Pneumolysin-induced autophagy contributes to inhibition of osteoblast differentiation through downregulation of Sp1 in human osteosarcoma cells

    Pneumolysin-induced autophagy contributes to inhibition of osteoblast differentiation through downregulation of Sp1 in human osteosarcoma cells Jinwook Kim, Hee-Weon Lee, Dong Kwon Rhee, James C. Paton, Suhkneung Pyo PII: DOI: Reference:

S0304-4165(17)30219-2 doi:10.1016/j.bbagen.2017.07.008 BBAGEN 28888

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

16 January 2017 12 July 2017 12 July 2017

Please cite this article as: Jinwook Kim, Hee-Weon Lee, Dong Kwon Rhee, James C. Paton, Suhkneung Pyo, Pneumolysin-induced autophagy contributes to inhibition of osteoblast differentiation through downregulation of Sp1 in human osteosarcoma cells, BBA - General Subjects (2017), doi:10.1016/j.bbagen.2017.07.008

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ACCEPTED MANUSCRIPT Pneumolysin-induced autophagy contributes to inhibition of osteoblast differentiation through

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downregulation of Sp1 in human osteosarcoma cells

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Jinwook Kim1#, Hee-Weon Lee1#, Dong Kwon Rhee1, James C. Paton2, Suhkneung Pyo 1*

School of Pharmacy, Sungkyunkwan University, Suwon, Gyunggi-do, 16419, Republic of Korea.

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School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, 5005, Australia

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These two authors contributed equally to this work

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*Correspondence to: Suhkneung Pyo, Phone: +82-31-290-7713, FAX: +82-31-292-8800,

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E mail: [email protected]

ACCEPTED MANUSCRIPT Highlight

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PLY inhibits the expression of osteogenesis related genes and ALP activity

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PLY inhibits osteoblast differentiation through lysosome-dependent downregulation of Sp1

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PLY induces autophagy through ROS-mediated AMPK/mTOR signaling pathway.

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ACCEPTED MANUSCRIPT Abstract

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Background information: The 53 kDa protein pneumolysin (PLY) is the main virulence factor of Streptococcus

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pneumoniae, a leading cause of invasive pneumococcal diseases. PLY forms pores in cholesterol-containing membranes, thereby interfering with the function of cells. Bone destruction is a serious matter in chronic

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inflammatory diseases such as septic arthritis and osteomyelitis. S. pneumoniae is increasingly being recognized as a common cause of septic arthritis, but its pathogenesis is poorly defined.

Method: We examined the effect of PLY on osteoblast differentiation and its mechanisms of action. The effect of

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PLY on osteoblast differentiation was evaluated by qRT-PCR, ALP activity assay, flow cytometric analysis, and Western blotting. We also examined the role of PLY-induced autophagy in osteoblast differentiation using RNA

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interference analysis.

Results: PLY inhibited osteoblast differentiation by decreasing the expression of osteoblast marker genes such as

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Runx2 and OCN, along with ALP activity. ROS production was increased by PLY during osteoblast

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differentiation. PLY induced autophagy through ROS-mediated regulation of AMPK and mTOR, which downregulated the expression of Sp1 and subsequent inhibition of differentiation. Treatment with autophagy

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inhibitors or Atg5 siRNA alleviated the PLY-induced inhibition of differentiation. Conclusion: The results suggest that PLY inhibits osteoblast differentiation by downregulation of Sp1 accompanied by induction of autophagy through ROS-mediated regulation of the AMPK/mTOR pathway.

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General significance: This study proposes a molecular mechanism for inhibition of osteoblast differentiation in response to PLY.

Keywords Osteoblast, Pneumolysin, Autophagy, Sp1, ROS, AMPK, mTOR

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ACCEPTED MANUSCRIPT 1. Introduction

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Streptococcus pneumoniae (pneumococcus) is a highly invasive Gram-positive bacterial pathogen that is

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responsible for high mortality and morbidity worldwide. The bacterium is a deadly threat to young children, the elderly, and others with weak immune systems [1]. In addition, an increase in antimicrobial resistance of

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pneumococcus, such as resistance to β-lactams and fluoroquinolones, has raised the risk of disease [2]. The mucosal surface of the upper respiratory tract is the main residence of pneumococcus. During colonization of the site, the organism can gain access to other organs, such as the lungs, ears, and brain, which causes

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pneumonia, otitis media, and meningitis, respectively [3]. Moreover, pneumococcus is an important causative agent of osteomyelitis and septic arthritis [4].

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Bone is the largest organ in the human body and is a major site of bacterial infection. Bone infection is terribly hard to treat with antibiotics. Furthermore, it can be worsened by infection with antibiotic resistant organisms

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[5]. During bacteremia, pathogens can reach the metaphyseal space of the bone through the blood stream.

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Because of the slow and turbulent blood flow in this region, bacteria can exit the metaphyseal vessels through endothelial gaps and then may reach the extravascular space [6]. The infiltrated bacteria damage bone cells

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either directly or by the release of factors that affect bone cells, resulting in pathological changes [5]. Increasing evidence has suggested that pneumococcus is one of the bone-infecting bacteria, and it is known to be closely related to chronic inflammation of bone [7, 8, 9]. Thus, bone destruction is a serious matter in chronic

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inflammatory diseases such as septic arthritis and osteomyelitis. However, the mechanism of pneumococcal bone infection has not yet been clearly revealed. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae; it is necessary for survival of pneumococcus and for bacterial spread to the bloodstream from the lungs. In addition, toxin-mediated pore formation elevates chemokine levels, which attracts neutrophils, resulting in stimulation of the inflammatory response [5]. Previous studies have shown that pneumococci in the bloodstream cause cardiac injury by the release of PLY [10], and that PLY plays a critical role in a murine model of S. pneumoniae corneal infection and meningitis [11, 12]. Thus, PLY is a key virulence factor in invasive pneumococcal diseases. PLY is a 53 kDa protein and a cholesterol-dependent cytolysin that binds to cholesterol-containing cell membranes and consequently creates pores. By forming the pores, the toxin alters the membrane’s electrical charge or causes a calcium influx. It disturbs osmotic balance so that cells are stressed and reactive oxygen species (ROS) are generated [3, 13].

