punicalagin on JNK Jun N-terminal kinase intracellular signaling during the OCL differentiation of Erk Extracellular signal-regulated kinase BMMs indicated that punicalagin-treated OCLs displayed MAPK Mitogen-activated protein kinase markedly reduced phosphorylation of Jun N-terminal HO-1 Heme oxygenase-1 kinase and Akt, and partially impaired phosphorylation of NFAT Nuclear factor of activated T cells extracellular signal-regulated kinase, p38 mitogen-acti- Abs Antibodies vated protein kinase, and inhibitor of nuclear factor SD Standard deviations PMSF Phenylmethylsulfonyl fluoride TBS-T Tris buffered saline with 0.1 % Tween 20 & Takayuki Tsukuba [email protected] 1 Division of Dental Pharmacology, Nagasaki University Introduction Graduate School of Biomedical Sciences, Sakamoto 1-7-1, Nagasaki 852-8588, Japan Osteoclasts (OCLs) are bone-resorbing multinucleated 2 Division of Natural Product Chemistry, Nagasaki University Graduate School of Biomedical Sciences, Bunkyo, 1-14, cells derived from hematopoietic precursors of monocyte- Nagasaki 852-8521, Japan macrophage lineage [1]. Bone resorption by OCLs is 123 Mol Cell Biochem required for the formation of a resorption lacuna, charac- including the ability to inhibit nuclear factor of activated T terized by the maintenance of an acidic pH and the cells (NFAT) in murine splenic CD4? T cells [20]. Since secretion of specific hydrolases, such as cathepsin K and NFATc1 is an essential factor in OCLs, we hypothesized tartrate-resistant acid phosphatase (TRAP). Cathepsin K, that punicalagin may inhibit OCL differentiation. There- an OCL-specific cysteine protease, is essential for bone fore, in this study, we investigated the effects of puni- matrix degradation [2, 3], while TRAP is required for calagin on osteoclastogenesis using in vitro culture specialized electrochemical reactions associated with bone systems. matrix resorption [4]. Therefore, resorption of the miner- alized bone matrix by OCL is closely related to the regu- lation of these OCL-specific lysosomal enzymes. Materials and methods OCL differentiation is mainly regulated by two impor- tant cytokines, macrophage colony-stimulating factor (M- Reagents CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). M-CSF is a secreted cytokine that pro- M-CSF was purchased from Kyowa Hakko Kogyo (Tokyo, motes the differentiation of hematopoietic stem cells into Japan). Recombinant RANKL was prepared as described macrophages and OCLs [5]. RANKL, a member of the previously [21]. Antibodies (Abs) were purchased as fol- tumor necrosis factor superfamily, is a key cytokine that lows: anti-b-actin (Cat. No. A5060, rabbit polyclonal Ab) regulates osteoclastogenesis and bone resorption [6, 7]. The was purchased from Sigma-Aldrich (St. Louis, MO, USA), RANKL-RANK triggers the activation of the essential Src (Cat. No. 05-184, mouse monoclonal Ab) was pur- signaling pathways for OCL differentiation: nuclear factor chased from Upstate Biotechnology (Lake Placid, NY, kappa-B (NF-jB), phosphatidylinositol 3-kinase (PI3 K)/ USA), and anti-c-fms (Cat. No. sc-692, rabbit polyclonal Akt, Jun N-terminal kinase (JNK), extracellular signal- Ab), anti-RANK (Cat. No. sc-9072, rabbit polyclonal Ab), regulated kinase (Erk), p38 mitogen-activated protein and anti-NFATc1 (Cat. No. sc-7294, mouse monoclonal kinase (MAPK) [8–10], and nuclear factor of activated T Ab) were purchased from Santa Cruz Biotechnology (Santa cells cytoplasmic-1 (NFATc1)/calcineurin-dependent Cruz, CA, USA). Abs specific for phospho-ERK1/2 (Cat. pathways [11]. Recent studies have shown that in addition No. 9101S, Thr202/Tyr204, rabbit polyclonal Ab), phos- to these signaling pathways oxidative stress plays impor- pho-JNK (Cat. No. 9751S, Thr183/Tyr185, rabbit poly- tant roles in osteoclastogenesis [12]. Indeed, the induction clonal