Glycogen synthase kinase-3 beta inhibitor suppresses Porphyromonas gingivalis lipopolysaccharide-induced CD40 expression by inhibiting nuclear factor-kappa B activation in mouse osteoblasts

Molecular Immunology 52 (2012) 38–49

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Glycogen synthase kinase-3 beta inhibitor suppresses Porphyromonas gingivalis lipopolysaccharide-induced CD40 expression by inhibiting nuclear factor-kappa B activation in mouse osteoblasts Liu Die, Peng Yan, Zhai Jun Jiang, Teng Min Hua, Wen Cai, Liang Xing ∗ Sichuan University, State Key Laboratory of Oral Disease, West China College of Stomatology, 14 Renminnan Road, Chengdu 610041, Sichuan, China

a r t i c l e

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Article history: Received 20 January 2012 Received in revised form 28 March 2012 Accepted 16 April 2012 Available online 14 May 2012 Keywords: CD40 Glycogen synthase kinase-3 beta Nuclear factor-kappa B Beta-catenin Osteoblast Lipopolysaccharide

a b s t r a c t Bone-forming osteoblasts have been recently reported capable of expressing the critical co-stimulatory molecule CD40 upon exposure to bacterial infection, which supports the unappreciated role of osteoblasts in modulating bone inflammation. Recent studies highlight the anti-inflammatory potential of glycogen synthase kinase-3␤ (GSK-3␤) inhibitors; however, their effect on osteoblasts remains largely unclear. In the present study, we showed that treatment with SB216763, a highly specific GSK-3␤ inhibitor, resulted in a dose-dependent decrease in the mRNA and protein expression of CD40, as well as production of pro-inflammatory cytokines IL-6, TNF-␣ and IL-1␤, in the Porphyromonas gingivalis-lipopolysaccharide (LPS)-stimulated murine osteoblastic-like MC3T3-E1 cells. Furthermore, inhibition of GSK-3␤ remarkably represses the LPS-induced activation of the nuclear factor kappa B (NF-␬B) signaling pathway by suppressing I␬B␣ phosphorylation, NF-␬Bp65 nuclear translocation, and NF-␬Bp65 DNA binding activity. Closer investigation by immunoprecipitation assay revealed that ␤-catenin can physically interact with NF-␬Bp65. The negative regulation effect of GSK-3␤ inhibitor on CD40 expression is mediated through ␤-catenin, for siRNA of ␤-catenin attenuated the GSK-3␤ inhibitor-induced repression of NF-␬B activation and, consequently, the expression of CD40 and production of pro-inflammatory cytokines in LPS-stimulated MC3T3-E1 cells. Thus our results elucidate the molecular mechanisms whereby GSK-3␤ inhibitor prevents the LPS-induced CD40 expression on osteoblasts and provide supportive evidence of the potential role of GSK-3␤ inhibitors in suppressing the immune function of osteoblasts in inflammatory bone diseases. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction CD40, a critical co-stimulatory molecule, is constitutively expressed on the surface of professional antigen-presenting cells, such as macrophages and dendritic cells (Suttles and Stout, 2009). The functional expression of CD40 on these cells confers upon them the ability to play a significant role in controlling inflammatory

responses. Interaction between CD40 and CD40 ligand (CD154) leads to the efficient induction of pro-inflammatory cytokines and chemokines, including IL-6, TNF-␣, IL-1, IL-8, CCL2, and CCL5 (Alderson et al., 1993; Kornbluth et al., 1998; Mukundan et al., 2005; Stout et al., 1996; Wagner et al., 1994). CD40 engagement on antigen-presenting cells induces up-regulated expression of major histocompatibility complex class II and co-stimulatory

Abbreviations: LPS, lipopolysaccharide; SB216763, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; Wnt, Wingless; GSK-3␤, glycongen synthase kinase-3 beta; APC, adenomatous polyposis coli; Tcf/Lef, T-cell factor protein and lymphoid enhancer factor protein; NF-␬B, nuclear factor-kappa B; I␬B-␣, inhibitor of kappa B-␣; IKK, I␬B kinase; STAT-1␣, signal transducer and activator of transcription 1␣; IL, interleukin; CCL, CC chemokine ligand; TNF-␣, tumor necrosis factor-␣; TNFRSF, tumor necrosis factor receptor superfamily; ATP, adenosine triphosphate; ␣-MEM, ␣-modified minimal essential media; FBS, fetal bovine serum; FACS, fluorescenceactivated cell sorting; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TFIIB, transcription factor IIB; RIPA, radioimmunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST, Tris-buffered saline tween; ECL, electrochemiluminescence; DAPI, 4 ,6-diamidino-2-phenylindole; EDTA, ethylenediaminetetraacetic acid; LiCl, lithium chloride; ELISA, enzyme-linked immunosorbant assay. ∗ Corresponding author. Tel.: +86 028 85501441. E-mail addresses: [email protected] (L. Die), [email protected] (P. Yan), [email protected] (Z. Jun Jiang), [email protected] (T. Min Hua), [email protected] (W. Cai), [email protected] (L. Xing). 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.04.005