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ACCEPTED MANUSCRIPT Previous studies indicated that the generation of ROS by PLY induces autophagy [12, 14]. However, the precise mechanism of action of PLY on the differentiation and function of bone cells is unclear.

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Autophagy is a natural self-degradative mechanism which plays important roles in recycling or clearing of

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useless proteins during cell development [15]. Many studies showed that autophagy is highly induced and is vital for cell differentiation during erythropoiesis, lymphopoeisis, adipogenesis, and osteogenesis [16, 17]. ROS

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generation is also considered to be important for the induction of autophagy [18]. Recent studies have shown that autophagy is induced by AMP activated protein kinase (AMPK) and is inhibited by the mammalian target of rapamycin (mTOR). ROS induces autophagy through phosphorylation of AMPK, which inhibits mTOR [18,

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19]. During induction of autophagy, autophagy related proteins (ATG) regulate the formation of autophagosomes via LC3-II and the ATG5-ATG12 complex. Subsequently, the proteins in autophagosomes are

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degraded by fusion with lysosomes [20]. In this way, cells are able to maintain intracellular homeostasis. However, over-induction of autophagy can cause cellular dysfunction and is significantly associated with many

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chronic diseases, including Alzheimer’s disease and rheumatoid arthritis (RA) [21, 22]. In rheumatoid arthritis,

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autophagy is continually activated and apoptosis is downregulated in synovial fibroblasts, which causes activation of the cells resulting in inflammation and joint destruction. Moreover, autophagy is activated in

destruction [22, 23].

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osteoclasts of patients with RA and activation of autophagy induces osteoclastogenesis, leading to bone

The transcription factor Sp1 (Sp1) is essential during cell proliferation and differentiation. Sp1 binds to GC-

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rich regions, which enhance various gene promoter activities [24]. Sp1 regulates the transcription of runt-related transcription factor (Runx2) by binding to a purine-rich region in the Runx2 promoter. Runx2 is known as a major transcription factor that induces alkaline phosphatase (ALP) activity and the expression of bone matrix genes, including osteocalcin (OCN), during osteoblast differentiation [25, 26]. In addition, overexpression of Sp1 enhances Runx2 promoter activity, whereas knockdown of Sp1 downregulates the expression of Runx2 [27]. Previous studies have also shown that ROS generation is associated with downregulation of Sp1 [28, 29]. The potential correlation between osteoblast differentiation, autophagy and PLY remains unknown. For the present study, we used Human osteoblast-like MG63 and HOS cells as an in vitro model to investigate the effect of PLY on osteoblast differentiation, because MG63 and HOS cells are the most frequently used substitute for primary osteoblast. We herein examined the effect of PLY on osteoblast differentiation. In this study, we reveal that PLY inhibits the differentiation of osteoblasts by early induction of autophagy and subsequent downregulation of Sp1 protein levels via ROS-mediated activation of the AMPK/mTOR cascade. 4

ACCEPTED MANUSCRIPT 2. Materials and Methods

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2.1 Reagents and chemicals

Unless otherwise mentioned, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

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3-Methyladenine (3-MA) was purchased from Tocris Bioscience (Bristol, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin G, and streptomycin were obtained from Life

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Technologies Inc. (Carlsbad, CA, USA). TRIzol reagent, SuperScript Ⅱ kits, and Lipofectamine RNAiMAX were purchased from Invitrogen (Cergy Pontoise, France). MG-132 was purchased from Calbiochem (Billerica,

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MA, USA). TOPreal qPCR 2X PreMIX was purchased from Enynomics (Daejeon, Korea). The Alkaline Phosphatase Activity Colorimetric Assay Kit was purchased from Biovision (Milpitas, CA, USA). DC protein assay reagent was from Bio-Rad Laboratories (Hercules, CA, USA). Antibodies against Sp1, p-AMPK (Thr

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172), pneumolysin and AMPK𝛼1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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The antibody against LC3B/MAP1LC3B was purchased from Novus Biological (Littleton, CO, USA). The antibodies against p-mTOR (Ser 2448) and mTOR were purchased from Cell Signaling Technology (Beverly,

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MA, USA). The antibody against p62 was purchased Enzo Life Science (Exeter, UK).

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2.2. Purification of recombinant pnuemolysin

Purification of recombinant PLY was performed as described previously [12]. Briefly, the PLY gene (Table 1) was amplified with the incorporation of BamHI and XhoI restriction sites at the 5’ and 3’ ends of the amplicon. The products were digested by the enzymes and cloned into the plasmid pET32b(+). The plasmid was inserted into BL21 and the bacteria was incubated in LB media supplemented with kanamycin (20 µg/ml) at 37°C in an incubator with shaking until OD600 reached 0.6-0.8. The proteins were overexpressed by providing IPTG at a final concentration of 0.5 mM. The bacteria were grown at 25°C in an incubator with shaking overnight. The cells were centrifuged at 4,000 rpm for 15 min at 4°C, and the pellet was disentangled in 10 ml of lysis buffer (50mM Tric-HCl pH 7.5, 10% glycerol, 1mM DTT, 1mM PMSF). The cell suspension was sonicated and centrifuged at 13,000 rpm for 30 min at 4°C. Histidine-tagged PLY in the supernatant was purified using a NiNTA column. L460D, which is a detoxified derivative of PLY was kindly provided by Prof. James C. Paton, University of Adelaide. L460D is unable to bind cholesterol or form pores and was constructed as described 5

ACCEPTED MANUSCRIPT previously [30]. In addition, native PLY was purified from a clinical strain of Streptococcus pneumoniae

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(serotype 19A) which was obtained from Samsung Medical center, Seoul, Korea, as described elsewhere [31].