Ab), phospho-p38 (Cat. No. 9211S, Thr180/Tyr182, of heme oxygenase-1 (HO-1), an anti-oxidative stress rabbit polyclonal Ab), phospho-inhibitor of nuclear factor enzyme, inhibits osteoclastogenesis [13]. We have recently kappa-B alpha (IjBa) (Cat. No. 2859S, Ser32), and phos- demonstrated that the RANKL-mediated suppression of pho-Akt (Cat. No. 9271S, Ser473, rabbit polyclonal Ab) HO-1 promotes OCL differentiation and, conversely, the were purchased from Cell Signaling Technology (Danvers, induction of HO-1 inhibits osteoclastogenesis [14]. Based MA, USA). Cathepsin K Abs were prepared as described on these findings, there is a strong possibility that com- previously [22]. The Osteo Assay Plate was purchased pounds interfering with OCL differentiation signaling from Corning (Corning, New York, NY, USA). All other pathways and/or HO-1 induction have inhibitory effects on reagents, including phenylmethylsulfonyl fluoride (PMSF) OCL differentiation. and the protease inhibitor cocktail, were obtained from Punicalagin (2,3-(S)-hexahydroxydiphenoyl-4,6-(S)-gal- Sigma-Aldrich. lagyl-D-glucose) is an ellagitannin, which is a high- molecular-weight bioactive polyphenol [15]. Punicalagin is mainly found in fruit pomegranates (Punica granatum)or Isolation of punicalagin in the leaves of Terminalia catappa. Punicalagin has been shown to have a variety of pharmacological effects, such as Isolation of punicalagin was performed as described pre- anti-oxidative, anti-inflammatory, and anti-tumor effects viously [23]. Briefly, an aqueous acetone extract of the [16–19]. For example, punicalagin attenuates oxidative fresh peel of P. granatum was separated by Sephadex LH- stress and apoptosis in human placental trophoblasts [17]. 20 column chromatography with H2O containing an In addition, punicalagin inhibits inflammation responses in increasing proportion of MeOH and finally with H2O- lipopolysaccharide (LPS)-induced macrophages through acetone (1:1, v/v). The fractions containing punicalagin Toll-like receptor 4-dependent NF-jB activation [18]. were collected and further separated by Diaion HP20SS Punicalagin suppresses the proliferation of H-ras-trans- chromatography with 20–40 % MeOH. Punicalagin was formed NIH3T3 cells, but only partially affects the pro- obtained as a yellow amorphous powder and identified by liferation of non-transformed NIH3T3 cells [19]. 1H NMR spectroscopic comparison with an authentic Interestingly, punicalagin has immunosuppressant activity, sample. The structure of punicalagin is shown in Fig. 1. 123 Mol Cell Biochem Cell culture Cell viability assay Five-week-old male BALB/c mice were obtained from Cells seeded in 96-well cell culture plates were incubated CLEA Japan, Inc. (Tokyo, Japan) and handled in our with the cell counting kit-8 (Dojindo, Kumamoto, Japan) facilities under the approved protocols of the Nagasaki for 1 h, and then the absorbance at 450 nm was measured University Animal Care Committee. The isolation of with a microplate reader (Bio-Rad iMarkTM, Hercules, CA, bone marrow-derived macrophages (BMMs) was per- USA). formed as described previously [14]. The BMMs were replated in culture plates and incubated in a-minimal Western blot analysis essential medium (a-MEM) (Wako Pure Chemicals, Code: 135-15175, bicarbonate buffered with L-glutamine) BMMs were stimulated with or without RANKL in the containing 10 % fetal bovine serum (FBS) with 100 presence of M-CSF for the indicated amount of time. Cells U/mL penicillin and 100 lg/mL streptomycin in the were rinsed twice with ice-cold PBS, and lysed in a cell presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) lysis buffer [50 mM Tris–HCl (pH 8.0), 1 % Nonidet P-40, for 72 h until the cells differentiated into multinucleated 0.5 % sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, mature OCLs. 1 mM PMSF,