L. Die et al. / Molecular Immunology 52 (2012) 38–49

molecules, resulting in enhanced ability to stimulate T cells in adaptive immune response (Diehl et al., 2000; Kiener et al., 1995; Noelle, 1996). Interestingly, recent studies demonstrated that boneforming osteoblasts can express functional CD40 co-stimulatory molecules following stimulation by bacteria or bacterial products (Ahuja et al., 2003; Schrum et al., 2003). This observation supports a previously unappreciated immunological role of osteoblasts in bone inflammation. Glycogen synthase kinase-3␤ (GSK-3␤) is a key regulator of the Wnt/␤-catenin signaling pathway (Freese et al., 2010). Normally, GSK-3␤ is constitutively active when the cell is in a resting state. The active form of GSK-3␤ phosphorylates cytoplasmic ␤-catenin, which induces it for proteosomal degradation, resulting in low cytoplasmic ␤-catenin levels. However, when Wnt/␤-catenin signaling is activated, GSK-3␤ is inactivated through phosphorylation at the Ser9 residue, resulting in the accumulation of cytoplasmic ␤catenin, which then translocates to the nucleus and interacts with T-cell factor protein and lymphoid enhancer factor protein (Tcf/Lef) to activate the expression of target genes (Logan and Nusse, 2004; Moon et al., 2004). GSK-3␤ is not yet exclusively involved in the regulation of the Wnt/␤-catenin signaling pathway. It has been revealed that GSK3 is a point of convergence of many signaling pathways (Jope and Johnson, 2004), including that of NF-␬B signaling pathway. A number of studies have confirmed that GSK-3␤ has a pivotal role in the regulation of the activation of NF-␬B signaling (Hoeflich et al., 2000; Ougolkov et al., 2007; Takada et al., 2004). Hoeflich et al. (2000) showed that GSK-3␤ is required for NF-␬B-mediated cell survival response after TNF-␣ stimulation, indicating that GSK-3␤ facilitates NF-␬B function. Takada et al. (2004) demonstrated that the genetic depletion of GSK-3␤ suppressed the activation of the NF␬B pathway induced by LPS or inflammatory cytokines. Ougolkov et al. (2007) reported that inhibition of GSK-3␤ abrogates NF-␬B binding to its target-gene promoters, thus enhancing apoptotic cell death in chronic lymphocytic leukemia B cells. NF-␬B is an important signaling pathway that participates in the induction of a wide variety of cellular genes involved in immunity and inflammation, including lots of co-stimulatory molecules and pro-inflammatory cytokines (Bonizzi and Karin, 2004; Liang et al., 2004). For these reasons, the involvement of GSK-3␤ in the regulation of NF-␬B activation has raised the possibility that this kinase may play an important role in modulating inflammatory process (Beurel et al., 2010; Dugo et al., 2007). Although GSK-3␤ inhibitors have been reported to exert anti-inflammatory effects in several inflammatory diseases (Gao et al., 2008; Gurrieri et al., 2010; Yuskaitis and Jope, 2009), little information is available about its effect in modulating bone inflammation. In particular, because the enhanced immune functions of osteoblasts in the presence of inflammatory substances have been observed, it is necessary to clarify the effects of GSK-3␤ inhibitors in regulating immune functions of osteoblasts. Based on these evidences, we postulated that inhibition of GSK3␤ may affect the CD40 expression in infected osteoblasts. The purpose of this study was to investigate whether the CD40 expression in LPS-stimulated murine osteoblastic-like MC3T3-E1 cells is suppressed by a well-characterized pharmacological GSK-3␤ inhibitor, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1Hpyrrole-2,5-dione (SB216763) (Sigma–Aldrich, St. Louis, MO, USA). SB216763, a maleimide derivative, was shown to inhibit GSK-3␤ potently in an adenosine triphosphate (ATP)-competitive manner (Coghlan et al., 2000; Gurrieri et al., 2010; Smith et al., 2001). Our results showed that SB216763 significantly down-regulated LPS-induced CD40 expression and pro-inflammatory cytokine production in MC3T3-E1 cells via inhibition of NF-␬B activation. We also identified a crucial role of ␤-catenin in mediating GSK-3␤ inhibitor-induced suppression of NF-␬B activity.

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2. Materials and methods 2.1. Cell culture Cloned osteoblast-like MC3T3-E1 cells were derived from newborn mouse calvaria (Sodo et al., 1983). MC3T3-E1 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Science. The cells were cultured in a growth medium composed of ␣-modified minimal essential media (␣-MEM, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS), 100 units/ml penicillin (Sigma–Aldrich), and 100 ␮g/ml streptomycin (Sigma–Aldrich) at 37 ◦ C in a humidified atmosphere of 5% CO2 /95% air. 2.2. Flow cytometry For flow cytometry analysis, single-cell suspensions were washed twice with fluorescence-activated cell sorting (FACS) buffer containing Ca2+ -, Mg2+ -free phosphate-buffered saline (PBS), 0.5% BSA, and 0.02% sodium azide. The cells were then stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD40 mAb or isotype control antibody (BD Pharmingen, San Jose, CA, USA) for 30 min at 4 ◦ C in the dark. After washing, the cells were fixed with 2% paraformaldehyde and analyzed with a Becton Dickinson FACScan flow cytometer using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). 2.3. Real-time PCR Total RNA was extracted from MC3T3-E1 cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentrations were quantified using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260 nm. One microgram of total RNA was reverse transcribed into cDNA using a PrimeScript RT Master Mix Kit (Takara, Tokyo, Japan), according to the manufacturer’s protocol. Quantification of mRNA was performed using real-time PCR with an MyiQ thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) and a STBR Premix Ex Taq II Kit (Takara), according to the manufacturer’s instructions. Primers for CD40, IL-6, TNF-␣, IL-1␤ and GAPDH were synthesized by Sangon (Shanghai, China), and the primer sequence follows: CD40, forward, 5 -GCCACTGAGACCACTGATAC3 , and reverse, 5 -TCTGACTCGTTCCTTTCTGTAG-3 ; IL-6, forward, 5 -TCTATACCACTTCACAAGTCGGA-3 , and reverse, 5 -GAATTGCCATTGCACAACTCTTT-3 ; TNF-␣, forward, 5 -CAGGCGGTGCCTATGTCTC-3 , and reverse, 5 -CGATCACCCCGAAGTTCAGTAG-3 ; IL-1␤, forward, 5 -TTCAGGCAGGCAGTATCACTC-3 , and reverse, 5 -CCACGGGAAAGACACAGGTAG-3 ; GAPDH, forward, 5 -ACCACAGTCCATGAAATCAC-3 , and reverse, 5 -AGGTTTCTCCAGGCGGCATG-3 . The PCR amplification was performed in triplicate, and the specificity of the PCR products was verified by melting curve analysis. The mRNA expression was calculated using the comparative Ct method after it was normalized to the level of GAPDH mRNA, which was used as an internal standard. The resulting data were analyzed using iQ5 Optical System Software (Bio-Rad Laboratories). 2.4. Measurement of pro-inflammatory cytokines production The amounts of IL-6, TNF-␣ and IL-1␤ released form MC3T3-E1 cells in the supernatant medium were determined using enzymelinked immunosorbant assay (ELISA) kits for mouse IL-6, TNF-␣ and IL-1␤ (R&D systems, Minneapolis, MN), respectively, according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, USA).