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2.3. Bacterial strains

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Streptococcus pneumoniae D39 (serotype 2) was grown overnight on 5% sheep blood agar (BA). D39 was cultured in Brain Heart Infusion media containing (per liter) 37 g of Brain Heart Infusion Broth (BD, New Jersey, USA), and 1.5% Agar (Merck, Darmstadt, Germany). The broth was sterilized for 15 min at 121°C. The

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pneumococcal growth was cultured as described previously [12]. The pneumococcus was cultured at 37°C overnight on BHI blood agar (supplemented with antimycin where appropriate). Subsequently, the

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pneumococcus grown on BHI agar plate was transferred into BHI broth and cultures were grown in broth at

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37°C without aeration. Cultures to be frozen were first brought to 5 to 10% glycerol and then stored at −80°C.

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2.4 In vivo infection and synovial fluid collection

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Male C57BL/6 mice at 6-7 weeks of age were used in infection experiments. All animal experimental procedures were approved and monitored by the Sungkyunkwan University Animal Ethical Committee. The mice were challenged i.p. with 1 × 104 CFU of pneumococci [D39 or pneumococcal isolate (serotype 19)]. Six

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mice were sacrificed 24 h after infection. Synovial fluid samples were obtained from mouse synovial joints by the modified method previously described [32]. Briefly, after the skin overlying the knee was excised within 1 h of sacrifice, the patellar ligament was cut to expose the synovial membrane. A 30-gauge needle (Micro-fine insulin syringe: BD, New Jersey, USA) was inserted through the membrane and the synovial cavity was washed twice by injecting and aspirating 200 l of phosphate buffered saline (PBS) containing FBS. The level of PLY was detected from the collected fluid by ELISA and Western blot, respectively. Synovial fluid samples were also used for analysis of neutrophil number. The neutrophil number was assessed by flow cytometry based on cell surface expression of Ly6G.

2.5. Cell culture

Human osteoblast-like MG63 and HOS cells which are commonly used for osteoblastic models were obtained 6

ACCEPTED MANUSCRIPT from the American Type Culture Collection (Manassas, VA, USA). MG63 and HOS cells were maintained in DMEM with 10% heat-inactivated FBS, 100 IU/ml of penicillin and 100 µg/ml of streptomycin in a 37°C

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humidified atmosphere of 5% CO2. To induce osteoblast differentiation, MG63 and HOS cells were seeded in 6-

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wells plate (2×105 cells/well). The cells were incubated in osteogenic differentiation medium (OM), which is DMEM with 5% FBS and antibiotics supplemented with 𝛽-glycerophosphate (10 mM), L-ascorbic acid

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(50µg/ml), and dexamethasone (0.5 µM). The media was changed every 2 to 3 days.

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2.6. Measurement of cell proliferation

MG63 cells at a density of 4×104 cells/well were plated in 96-well culture plates and cultured in OM in the

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absence or presence of 5, 50, 125, 250, or 500 ng/ml of PLY for 1, 3, 5, or 7 days. At the end of treatment, cell proliferation was measured by the addition of 25 µg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) for an additional 4 hrs. The supernatant was removed and 150 µl of Dimethyl sulfoxide (DMSO)

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was added for formazan-crystal solubilization. The absorbance was measured at 540 nm using a scanning multi-

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well plate reader. This experiment was performed in triplicate.

2.7. RNA isolation and qRT-PCR

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Total RNA was isolated from cultured MG63 and HOS cells with TRIzol reagent as per the manufacturer’s protocol and cDNA was prepared using the SuperScript Ⅱ kit. Quantitative real-time RT-PCR was carried out using TOPreal qPCR 2X PreMIX. The expression level of GAPDH was used as the standard to normalize the sample quantification. The sequences of the primers used are listed in Table 2. The data are relative expression levels shown as the fold difference compared with the control group at day 0 and are normalized to the expression level of the housekeeping gene (GAPDH).

2.8. Measurement of ROS production

For quantification of intracellular reactive oxygen species (ROS) generation, MG63 cells were incubated with 2’,7’-Dichlorofluorescin diacetate (DCF-DA). The cells were incubated for 2 hrs with 10 mM of N-Acetyl Cysteine (NAC) followed by PLY treatment for 1 hr. The cells were then incubated with 10 µM DCF-DA for 30 7

ACCEPTED MANUSCRIPT min at 37°C, suspended in PBS and analyzed using flow cytometry to determine the fluorescence intensity of cells. The spontaneous fluorescence intensity of the negative control tube without DCF-DA was defined as 1,

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and the fluorescence intensity values of OM-treated or OM+PLY-treated groups were the values relative to that

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of the negative control (detected fluorescence intensity/the fluorescence intensity of the negative control).

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2.9. Alkaline phosphatase activity measurement

ALP activity was measured using the Alkaline Phosphatase Activity Colorimetric Assay Kit according to the manufacturer’s protocol. Briefly, MG63 and HOS cells were plated in 12-well plates at 5×104 cells/well and

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cultured in OM for 7 days. The cells were harvest and homogenized in the assay buffer, then centrifuged at 13,000 rpm for 5 min at 4°C to remove insoluble material. The supernatant was added to 96-well plates, mixed

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Nitrophenol (pNP) was measured at 405 nm.

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with p-Nitrophenylphosphate (pNPP) substrate, and incubated at 25°C for 60 min. The amount of colored p-

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2.10. Western blot analysis

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Western blot analysis was performed to confirm protein expression. MG63 cells were pre-treated with inhibitors (NAC, rapamycin, CQ, 3-MA, and MG132), then incubated with OM in the absence or presence of PLY, or co-treated with Mith A. After washing twice with PBS, total cell lysates were lysed in lysis buffer (150

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mM NaCl, 50 mM Tris-HCl pH8.0, 5 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) supplemented with protease inhibitors then centrifuged at 13,000 rpm for 10 min at 4°C, after which the supernatants were taken. The proteins were quantified using the DC protein assay (Bio-Rad Lab, CA, USA). An equivalent amount of protein from each sample was loaded onto SDS-PAGE gels, followed by transfer to a NC/PVDF membrane (Millipore Corporation, MA, USA). After blocking with 5 % skim milk (Millipore Corporation,

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in

Tris-buffered

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containing

0.05%

Tween-20,

nitrocellulose

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polyvinylidenedifluoride membranes were probed with primary antibodies against Sp1, p-AMPK (Thr 172), AMPK𝛼1/2, LC3B/MAP1LC3B, p-mTOR (Ser 2448), mTOR, p62, and PLY, and the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Specific protein bands were detected using the ‘AbSignal’ Western blotting detection reagent (AbClon, Korea). The amount of protein was assessed by densitometry using ImageJ software and quantified relative to 𝛽-actin (sp1, p62, LC3) or the corresponding total protein band (p-AMPK, p-mTOR). 8

ACCEPTED MANUSCRIPT 2.11. RNA interference

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Nonsilencing control siRNA and siRNA targeting human Atg5 were chemically synthesized (Genolution

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Pharmaceuticals Inc.) MG63 cells were seeded in 6-well culture plates (1×105 cells/well) and then transiently transfected with siRNA for 24 hrs at a final concentration of 100nM using Lipofectamine RNAiMAX according

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to manufacturer’s instructions. The sequences of the siRNAs used are listed in Table 3.

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2.12. ELISA assays

A 96-well microplate was coated with mouse monoclonal anti-PLY antibody (Invitrogen, MA) and incubated

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overnight. After blocking nonspecific protein binding, 100 l of synovial fluid was added and the plate was incubated for 2 hrs at room temperature. The rabbit IgG polyclonal anti-PLY was added followed by anti-rabbit

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IgG alkaline phosphatase conjugate (Sigma-Aldrich). ELISA was developed by addition of p-

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2.13. Statistical analysis

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nitrophenylphosphate (Sigma-Aldrich). The absorbance was measured at 405 nm.

Unless otherwise indicated, all reported results were reproduced at least three independent experiments and

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were expressed as means ± SEM. The representative data are shown. The statistical significance of the differences between each of the results was evaluated using one way analysis of variance (ANOVA) followed by Tukey's post-test in GraphPad Prism 5 and significant differences are indicated when a p-value is less than 0.05.

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ACCEPTED MANUSCRIPT 3. Results

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3.1. PLY inhibits osteoblast differentiation in MG63 cells

To investigate the effects of PLY on osteoblast differentiation, we used MG63 and HOS cells which are

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capable of differentiating into functioning osteoblast-like cells [34, 35]. To find concentration and treatment that do not affect cell proliferation, the MTT assay was performed by treating the cells with 5, 50, 125, 250, or 500 ng/ml of PLY for 1, 3, 5, or 7 days, respectively, in osteogenic conditions. The results showed that only 500

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ng/ml of PLY negatively influenced the proliferation of MG63 cells through 7 days of culture (Fig. 1A). Thus, in all subsequent experiments, we used PLY at concentrations of 50, 125 or 250 ng/ml and investigated the effect

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of PLY on osteoblast differentiation. Similar results were observed in the HOS cells (data not shown). Next, we determined whether PLY affects differentiation of osteoblast by measuring ALP activity and mRNA expression

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of Runx2 and OCN which are commonly used as markers of osteogenic differentiation. MG63 and HOS cells

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were incubated in osteogenic differentiation media (OM) in the absence or presence of various concentrations of PLY for the indicated number of days. The results showed that PLY decreased ALP activity and mRNA

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expression of Runx2 and OCN in a time- and concentration-dependent manner (Fig. 1B-D), indicating that PLY suppresses the differentiation of osteoblast. Additional support for the conclusion that inhibitory effect of PLY on the differentiation of osteoblast came from the observation that native PLY from a clinical isolate (serotype

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19A) was able to decrease ALP activity and mRNA expression of Runx2 and OCN (Supplementary 1).

3.2. PLY-induced ROS production is sufficient to impair osteoblast differentiation

Because PLY is known to induce intracellular reactive oxygen species (ROS) [12, 36], we examined whether PLY-induced ROS affects osteoblast differentiation. First, we investigated the production of ROS in the presence of 250 ng/ml PLY. We also determined the effect of PLY on the ROS production by comparing PLY to L460D, which is a non-cytolytic derivative of PLY that cannot bind to cell membrane or create pore [30]. The results showed that the production of ROS by PLY-treated MG63 cells is approximately 4-fold higher than that by OM-treated control, whereas L460D did not significantly induce production of ROS and pretreatment with 10 mM NAC prevented ROS production (Fig. 2A, B). To further confirm the involvement of PLY-driven ROS production in osteoblast differentiation, we examined ALP activity and mRNA expression of Runx2 and OCN. 10

ACCEPTED MANUSCRIPT The results showed that the inhibitory effect of PLY on ALP activity and Runx2 and OCN expression was alleviated by the presence of 1 mM NAC, while L460D did not significantly decrease these differentiation

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markers (Fig. 2C, D). These data indicate that the production of ROS induced by PLY is involved in the

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inhibition of osteoblast differentiation.