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2.5. Western blotting Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer: 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 10 ␮g/ml leupeptin, 100 ␮g/ml aprotinin, 150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1% Nornidet P-40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). The solution was left standing on ice for 20 min. After centrifugation, the total protein supernatant was then collected for western blotting analysis. Nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA). The protein concentration was quantified using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories), following the manufacturer’s protocol. Aliquots (30 ␮g) were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The proteins were then electrophoretically transferred from the gels onto nitrocellulose membranes and blotted in Tris-buffered saline tween-20 (TBST) with 5% non-fat milk and were incubated overnight at 4 ◦ C with the corresponding primary antibodies against ␤-catenin, p65, STAT-1␣, phospho-GSK-3␤Ser9 , phospho-I␬B-␣Ser32/36 , phosphoSTAT-1␣Tyr701 , phospho-STAT-1␣Ser727 , ␤-actin, and TFIIB (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed with TBST: 0.05% (v/v) Tween-20 in PBS, pH 7.4. Then they were incubated with a 1:2000 dilution of secondary antibodies linked to horseradish peroxidase. The protein bans were visualized using an ECL system (Millipore, Billerica, MA, USA).

37 ◦ C. After further washing, nuclei were counterstained with 4 ,6diamidino-2-phenylindole (DAPI) (Sigma–Aldrich) for 10 min. The slides were then washed again and mounted using a ProLong Antifade Kit (Molecular Probes). Specimens were viewed and photographed using a fluorescence microscope (Leica Microsystems Ltd., Heerbrugg, Switzerland). The magnification of immunofluorescence images is 400×. 2.8. Immunoprecipitation The cells were harvested and lysed in cold immunoprecipitation lysis buffer comprised of 20 mM Tris (pH 7.5), 100 mM NaCl, 0.5% Nonidet P-40, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% protease inhibitor cocktail (Sigma–Aldrich). Lysates were then precleared with rabbit IgG and protein A/G-agarose (Invitrogen) for 2 h at 4 ◦ C. Next, precleared lysates were incubated with anti-P65 or a control rabbit IgG (Cell Signaling Technology) overnight at 4 ◦ C on a rocking platform, followed by 2 h incubation with protein A/G agarose at 4 ◦ C. After three washes with the IP lysis buffer, the pellets were suspended in SDS sample buffer, boiled for 5 min, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane and blotted with anti-␤-catenin and anti-P65 antibodies (Cell Signaling Technology). 2.9. siRNA transfection

2.6. NF-B DNA-binding assay Nuclear extracts from cells were prepared using a nuclear protein extraction kit (NEPER, Pierce Biotechnology) according to the manufacturer’s protocol. The DNA-binding activity of NF-␬B was measured using a TransAM NF-␬B p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, USA), which specifically measures NF-␬B binding to its consensus site (5 -GGGACTTTCC-3 ), according to the manufacturer’s instructions. Briefly, the nuclear extracts were incubated in 96-well plates coated with an oligonucleotide containing the NF-␬B consensus sites for 1 h at room temperature. After three washes, the primary antibody specific for the activated form of p65 was added to each well and was incubated for 1 h, followed by incubation with anti-IgG horseradish peroxidaseconjugated secondary antibody and developing solution. The level of nuclear NF-␬B p65 activation was expressed as the optical density emitted at 450 nm with a reference at 650 nm.

siRNA targeting GSK-3␤, ␤-catenin and scrambled control were commercially obtained from Cell Signaling Technology. A total of 4 × 104 cells/well were seeded in 24-well plates and then were allowed to grow until reaching ∼30–50% confluency. Cells were then transfected with 100 nM siRNA using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. After transfection, cells were cultured for 48 h before treatment. The efficiency of siRNA transfection was confirmed by western blotting analyses. 2.10. Statistics Statistical analyses were conducted using SPSS 13.0 software (IBM, Armonk, NY, USA). The experiments were repeated at least three times. The results are presented as mean ± SEM. Data were analyzed using the Student’s t test or ANOVA, and a difference of P < 0.05 was considered statistically significant.

2.7. Immunofluorescence 3. Results MC3T3-E1 cells were seeded into six-well culture plates, with a coverslip for each well, at a density of 1 × 105 cells/well. After overnight incubation, the cells were serum-starved for 6 h and then cultured in the presence or absence of SB216763 for 2 h. Next, 10 ␮g/ml of LPS (Invivogen, San Diego, CA, USA) was added to the medium for 24 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 10 min, and then were rinsed again with PBS. Cells were permeabilized with 0.1% Triton X-100 for 20 min and incubated in blocking buffer (1% BSA, 0.05% Tween-20 in PBS) for 10 min to block nonspecific binding. After washing three times with PBS, cells were incubated with the rabbit polyclonal anti-␤-catenin antibody at 1:800 dilution and the mouse monoclonal anti-NF-␬Bp65 antibody (Cell Signaling Technology, Danvers, MA) at 1:400 dilution for 1 h at 37 ◦ C. The cells were then washed three times for 5 min with PBS and subsequently were incubated with donkey anti-rabbit FITCconjugated secondary antibodies and goat anti-mouse tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies at 1:400 dilution (Abcom, Cambridge, MA, USA) for 1 h at

3.1. GSK-3ˇ inhibitor suppresses LPS-induced CD40 expression in osteoblasts To examine whether osteoblasts can express the surface molecular CD40 in response to LPS stimulation, MC3T3-E1 cells were cultured in the presence of 10 ␮g/ml Porphyromonas gingivalisderived LPS (Invivogen, San Diego, CA, USA) for 24 h. Results from real-time PCR revealed a constitutive level of CD40 mRNA in unstimulated MC3T3-E1 cells; however, after exposure to 10 ␮g/ml LPS for 24 h, the CD40 mRNA level significantly increased in MC3T3E1 cells (7.4 ± 0.8-fold) (Fig. 1D). Similar to the changes observed in mRNA levels, a notable increase in surface expression of CD40 was detectable by flow cytometry after treatment with LPS as compared with the unstimulated cells (Fig. 1A–C). These results indicated that CD40 expression in murine osteoblast-like MC3T3-E1 cells is significantly induced by LPS stimulation. To investigate the influence of GSK-3␤ inhibitor on LPSinduced CD40 expression in MC3T3-E1 cells, a specific GSK-3␤

L. Die et al. / Molecular Immunology 52 (2012) 38–49

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Fig. 1. The GSK-3␤ inhibitor suppresses LPS-induced CD40 expression on MC3T3-E1 cells in a concentration-dependant manner. MC3T3-E1 cells were pretreated with different concentrations of SB216763: 0 ␮M, 5 ␮M, 10 ␮M, and 20 ␮M for 2 h. Cells were then cultured in the presence of 10 ␮g/ml LPS for 24 h. (A) CD40 protein expression in MC3T3-E1 cells was determined by flow cytometry. 2 × 105 of cells were incubated for 30 min at 4 ◦ C with FITC-conjugated anti-CD40 or isotype control antibody for analysis by flow cytometry. Data are representative of three independently repeated experiments. (B) Histogram represents the changes in percentage of CD40-positive cells. (C) Histogram represents the fold changes of CD40 mean fluorescence intensity (MFI) values. The fold changes of CD40 MFI values were calculated relative to the value of control cells. (D) Changes in the mRNA level of CD40 in MC3T3-E1 cells. Total RNA was isolated from cultures and was assayed for the CD40 mRNA expression using real-time PCR. Each result was normalized to GAPDH mRNA. Fold changes of CD40 mRNA were calculated relative to the expression value in control cells. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. *P < 0.05 compared to the control cells; # P < 0.05 compared to the cells treated with LPS alone.