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3.3. PLY down-regulates Sp1 protein in MG63 cells

Sp1 is an essential transcription factor during osteoblast differentiation [27], and the expression of Sp1 is

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downregulated by ROS [28, 37]. Based on these findings and our data, we investigated whether PLY downregulates the expression of Sp1 during osteoblast differentiation. MG63 cells were incubated in OM in the

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absence or presence of 250 ng/ml PLY. The expression of Sp1 was significantly downregulated in the presence of PLY (Fig. 3A). To confirm the role of Sp1 in the differentiation of MG63 cells, the cells were incubated in

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OM with PLY (250 ng/ml) or mithramycin A (Mith A) (100 nM), an inhibitor of Sp1. Sp1 expression, ALP

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activity, and mRNA expression of Runx2 and OCN were measured. The obtained results showed that PLY downregulates the expression of Sp1 and also inhibits osteoblast differentiation markers, similar to the effect of

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Mith A (Fig. 3B-D). These data suggest that the downregulation of Sp1 by PLY is associated with inhibition of osteoblast differentiation.

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3.4. PLY-induced autophagy inhibits osteoblast differentiation through the downregulation of Sp1

Since previous studies demonstrated that PLY plays an important role in the induction of autophagy during pneumococcal infection [12, 14], autophagy was investigated in the absence or presence of 3-MA or chloroquine (CQ) in PLY-treated MG63 cells. It is well known that 3-MA inhibits an early stage of autophagy by blocking type III Phosphatidylinositol 3-kinases (PI-3K) and CQ blocks autophagy at late stage. Our experiments showed that pretreatment with 3-MA reduced the turnover of LC3-II induced by PLY, whereas pretreatment with CQ elevated LC3-II levels and the combination of PLY with CQ caused more pronounced conversion of LC3-1 toLC3-II (Fig. 4A). To further examine the role autophagy played in the inhibition of osteoblast differentiation caused by PLY, Atg5 RNAi-mediated inhibition of autophagy was performed. This is because Atg5 is essential for the initiation of autophagosome formation and Atg5 deletion is known to complete inhibits autophagy. The results showed that knockdown of Atg5 effectively inhibited autophagy (Fig. 4B). Moreover, the obtained 11

ACCEPTED MANUSCRIPT results showed that inhibiting autophagy by 3-MA and CQ significantly restores the mRNA expression of Runx2 and OCN, as well as ALP activity in PLY-treated cells (Fig. 4C, D), Additionally, the knockdown of Atg5

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reduced mRNA expression of Runx2 and OCN, as well as ALP activity (Fig. 4C, D). Taken together, these data

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suggest that PLY-induced autophagy inhibits osteoblast differentiation.

We next determined whether the downregulation of Sp1 is proteasome- or lysosome-dependent in PLY-treated

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cells, because lysosome and proteasome systems are major proteolytic pathways. MG63 cells were pretreated with the lysosome inhibitor CQ (10µM), the proteasome inhibitor MG-132 (10µM) or the autophagy inhibitor 3MA (5mM) for 2 hrs followed by treatment with OM in the absence or presence of 250 ng/ml PLY for 12 hrs.

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The results of this study showed that the decrease in the expression of Sp1 induced by PLY is restored by 3-MA or CQ pretreatment but not pretreatment with MG-132 (Fig. 4E), indicating that the downregulation of Sp1 is

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lysosome-dependent. Collectively, our investigation revealed that PLY inhibits osteoblast differentiation by

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downregulating Sp1 through lysosomal-dependent autophagy pathway.

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3.5. PLY induces autophagy through ROS-mediated AMPK activation and mTOR inhibition signaling pathway

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AMPK and mTOR regulate autophagy by regulation of Ulk1/2 kinase activity, which is an autophagy initiator. Activated AMPK phosphorylates Ulk1 and inhibits phosphorylation of mTOR, leading to induction of autophagy [19, 38]. ROS is also well known to be an important mediator of autophagy via activation of AMPK

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[18]. To investigate the detailed signaling pathway which may contribute to the induction of autophagy in PLYtreated cells, MG63 cells were incubated in OM with or without 250 ng/ml PLY for the indicated times. The results show that phosphorylation of AMPK was increased and phosphorylation of mTOR was suppressed by PLY (Fig. 5A, B). Furthermore, an increase in the levels of LC3-II conversion and Atg5-Atg12 complex expression and a decrease in the level of p62 expression were observed in PLY-treated cells (Fig. 5A, B). To support the notion that a decrease in phosphorylation of mTOR by PLY causes autophagy, MG63 cells were pretreated with rapamycin and then incubated with OM in the absence or presence of PLY. Rapamycin effectively blocked phosphorylation of mTOR and induced the conversion of LC3-II. Additionally, the conversion of LC3-II was more increased by a combination of rapamycin and PLY, compared with rapamycin alone (Fig. 5C, D). Together, these results suggest that AMPK and mTOR are involved in the induction of autophagy by PLY. Furthermore, we examined whether ROS generated by PLY induces autophagy through the AMPK/mTOR signaling pathway. MG63 cells were pretreated with 10 mM NAC for 2 hrs and then incubated 12

ACCEPTED MANUSCRIPT with OM in the absence or presence of PLY or L460D. The results show that NAC pretreatment reversed the phosphorylation of mTOR, which was inhibited by PLY-activated AMPK. Moreover, NAC pretreatment not

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only reduced the proportion of LC3-II/LC3-I that was increased by PLY, but also restored Sp1 expression, which

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was reduced by PLY (Fig. 5E, F). Similar results are also observed in HOS osteoblast-like cells (Fig. 6A-D). Thus, results of the present study imply that PLY induces autophagy through ROS-mediated activation of

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AMPK and inhibition of mTOR signaling pathway.

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3.6. PLY is present in synovial fluid from S. pneumoniae-infected mice.

Bacterial infections and their components can be localized in joints, causing bone cell damage leading to

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pathological changes. It is intriguing that PLY might be found in synovial fluid in cavities of synovial joints and PLY concentrations in these cell experiments are compared with concentrations detected in in vivo experiments. To examine this question, we investigate whether PLY would be present in the synovial fluid of S. pneumoniae-

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infected mice and served as one of virulence factors of the disease severity. Mice were infected with S.

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pneumoniae (D39) or pneumococcal isolate (serotype 19) via the intraperitoneal route. ELISA and Western blot assays were used to detect the level of PLY released by the bacteria at 24 h post-infection. When compared to

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uninfected control, the level of PLY was increased in the synovial fluid from mice infected with S. pneumoniae (D39) (Fig. 7A). Additionally, the number of recoverable viable bacteria was significantly increased in the synovial fluid of pneumococcal-infected mice (Fig. 7B). Moreover, an increased number of Ly6G+ neutrophils

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were observed, implicating that the cells are recruited into joints and increased numbers positively correlate with the development of inflammatory process in joints (Fig. 7C). Further support for these results comes from experiments using pneumococcal isolate (serotype 19A), which showed similar findings (Supplementary 2). It should be noted that the concentration used in the present study is slightly higher than that achieved in in vivo. Nonetheless, our data suggest that bone cell damage and inflammation could be induced by PLY and thereby related to an increase in PLY.

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ACCEPTED MANUSCRIPT 4. Discussion

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Bone infection is characterized by bone destruction and irreversible loss of function. Bone is regenerated by

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osteoblasts and resorbed by osteoclasts, and an imbalance of the activity of these cells causes bone destruction [5, 7]. Previous studies showed that S. pneumoniae is a common cause of bone infection [7-9, 39, 40] and

For

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the present study, human osteoblast-like MG63 and HOS cells were used to examine the effect of PLY on cell differentiation, because although some difference are observed between primary osteoblast and cell lines, human osteoblast-like cells are used frequently as an attractive model for research focused on basic bone biology and a

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substitute for primary osteoblast. The present study revealed the inhibitory effect of the S. pneumoniae toxin PLY on osteoblast differentiation. It was also found that treatment with PLY blocked osteoblast differentiation

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by decreasing osteogenic differentiation-related gene expression such as Runx2 and osteocalcin and ALP activity but not OPG and RANKL expression (data not shown) in osteogenic conditions. Additionally, our data

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PLY could induce bone cell damage.

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showed that the level of PLY was increased in the synovial fluid of S. pneumoniae-infected mice, suggesting

PLY induces the production of intracellular ROS, which induces activation of neutrophils and triggers

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antibacterial defenses [36]. Moreover, PLY-induced ROS production induces autophagy, which enhances clearance of bacterial pathogens [14]. However, the production of ROS results in insufficient osteogenic differentiation potential [41]. For this reason, we were prompted to test whether PLY generates a significant

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level of ROS in MG63 cells. Interestingly, the production of ROS was significantly induced in the presence of PLY but not L460D. Furthermore, L460D had no inhibitory effect on osteoblast differentiation and the inhibition of osteoblast differentiation by PLY was recovered by treatment with NAC. Thus, it can be suggested that the production of ROS induced by PLY is involved in the inhibition of osteoblast differentiation. Autophagy is associated with an important adaptive cellular strategy involved in cell differentiation [16]. It is also known that autophagy is a possible cellular intermediate and related to the regulation of differentiation of osteoblast [42], although the role of autophagy for osteogenesis remains to be fully elucidated. During bacterial infection, autophagy is induced by bacterial components such as toxin. A recent study demonstrated that PLY is a cause of S. pneumoniae infection-induced autophagy in human pulmonary epithelial cells [14]. Our data indicated that PLY is able to induce autophagy in osteoblast and the autophagy inhibitors 3-MA and CQ markedly reverse the inhibition of osteoblast differentiation. Additional support for this conclusion comes from the observation that transfection with Atg5 siRNA inhibited PLY-induced autophagy and knockdown of Atg5 14

ACCEPTED MANUSCRIPT reduced osteoblast differentiation. These findings suggest that PLY-induced autophagy is a critical factor in the inhibition of osteoblast differentiation. It is interesting to note that knockdown of Atg5 also inhibits autophagy

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in OM-treated cells, indicating that autophagy is essential during the differentiation process. This is consistent

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with previous study which shows that autophagy is essential during human mesenchymal stem cell differentiation into osteoblasts [17].

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It is widely accepted that various transcription factors play important roles in the differentiation of many cell types including osteoblast. Runx2 is a key transcription factor in osteoblast differentiation, which induces ALP activity and the activation of osteogenic differentiation-related genes, including osteocalcin [43, 44]. In addition,

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it was known that Runx2 gene transcription is regulated by Sp1 [27]. Previous studies also showed that oxidative stress-induced ROS downregulates Sp1 expression and that this degradation is proteasome-dependent

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[28, 37]. Interestingly, our data showed that PLY downregulates the expression of Sp1 during osteoblast differentiation. Hence, we hypothesized that the production of ROS by PLY leads to proteasome-dependent

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degradation of Sp1. However, MG132, a proteasome inhibitor, could not rescue the downregulation of Sp1.