inhibitor, SB216763, was used. After 6 h serum-starved incubation, MC3T3-E1 cells were pretreated with different concentrations of SB216763: 0 ␮M, 5 ␮M, 10 ␮M, and 20 ␮M for 2 h. Then 10 ␮g/ml of LPS was added to the culture media for 24 h. The CD40 mRNA and protein expression at each concentration were determined using real-time PCR and flow cytometry analysis. Results of real-time PCR analysis showed that the mRNA level of CD40 in LPS-stimulated MC3T3-E1 cells was inhibited by SB216763 treatment in a dosedependent fashion. However, treatment with SB216763 alone had little effect on CD40 mRNA level. As shown in Fig. 1D, 20 ␮M of SB216763 significantly reduced the mRNA expression level of CD40 in LPS-stimulated MC3T3-E1 cells. Similar effects were observed using flow cytometry analysis; the results of flow cytometry analysis further confirmed that SB216763 resulted in a dose-dependent suppression of LPS-stimulated CD40 expression in MC3T3-E1 cells (Fig. 1A–C), indicating that GSK-3␤ inhibitor negatively regulates LPS-induced CD40 expression in MC3T3-E1 cells.

3.2. GSK-3ˇ inhibitor suppresses LPS-induced pro-inflammatory cytokines production in osteoblasts To further ascertain the anti-inflammatory property of GSK-3␤ inhibitor in infected osteoblasts, we determined the effect of GSK3␤ inhibitor SB216763 on the mRNA levels and protein secretion of the pro-inflammatory cytokines IL-6, TNF-␣ and IL-1␤. The intracellular mRNA levels of IL-6, TNF-␣ and IL-1␤ were determined by real-time PCR. As illustrated in Fig. 2A–C, upon stimulation with 10 ␮g/ml LPS, the mRNA levels of IL-6, TNF-␣ and IL-1␤ were significantly elevated in MC3T3-E1 cells; however, the LPSinduced upregulation in mRNA levels of the three inflammatory cytokines were dose-dependently suppressed by SB216763 pretreatment. Additionally, the amounts of IL-6, TNF-␣ and IL-1␤ in the culture supernatants were measured by ELISA. In agreement with the results from real-time PCR, LPS stimulation significantly increased the protein production of IL-6, TNF-␣ and IL-1␤; however, after pretreatment with various concentration of SB216763, protein secretions of the three inflammatory cytokines were significantly inhibited in a dose-dependent manner (Fig. 2D–F).

3.3. Inhibition of GSK-3ˇ suppresses LPS-induced activation of NF-B signaling rather than STAT-1˛ signaling in osteoblasts To investigate the inhibitory mechanism of the GSK-3␤ inhibitor on CD40 expression in LPS-stimulated MC3T3-E1 cells, we examined the activity of the NF-␬B and STAT-1␣ signaling pathway. Since the NF-␬B signaling has been reported to predominantly modulate CD40 gene expression (Harhaj et al., 2005; Qin et al., 2005; Tone et al., 2002; Wu et al., 2009), we firstly tested the influence of SB216763 on NF-␬B signaling activity by measuring the expression of phosphorylated I␬B␣ and nuclear NF-␬Bp65 in LPSstimulated MC3T3-E1 cells with or without SB216763 treatment (Fig. 3A and B). Western blotting showed that 10 ␮g/ml LPS stimulation for 24 h significantly increased I␬B-␣ phosphorylation and NF-␬Bp65 protein expression in MC3T3-E1 cells. Pretreatment with 20 ␮M SB216763 and subsequent stimulation with 10 ␮g/ml LPS in MC3T3-E1 cells, however, significantly attenuated the LPS-induced increase in phosphorylated I␬B-␣ and nuclear NF-␬Bp65 protein expression. In addition, treatment with 20 ␮M SB216763 alone failed to affect the I␬B-␣ phosphorylation and nuclear NF-␬Bp65 protein expression. Moreover, consistent with these observations, results from the NF-␬B DNA-binding assay also demonstrated that 10 ␮g/ml LPS stimulation for 24 h significantly increased the NF-␬B DNA binding activity in MC3T3-E1 cells; however, this increase was reversed when MC3T3-E1 cells were treated with 20 ␮M SB216763 in conjunction with 10 ␮g/ml LPS. Treatment with 20 ␮M SB216763 alone had no effect on the NF-␬B DNA binding activity in MC3T3E1 cells (Fig. 3H). These results indicated that GSK-3␤ inhibitor represses the LPS-induced activation of NF-␬B signaling pathway. In addition to NF-␬B, it’s been shown that the activation of the signal transducer and activator of transcription 1␣ (STAT-1␣) signaling is also involved in regulating CD40 expression (D’Alimonte et al., 2007; Lam et al., 2007; Qin et al., 2005; Rezai-Zadeh et al., 2008; Singh et al., 2010). We next tested the influence of GSK3␤ inhibitor on the activity of the STAT-1␣ signaling (Fig. 3C and D). In response to LPS stimulation, the enhancement in the protein expression of phosphorylated STAT-1␣ and nuclear STAT-1␣ was observed by Western blotting, whereas no detectable difference was found in the phosphorylation level or nuclear translocation of

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Fig. 2. The GSK-3␤ inhibitor suppresses LPS-induced pro-inflammatory cytokines production in MC3T3-E1 cells in a dose-dependant manner. The cells were pretreated with various concentrations of SB216763 (0 ␮M, 5 ␮M, 10 ␮M, 20 ␮M) for 2 h before the addition of 10 ␮g/ml LPS, and the cells were further incubated for 24 h. (A–C) Effect of the GSK-3␤ inhibitor on LPS-induced pro-inflammatory cytokines IL-6, TNF-␣ and IL-1␤ mRNA expression in MC3T3-E1 cells was measured by real-time PCR. Total RNA was isolated from cultures and was assayed for the mRNA expression of the three pro-inflammatory cytokines using real-time PCR. Each result was normalized to GAPDH mRNA. Fold changes of mRNA expression were calculated relative to the expression value in control cells. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. (D–F) Effect of the GSK-3␤ inhibitor on production of pro-inflammatory cytokines IL-6, TNF-␣ and IL-1␤ released from LPS-induced MC3T3-E1 cells in the culture medium was evaluated by ELISA. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. *P < 0.05 compared to the control cells; # P < 0.05 compared to the cells treated with LPS alone.

STAT-1␣ by SB216733 treatment in the presence of LPS, as compared to cells stimulated with LPS alone. Thus our data suggested that GSK-3␤ inhibition may have no effect on the LPS-induced activation of STAT-1␣ signaling. To confirm the effect of the pharmacological GSK-3␤ inhibitor, we knockdown GSK-3␤ in MC3T3-E1 cells by siRNA and determined the activity of the NF-␬B and STAT1␣ signaling pathway (Fig. 3F, G and I). Consistent with the results by using SB216763, the LPS-induced upregulation in the I␬B-␣ phosphorylation, nuclear NF-␬Bp65 protein expression and the NF-␬B DNA binding activity was reversed in siRNA-GSK-3␤-transfected cells, whereas siRNA of GSK-3␤ did not alter the LPS-induced increase in the phosphorylation level or nuclear translocation of STAT-1␣. These results provide evidence that inhibition of GSK-3␤ by pharmacological inhibitor or siRNA suppresses the LPS-induced activation of NF-␬B rather than STAT-1␣ signaling in MC3T3-E1 cells. 3.4. GSK-3ˇ inhibitor induces activation of the Wnt/ˇ-catenin signaling in osteoblasts The pharmacological inhibitor for GSK-3␤, SB216763, reportedly inactivates GSK-3␤ and prevents ␤-catenin degradation, resulting the activation of the Wnt/␤-catenin signaling (Bergmann et al., 2011; Xing et al., 2010). Therefore, we also determined the activity of Wnt/␤-catenin signaling in MC3T3-E1 cells upon SB216763 treatment using Western blotting. In complete agreement with the previous studies, our results showed that SB216763 treatment significantly increased GSK-3␤ phosphorylation at the Ser9 residue and nuclear ␤-catenin expression in MC3T3-E1 cells, suggesting that the pharmacological GSK-3␤ inhibitor SB216763 effectively activates of the Wnt/␤-catenin signaling (Fig. 3A and B). 3.5. The involvement of NF-B and Wnt/ˇ-catenin signaling pathways in the inhibitory mechanism of GSK-3ˇ inhibitor We further performed immunofluorescence experiments to study the subcelluar localization of ␤-catenin and NF-␬Bp65

protein in LPS-stimulated MC3T3-E1 cells with or without SB216763 treatment. As shown in Fig. 4, in unstimulated MC3T3E1 cells, ␤-catenin proteins resided in the cytoplasm near the cell membrane, and NF-␬B p65 was mainly dispersed throughout the cytoplasm in an inactive state. In cells treated with 20 ␮M SB216763 alone, obvious nuclear staining of ␤-catenin was observed, suggesting that SB216763 activated Wnt/␤-catenin signaling by translocating ␤-catenin to the nucleus, whereas nuclear staining of NF-␬Bp65 was barely invisible. In contrast, in LPSstimulated cells, obvious nuclear staining of NF-␬B p65 was seen, indicating that LPS stimulation induced translocation of NF-␬Bp65 to the nucleus, whereas no nuclear staining of ␤-catenin was detected. As we expected, pretreatment with 20 ␮M SB216763 and subsequent stimulation with 10 ␮g/ml LPS reversed the increase of LPS-induced NF-␬Bp65 nucleus translocation. Taken together with our results form western blotting, these data implied that the inhibitory mechanism of GSK-3␤ inhibitor involves both of the Wnt/␤-catenin and NF-␬B pathways in MC3T3-E1 cells.

3.6. ˇ-Catenin physically interacts with NF-B in osteoblasts Recent studies have shown the physical interaction between ␤-catenin and NF-␬B in multiple cell types (Deng et al., 2002, 2004; Du et al., 2009; Duan et al., 2007; Sun et al., 2005). By performing an immunoprecipitation assay, we found that ␤-catenin is capable of forming a complex with NF-␬Bp65 in untreated MC3T3-E1 cells. We next tested whether a GSK-3␤ inhibitor or LPS stimulation might alter the physical interaction between ␤catenin and NF-␬Bp65. Treatment with 20 ␮M SB216763 alone significantly increased the immunoprecipitation of ␤-catenin by NF-␬Bp65. On the contrary, a dramatic decrease in the amount of ␤-catenin pulled down by NF-␬Bp65 was found in MC3T3-E1 cells after exposure to 10 ␮g/ml LPS for 24 h. However, treatment of 20 ␮M SB216763 reversed the decrease in the formation of the ␤-catenin and NF-␬B complex induced by LPS stimulation (Fig. 5A and B).

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Fig. 3. The activity of signaling pathways involved in the suppression mechanism of GSK-3␤ inhibitor in LPS-induced CD40 expression. (A and C) Serum-starved MC3T3-E1 cells were pretreated with 20 ␮M SB216763 for 2 h and were then cultured with treated medium for 24 h in the presence or absence of 10 ␮g/ml LPS. Cultures were harvested, and cytoplasmic and nuclear protein extracts were isolated. Cytoplasmic fractions were analyzed using western blotting for expression of phosphorylated GSK-3␤Ser9 (A), phosphorylated I␬B-␣Ser32/36 (A), phosphorylated STAT-1␣Tyr701 (C) and phosphorylated STAT-1␣Ser727 (C). ␤-Actin expression served as a cytoplasmic control. Nuclear fractions were analyzed using western blotting for expression of nuclear ␤-catenin (A), nuclear NF-␬Bp65 (A) and nuclear STAT-1␣ (C). TFIIB served as a nuclear control. Data are representative of three independently repeated experiments. (B and D) Relative band intensity of phosphorylated GSK-3␤Ser9 (A), phosphorylated I␬B-␣Ser32/36 (A), phosphorylated STAT-1␣Tyr701 (C), phosphorylated STAT-1␣Ser727 (C), nuclear ␤-catenin (A), nuclear NF-␬Bp65 (A) and nuclear STAT-1␣ (C) protein expression. Values were expressed relative to the control cells with no treatment. Data are averaged from three blots and are expressed as the mean ± SD. (E) MC3T3-E1 cells were transfected with GSK-3␤ siRNA or scramble siRNA. At 48 h after transfection, cells were cultured with or without LPS stimulation for 24 h. The efficiency of siRNA-GSK-3␤ transfection was confirmed by western blotting. (F) The GSK-3␤ siRNA-transfected cells were harvested for western blotting. Cytoplasmic protein extracts were detected for expression of phosphorylated I␬B-␣Ser32/36 , STAT-1␣Tyr701 and phosphorylated STAT-1␣Ser727 . Nuclear protein extracts were analyzed for expression of nuclear NF-␬Bp65 and STAT-1␣ protein. Data are representative of three independently repeated experiments. (G) Relative band intensity of phosphorylated I␬B-␣Ser32/36 , phosphorylated STAT-1␣Tyr701 , phosphorylated STAT-1␣Ser727 , and nuclear NF-␬Bp65, and nuclear STAT-1␣ protein expression. Values were expressed relative to the control cells with no treatment. Data are averaged from three blots and are expressed as the mean ± SD. (H and I) Nuclear extracts of MC3T3-E1 cells treated by GSK-3␤ inhibitor (H) or transfected by GSK-3␤