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Meanwhile, it was found that the lysosome inhibitor CQ and the autophagy inhibitor 3-MA effectively prevented the PLY-induced downregulation of Sp1 and significantly recovered osteoblast differentiation in the

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presence of PLY. The results of this study imply that the downregulation of Sp1 by PLY is lysosome-dependent and PLY inhibits osteoblast differentiation by downregulating Sp1 through lysosomal-dependent autophagy pathway. However, our data don’t totally rule out the possibility that other molecules are involved in regulation

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of Rnx2. It has been known that the Smad and HES-1 enhance the activity Runx2 and TLE-2 inhibits its activity [45]. Thus, it is plausible that PLY regulates these molecules. It has been known that autophagy generally linked to a number of signaling pathways which are activated by various stimuli and such activation results in the activation of diverse transcription factors involving in the process of autophagy and osteoblast differentiation [45, 46]. Recent studies have reported that activation of AMPK is related to autophagy which is inhibited by mTOR [47]. AMPK activation and mTOR inhibition regulate autophagy by inducing initiation complexes, including the Ulk and Class III PI3K complexes. In addition, previous studies using the PLY mutant demonstrated that mTOR pathway inhibition leads to activation of autophagy [14]. Thus, we investigated the possible role of AMPK/mTOR in PLY-triggered activation of autophagy. The present data demonstrate that PLY inhibited the phosphorylation of mTOR following rapid activation of AMPK, and autophagy was induced at the same time. To more clearly verify the involvement of inhibition of mTOR by PLY in autophagy induction, we used rapamycin, an mTOR inhibitor. In the present 15

ACCEPTED MANUSCRIPT work, our data indicate that rapamycin and PLY act synergistically to induce autophagy by inhibition of mTOR. Together, these findings suggest that AMPK activation and mTOR inhibition contribute to the induction of

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autophagy, leading to the inhibition of osteoblast differentiation in PLY-treated cells. It is widely accepted that

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ROS is an initiator of autophagy [12, 14, 48]. These findings lead us to examine whether PLY-driven ROS production induces autophagy through the AMPK/mTOR signaling pathway. Our data showed that a ROS

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scavenger was able to effectively prevent the change of a signaling pathway affected by PLY, suggesting that PLY-induced overproduction of ROS is mainly responsible for the induction of autophagy and the inhibition of

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osteoblast differentiation.

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5. Conclusions

Our studies, for the first time, demonstrate that PLY inhibits osteoblast differentiation by downregulating the

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expression of Sp1, and that this is the reason for PLY-induced autophagy via ROS-mediated AMPK/mTOR

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signaling during osteoblast differentiation. Taken together, our data suggest that the S. pneumoniae toxin PLY

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contributes to inhibition of osteoblast differentiation in pneumococcal bone infections.

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ACCEPTED MANUSCRIPT 7. Figure legends

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Figure 1. PLY inhibits osteoblastic differentiation.

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(A) MG63 cells were incubated with OM in the absence or presence of the indicated concentrations of PLY for 1, 3, 5, or 7 days, then the MTT assay was performed. *p<0.05, significantly different from cells treated with OM

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alone (control) at 1, 3, 5 or 7 days, respectively. (B) MG63 cells were incubated with OM in the absence or presence of 250 ng/ml PLY for 1, 3, 5, or 7 days, then the mRNA expression levels of Runx2 and OCN were determined by qRT-PCR. *p<0.05, significantly different from OM-treated cells at 1 day. (C) MG63 and HOS

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cells were incubated with OM or not (con) in the absence or presence of the indicated concentrations of PLY for 7 days, then the mRNA expression levels of Runx2 and OCN were determined by qRT-PCR and (D) ALP

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activity was measured. These data are representative of three independent experiments performed in triplicate.

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*p<0.05, significantly different from OM-treated cells.

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Figure 2. PLY-induced ROS production is sufficient to impair osteoblast differentiation. (A) MG63 cells were pretreated with NAC (10 mM) for 2 hrs, then incubated with OM in the absence or

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presence of 250 ng/ml PLY or 250 ng/ml L460D for 1 hr. The cells were stained with DCF-DA (10 µM) for 30 min. ROS production was measured by flow cytometry. Percent (%) positive refers to the fraction of fluorescent cells, indicating ROS. (B) Bar graphs of FACS data for DCF-DA. Bar graph shows results of three independent

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experiments and histograms show one representative experiment. Error bars represent the mean ± SEM. *p<0.05, significantly different from OM-treated cells. #p<0.05, significantly different from OM+PLY-treated cells. (C) MG63 cells were incubated with OM in the presence or absence of 250 ng/ml PLY, L460D, or PLY in the presence of NAC (1 mM) for 7 days, then expression of osteogenic differentiation-related genes and (D) an ALP activity assay were analyzed. These data are representative of three independent experiments performed in triplicate. *p<0.05, significantly different from OM-treated cells. #p<0.05, significantly different from OM+PLY-treated cells.

Figure 3. PLY down-regulates Sp1 protein expression in MG63 cells. (A) MG63 cells were incubated with OM in the absence or presence of 250 ng/ml PLY for the indicated times. Cell lysates were subjected to Western blot analysis to detect the expression of Sp1. (B) MG63 cells were incubated with OM in the absence or presence of 250 ng/ml PLY or 100 nM Mith A for 12 hrs. Cell lysates were 21

ACCEPTED MANUSCRIPT subjected to Western blot analysis. The amount of the proteins was normalized to β-actin levels. These data are representative of three independent experiments. *p<0.05, significantly different from OM-treated cells. (C)

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MG63 cells were incubated with OM in the absence or presence of 250 ng/ml PLY or 100 nM Mith A for 7 days,

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then the expression of osteogenic differentiation-related genes and (D) an ALP activity assay were analyzed. These data are representative of three independent experiments performed in triplicate. *p<0.05, significantly

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different from OM-treated cells.