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Fig. 4. Colocation of ␤-catenin and NF-␬Bp65 in MC3T3-E1 cells. MC3T3-E1 cells were plated on coverslips. The cells were serum-starved for 6 h followed by pretreatment with 20 ␮M SB216763 for 2 h. Next, cells were cultured in the presence or absence of 10 ␮g/ml LPS for 24 h. Immunofluorescence staining was performed for ␤-catenin (green) and NF-␬Bp65 (red). DAPI (blue) counterstain was used to localize the nuclei. Cells were examined under a fluorescent microscope. (A) Control cells were left untreated. (B) Cells stimulated with 10 ␮g/ml LPS alone. (C) Cells treated with 20 ␮M SB216763 alone. (D) Cells treated with 20 ␮M SB216763 in conjunction with 10 ␮g/ml LPS. Three independently repeated experiments were performed, and a representative result is shown (magnification 400×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3.7. The GSK-3ˇ inhibitor-induced suppression of NF-B activation and inflammatory response is mediated through ˇ-catenin To confirm the importance of ␤-catenin in mediating the inhibitory effect of GSK-3␤ inhibitor on NF-␬B activity, we used RNA interference to deplete ␤-catenin in MC3T3-E1 cells and investigated its influence on nuclear NF-␬Bp65 expression and NF-␬B DNA-binding activity. As shown in Fig. 5D and E, silencing ␤catenin by siRNA restored the decrease of LPS-induced nuclear NF-␬Bp65 expression that was suppressed by the GSK-3␤ inhibitor. Consistent with the result from western blotting, NF-␬B DNAbinding assay showed that the decrease of LPS-induced NF-␬B DNA binding activity repressed by the GSK-3␤ inhibitor was also reversed in siRNA-␤-catenin-transfected cells (Fig. 5F). Our results showed that the suppression effect of the GSK-3␤ inhibitor on LPS-induced NF-␬B pathway activity was attenuated in siRNA-␤catenin-transfected MC3T3-E1 cells. Furthermore, to determine whether silencing ␤-catenin in MC3T3-E1 cells influences GSK-3␤ inhibitor-induced suppression of inflammatory response, we investigated CD40 expression and pro-inflammatory cytokines production in siRNA-␤-catenintransfected MC3T3-E1 cells. As shown in Fig. 6A–D, real-time PCR and flow cytometry analysis indicated that GSK-3␤ inhibitormediated suppression in LPS-induced CD40 expression was restored in siRNA-␤-catenin-transfected MC3T3-E1 cells. Besides, the mRNA levels and protein production of IL-6, TNF-␣ and IL1␤ were determined using real-time PCR and ELISA. As shown in Fig. 6E–J, it was found that the repressed expressions of IL-6,

TNF-␣ and IL-1␤ by the GSK-3␤ inhibitor was also reversed in siRNA-␤-catenin-transfected cells. Taken together, these findings suggested that depletion of ␤-catenin by siRNA interrupted the signal connection between the Wnt/␤-catenin and NF-␬B pathways, and thus reversed the anti-inflammatory effect of GSK-3␤ inhibitor. 4. Discussion In the present study, we demonstrate that the GSK-3␤ inhibitor dose-dependently suppresses the co-stimulatory molecular CD40 expression on P. gingivalis-LPS-induced murine osteoblast-like MC3T3-E1 cells. Furthermore, we have elucidated the molecular mechanisms underlying the negative regulation effect of the GSK3␤ inhibitor on CD40 expression. We show that GSK-3␤ inhibitor represses the LPS-induced activation of NF-␬B signaling pathway via ␤-catenin, which can physically interact with NF-␬B, and consequently prevents CD40 expression and pro-inflammatory cytokines production in osteoblast. Surface molecular CD40 is a crucial co-stimulator in immune response. Several lines of evidence have shown that CD40 is also expressed in cells other than antigen-presenting cells (Gelbmann et al., 2003; Lin and Levison, 2009; Poggi et al., 2009). In our study, MC3T3-E1 cells, a murine osteoblastic-like cell line, were stimulated with P. gingivalis-derived LPS (Invivogen, San Diego, CA, USA). P. gingivalis is a well-established periodontopathic bacterium. Chronic infection by P. gingivalis results in inflammatory response and bone resorption in periodontal inflammatory disease such as periodontitis and dental implantitis (Amano, 2003;

siRNA (I) were subjected to analyses for NF-␬Bp65 DNA-binding activity using a TransAM NF-␬B p65 Transcription Factor Assay Kit. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. *P < 0.05 compared to the control cells; # P < 0.05 compared to the cells treated with LPS alone; P < 0.05 compared to the siRNA-GSK-3␤-transfected cells.