Figure 4. PLY-induced autophagy inhibits osteoblast differentiation and downregulates the expression of

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Sp1.

(A) After MG63 cells were pretreated with 3-MA (5 mM) or CQ (10 µM) for 2 hrs, cells were washed with PBS

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and incubated with OM in the absence or presence of 250 ng/ml PLY for 12 hrs. Cell lysates were subjected to Western blot analysis. These data are representative of three independent experiments. The conversion of LC3-I

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to LC3-II was quantified by densitometry. *p<0.05, significantly different from PLY-treated cells. #p<0.05,

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significantly different from CQ- and OM-treated cells. (B) MG63 cells were transfected with 30 pM of control siRNA or Atg5 siRNA for 24 hrs and incubated with OM in the absence or presence of PLY for 12 hrs. Cell

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lysates were subjected to Western blot analysis. The amount of proteins was normalized to β-actin levels and the conversion of LC3-I to LC3-II was quantified by densitometry. These data are representative of three independent experiments. *p<0.05, significantly different from control siAtg5-transfected PLY-treated cells.

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(C) MG63 cells were pretreated with 3-MA (5 mM) or CQ (10 µM) for 2 hrs, then incubated with OM or not (con) in the absence or presence of 250 ng/ml PLY for 7 days, or the cells were transfected with 30 pM of control siRNA or Atg5 siRNA for 24 hrs and incubated with OM in the absence or presence of PLY for 7 days. Then, mRNA expression of Runx2 and OCN were determined by qRT-PCR and (D) ALP activity was measured. These data are representative of three independent experiments performed in triplicate. *p<0.05, significantly different from OM-treated cells. #p<0.05, significantly different from PLY-treated cells. †p<0.05, significantly different from control siAtg5-transfected PLY-treated cells. (E) MG63 cells were pretreated with 3-MA (5 mM), CQ (10 µM), or MG132 (10 µM) for 2 hrs, then incubated with OM in the absence or presence of 250 ng/ml PLY for 12 hrs. Cell lysates were subjected to Western blotting. The amount of proteins was normalized to βactin levels. These data are representative of three independent experiments. *p<0.05, significantly different from OM-treated cells.

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ACCEPTED MANUSCRIPT Figure 5. PLY induces autophagy through ROS-mediated activation of AMPK and inhibition of the mTOR signaling pathway.

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(A) MG63 cells were incubated with OM in the absence or presence of 250 ng/ml PLY for the indicated times.

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Cell lysates were subjected to Western blot analysis. (B) The amount of proteins was normalized to levels of the non-phosphorylated form of the protein (AMPK, mTOR) or β-actin (Atg5-Atg12, p62) and the conversion of

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LC3-I to LC3-II was quantified by densitometry. These data are representative of three independent experiments. *p<0.05, significantly different from OM-treated cells at the same time points. (C) MG63 cells were pretreated with rapamycin (100 nM) for 2 hrs, then incubated with OM in the absence or presence of 250 ng/ml PLY for 12

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hrs. Cell lysates were subjected to Western blot analysis. (D) The amount of proteins was normalized to levels of the non-phosphorylated form of the protein and the conversion of LC3-I to LC3-II was quantified by

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densitometry. *p<0.05, significantly different from rapamycin-treated cells. (E) MG63 cells were pretreated with NAC (10 mM) for 2 hrs, then incubated with OM in the absence or presence of 250 ng/ml PLY or 250

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ng/ml L460D for 12 hrs. Cell lysates were subjected to Western blot analysis. (F) The amount of the proteins

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was normalized to levels of the non-phosphorylated form of the protein (AMPK, mTOR) or β-actin (Sp1) and the conversion of LC3-I to LC3-II was quantified by densitometry. *p<0.05, significantly different from PLY-

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treated cells.

Figure 6. ROS generation induces autophagy and inhibits differentiation in PLY-treated HOS cells.

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(A) HOS cells were pretreated with NAC (10 mM) for 2 hrs, then incubated with OM in the absence or presence of 250 ng/ml PLY for 12 hrs. Cell lysates were subjected to Western blot analysis. (B) The amount of the proteins was normalized to levels of the non-phosphorylated form of the protein (AMPK, mTOR) or β-actin (Sp1) and the conversion of LC3-I to LC3-II was quantified by densitometry. *p<0.05, significantly different from PLY-treated cells. (C) HOS cells were incubated with OM in the absence or presence of the indicated concentrations of PLY or PLY plus NAC (1 mM) for 7 days, then mRNA expression of Runx2 and OCN were determined by qRT-PCR and (D) ALP activity was measured. These data are representative of three independent experiments performed in triplicate. *p<0.05, significantly different from OM-treated cells.

Figure 7. Expression level of pneumolysin, bacterial viability and the number of neutrophils in synovial fluid from S. pneumoniae-infected mice. The mice were challenged i.p. with 1 × 104 CFU of pneumococci (D39). Six mice were sacrificed 24 h after 23

ACCEPTED MANUSCRIPT infection. Synovial fluid samples were obtained from mouse synovial joints. (A) The level of PLY in synovial fluid was measured by Western blot and ELISA, respectively. The amount of the proteins was normalized to β-

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actin levels. *p<0.05, significantly different from uninfected control (B) The survival and growth of S.

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pneumoniae from synovial fluids of uninfected control and infected mice. (C) Representative histogram depicting expression of the Ly6G (blue) was gated on neutrophils in synovial fluid. Uninfected control is

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PLY inhibits the expression of osteogenesis related genes and ALP activity

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PLY inhibits osteoblast differentiation through lysosome-dependent downregulation of Sp1

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PLY induces autophagy through ROS-mediated AMPK/mTOR signaling pathway.

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