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Fig. 5. ␤-Catenin mediates the GSK-3␤ inhibitor-induced suppression in NF-␬B activation. (A) Physical interaction between ␤-catenin and NF-␬Bp65 in MC3T3-E1 cells. Serum-starved MC3T3-E1 cells were pretreated with 20 ␮M SB216763 for 2 h and were subsequently stimulated with or without 10 ␮g/ml LPS for 24 h. Cell lysates were harvested and immunoprecipitated with antibodies against NF-␬Bp65 and then were immunoblotted with antibodies against ␤-catenin and NF-␬Bp65. Data are representative of three independently repeated experiments. (B) Relative intensity of IP ␤-catenin band. Values were expressed relative to the control cell with no treatment. Results are representative of three independently repeated experiments. Data are expressed as the mean ± SD. (C) MC3T3-E1 cells were transfected with ␤-catenin siRNA or scramble siRNA. At 48 h after transfection, cells were incubated with treated medium for 24 h, as previously described. The efficiency of siRNA-␤-catenin transfection was confirmed by western blotting analyses. (D) Nuclear extracts of the siRNA-␤-catenin-transfected cells were submitted to analyses of western blotting for the expression of nuclear NF-␬Bp65 protein. Data are representative of three independently repeated experiments. (E) Relative band intensity of nuclear NF-␬Bp65 protein. Values were expressed relative to the control cell with no treatment. Data are averaged from three blots and are expressed as the mean ± SD. (F) Nuclear extracts of the siRNA-␤-catenin-transfected cells were submitted to analyses for NF-␬Bp65 DNA-binding activity. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. *P < 0.05 compared to the control cells; # P < 0.05 compared to the cells treated with LPS alone;

Hultin et al., 2002; Nonnenmacher et al., 2005). Our results show that unstimulated MC3T3-E1 cells express low level of CD40; however, a significant increase of CD40 expression was observed on MC3T3-E1 cells upon exposure to P. gingivalis-LPS. In agreement with our findings, Schrum et al. (2003) demonstrated that primary osteoblasts express the functional CD40 surface molecular upon exposure to two important pathogens of bone, Staphylococcus and Salmonella, and as well as Salmonella-derived LPS. Similarly, in a study by Ahuja et al. (2003), osteoblast-like cell lines, MC3T3-E1, OCT-1, and 2T3 were also found capable of expressing the CD40 on their surface. Considering these evidences and our results, we confirm that CD40, a key immunoregulatory molecular, is abundantly expressed on bone-forming osteoblasts upon stimulation of bacteria or bacterial products. Besides, we also observed notable increases in secretion of pro-inflammatory cytokines IL-6, TNF␣ and IL-1␤ in LPS-stimulated MC3T3-E1 cells. Interestingly, it is noteworthy that in response to inflammatory stimulation the immune activity of osteoblasts is greatly enhanced, including the upregulation in the co-stimulatory molecular expression and proinflammatory cytokines production, and this behavior is similar to the biological characteristics of dendritic cells (Banchereau and Steinman, 1998; Mellman and Steinman, 2001; Revy et al., 2001).

P < 0.05 compared to the siRNA-scramble-transfected cells.

Indeed, studies have addressed the improved immunological role of osteoblasts such as cytokine secretion, antigen presentation and stimulation of T cells (Ohno et al., 2006; Perez et al., 2006; Stanley et al., 2006), and conversely the depressed bone-forming capacity of osteoblasts under inflammatory situation (Bandow et al., 2010; Lee et al., 2010; Wang et al., 2010). Altogether, our results further support the previously unexpected immunological function of osteoblasts in inflammatory bone disease. We propose that the immunocompetent property of osteoblasts provides a new insight into the exploration of the pathophysiological mechanism and development of targeted drugs for inflammatory bone disease. Recent discoveries highlight the anti-inflammatory potential of GSK-3␤ inhibitors (Gao et al., 2008; Gurrieri et al., 2010; Yuskaitis and Jope, 2009). However, little is known about their anti-inflammatory role in infected osteoblasts. For this purpose, we sought to determine whether LPS-induced CD40 expression could be regulated by a GSK-3␤ inhibitor. Our results demonstrated that SB216763 treatment significantly inhibited LPS-stimulated CD40 expression in MC3T3-E1 cells in a dose-dependent manner. Besides, the release of pro-inflammatory cytokines is also a key factor involved in the process of inflammation. The proinflammatory cytokines IL-6, TNF-␣ and IL-1␤ are implicated in

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Fig. 6. siRNA-␤-catenin recovers the GSK-3␤-inhibitor-induced suppression of inflammatory response in LPS-stimulated MC3T3-E1 cells. (A) CD40 protein expression in MC3T3-E1 cells were determined by flow cytometry. Data are representative of three independently repeated experiments. (B) Histogram represents the changes in percentage of CD40-positive cells. (C) Histogram represents the fold changes of CD40 Mean Fluorescence Intensity (MFI) values. (D) Changes in the mRNA level of CD40 in MC3T3-E1 cells were measured by real-time PCR. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. (E–G) Changes in the mRNA level of pro-inflammatory cytokines IL-6, TNF-␣ and IL-1␤ were measured by real-time PCR. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. (H–J) Changes in the secretion of pro-inflammatory cytokines IL-6, TNF-␣ and IL-1␤ in the culture medium were evaluated by ELISA. Data are expressed as the mean ± SD. Results are representative of three independently repeated experiments. *P < 0.05 compared to the control cells; # P < 0.05 compared to the cells treated with LPS alone;

P < 0.05 compared to the siRNA-scramble-transfected cells.

many inflammatory bone disease, such as rheumatoid arthritis, periodontitis, dental implantitis (Javed et al., 2011; Schytte Blix et al., 1999; Verbruggen et al., 1999). Our results also showed concentration-dependant reductions by SB216763 treatment in both mRNA levels and protein expression of IL-6, TNF-␣ and IL-1␤ in LPS-stimulated MC3T3-E1 cells, further ascertaining that GSK3␤ inhibitor may prevents the inflammatory response in infected osteoblasts. In agreement with our findings, Natsume et al. (2011) showed that lithium chloride (LiCl), another inhibitor of GSK-3␤, significantly repressed IL-6 release in TNF-␣-induced MC3T3-E1 cells. Thus far, relatively little information is available about the effect of GSK-3␤ inhibitor on modulating the immune activities of osteoblasts. We provide important evidence supporting the speculation that the GSK-3␤ inhibitor may repress the immune activity of osteoblasts and thus possess anti-inflammatory potential in inflammatory bone diseases. More importantly, there is a

special significance to study the anti-inflammatory effect of GSK3␤ inhibitor in infected osteoblasts. It is known that inflammatory bone disease (e.g. periodontitis and dental implantitis) are characterized by localized inflammatory response and bone loss, which are induced by pathological bacteria colonization (Amano, 2003; Hultin et al., 2002; Nonnenmacher et al., 2005). Accumulating evidences have indicated that GSK-3␤ inhibitors can effectively induce osteoblast differentiation in vitro and increase bone mass in vivo (Kulkarni et al., 2006; Xing et al., 2010; Wang et al., 2009). Taken together with our findings, GSK-3␤ might represent a novel therapeutic target for bone inflammatory disease, with dual roles in suppressing inflammatory response as well as promoting bone formation. Thus, it is of great importance to clarity the regulatory mechanism of GSK-3␤ inhibitor in infected osteoblasts. It is well-recognized that CD40 is a tumor necrosis factor receptor superfamily member (TNFRSF5) with predominant activation

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through the NF-␬B signaling pathway (Benveniste et al., 2004; Munroe and Bishop, 2007; Song et al., 2012). Several lines of evidence have shown that the activation of the NF-␬B has a critical role in up-regulating CD40 gene expression following LPS stimulation in macrophages, dendritic cells, and other non-immune cell types (Qin et al., 2005; Tone et al., 2002; Wu et al., 2009). However, in addition to the NF-␬B signaling, increasing recent evidences suggest that the expression of CD40 is also regulated through a mechanism involving the activation of the STAT-1␣ signaling pathway (D’Alimonte et al., 2007; Lam et al., 2007; Qin et al., 2005; Rezai-Zadeh et al., 2008; Singh et al., 2010). Qin and colleagues suggested that LPS induces CD40 expression in macrophages and microglia at the transcriptional level and involves activation of the transcription factors NF-␬B and STAT-1␣. Similarly, Lam and colleagues demonstrated Leptin alone or in cooperation with LPS induce CD40 expression through the activation of transcription activators, STAT-1␣ and NF-␬Bp65, to target the CD40 promoter. Our results are in agreement with these previous findings showing that LPS stimulation induces the activation of NF-␬B and STAT1␣. However, the effects of GSK-3␤ inhibition on modulating the activities of the two signaling pathways are totally different. Inhibition of GSK-3␤ by inhibitor or siRNA repressed the LPS-induced activation of the NF-␬B by suppressing I␬B␣ phosphorylation, NF␬Bp65 nuclear translocation, and NF-␬Bp65 DNA binding activity in MC3T3-E1 cells, whereas inhibition of GSK-3␤ by inhibitor or siRNA failed to influence the LPS induced phosphorylation or nuclear translocation of STAT-1␣. Consistent with our data, previous study by Beurel and Jope have demonstrated that STAT1 activation was completely independent of GSK-3 in the IFN␥-induced RAW264.7 cells. LiCl or knockdown of the GSK-3 strongly reduced the activation of STAT3 but not STAT1 (Beurel and Jope, 2009). Accordingly, we suggest that STAT-1␣ is not involved in the suppression mechanism of LPS-induced CD40 expression by GSK-3␤ inhibitor. I␬B␣ is a major regulator of the NF-␬B signaling pathway. The phosphorylation and subsequent degradation of I␬B is indicative of the activation of NF-␬B signaling (Bonizzi and Karin, 2004; Liang et al., 2004). Our results revealed a significant decrease in LPS-induced I␬B␣ phosphorylation at serine residue 32/36 in GSK-3␤-inhibitor-treated MC3T3-E1 cells, implying that I␬B␣ is involved in the inhibition mechanism of the GSK-3␤ inhibitor. Consistent with our results, a number of previous studies also revealed an I␬B␣-related suppression effect by GSK-3␤ inhibitor treatment or GSK-3␤ knockdown (Duan et al., 2007; Sanchez et al., 2003; Takada et al., 2004). However, in a study by Steinbrecher et al. (2005), no major change was found in cytokine-induced I␬B kinase activity and subsequent phosphorylation of I␬B␣ in GSK3␤-null cells, although the loss of GSK-3␤ specifically affects a subset of NF-␬B-regulated genes. Similarly, Schwabe and Brenner (2002) reported that LiCl treatment resulted in a downregulation of the NF-␬B-dependent gene transcription without affecting the degradation of I␬B␣ in primary hepatocytes. Nevertheless, these controversial findings may be due to, at least in part, the differences in cell types or inhibitor types. Further investigation is required to determine whether the GSK-3␤ inhibitor suppresses activation of the NF-␬B pathway in an I␬B␣-dependent way. Data from our immunoprecipitation assay showed that ␤-catenin physically interacts with NF-␬Bp65 in osteoblasts, suggesting that ␤-catenin is a key mediator to bridge the crosstalk between the Wnt/␤-catenin and the NF-␬B signaling pathways. To confirm the importance of ␤-catenin, we used RNA interference to deplete ␤-catenin and showed that GSK-3␤ inhibitor-mediated suppression in LPS-induced NF-␬B activation, CD40 expression and pro-inflammatory cytokines production were recovered by silencing ␤-catenin in MC3T3-E1 cells. Consistent with our findings, Deng et al. (2004) showed that inhibition of GSK-3␤ suppresses TNF-␣-induced NF-␬B activity in cancer cells, whereas depletion

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of ␤-catenin with siRNA reverses the effect. In light of these findings, we further ascertain that the suppression mechanism of the GSK-3␤ inhibitor on NF-␬B activity is mediated through ␤-catenin. Moreover, results of western blotting and immunofluorescence indicated that GSK-3␤ inhibitor greatly induces translocation of ␤-catenin into the nucleus and thus activates the Wnt/␤-catenin signaling. Interestingly, in bone, the Wnt/␤-catenin signaling plays a critical role in regulation bone homeostasis. Enhanced Wnt/␤-catenin signaling promotes osteoblastic differentiation and bone formation (Kubota et al., 2009; Westerndrof et al., 2004; Yavropoulou and Yovos, 2007). Taken together with our findings, it is highly probable that the physical interaction of ␤-catenin and NF-␬Bp65 may provide molecular connection between the Wnt/␤-catenin-mediated bone formation and the NF-␬B-mediated inflammation. Thus, the GSK-3␤ inhibitor, which activates the Wnt/␤-catenin signaling and represses the NF-␬B signaling through ␤-catenin, appears to be an emerging target of the therapy for inflammatory bone-resorpting disease, With distinct advantage in bone protection and anti-inflammation.

5. Conclusion In conclusion, we confirmed the possibility that GSK-3␤ inhibitor is able to attenuate LPS-induced CD40 expression and pro-inflammatory cytokines production in murine osteoblast-like MC3T3-E1 cells by inhibiting NF-␬B activation through ␤-catenin. Although the exact molecular mechanisms require further investigation, our results provide supportive evidence of the potential anti-inflammatory role of GSK-3␤ inhibitors in inflammatory bone disease.

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