Toll-like receptor 2 activation primes and upregulates osteoclastogenesis via lox-1

Toll-like receptor 2 activation primes and upregulates osteoclastogenesis via lox-1 Background: Lectin-like oxidized low-density-lipoprotein receptor 1 (Lox-1) is the receptor for oxidized low-density lipoprotein (oxLDL), a mediator in dyslipidemia. Toll-like receptor (TLR)-2 and − 4 are receptors of lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major pathogen of chronic periodontitis. Although some reports have demonstrated that periodontitis has an adverse effect on dyslipidemia, little is clear that the mechanism is explained the effects of dyslipidemia on osteoclastogenesis. We have hypothesized that osteoclast oxLDL has directly effect on osteoclasts (OCs), and therefore alveolar bone loss on periodontitis may be increased by dyslipidemia. The present study aimed to elucidate the effect of Lox-1 on osteoclastogenesis associated with TLRs in vitro. Methods: Mouse bone marrow cells (BMCs) were stimulated with macrophage colony-stimulating factor into bone marrow macrophages (BMMs). The cells were also stimulated with synthetic ligands for TLR2 (Pam3CSK4) or TLR4 (Lipid A), with or without receptor activator of nuclear factor kappa-B ligand (RANKL), and assessed for osteoclastogenesis by tartrate-resistant acid phosphatase (TRAP) staining, immunostaining, western blotting, flow activated cell sorting (FACS) analysis, real-time polymerase chain reaction (PCR), and reverse transcription PCR. Results: Lox-1 expression was significantly upregulated by Pam3CSK4 and Lipid A in BMCs (p < 0.05), but not in BMMs. FACS analysis identified that Pam3CSK4 upregulated RANK and Lox-1 expression in BMCs. TRAP-positive cells were not increased by stimulation with Pam3CSK4 alone, but were increased by stimulation with combination combined Pam3CSK and oxLDL. Expression of both Lox-1 and myeloid differentiation factor 88 (MyD88), an essential adaptor protein in the TLR signaling pathway, were suppressed by inhibitors of TLR2, TLR4 and mitogen-activated protein kinase (MAPK). Conclusions: This study supports that osteoclastogenesis is promoted under the coexistence of oxLDL by TLR2-induced upregulation of Lox-1 in BMCs. This indicates that periodontitis could worsen with progression of dyslipidemia. Keywords: Dyslipidemia, Osteoclastogenesis, Toll-like receptor, Lectin-like oxidized low-density-lipoprotein receptor 1 Background the bone matrix [5]. Periodontitis is a chronic disease Osteoclasts (OCs) are multinucleated, bone-adhering cells, caused by bacterial infection in the gingiva [6, 7], resulting formed by fusion of mononuclear monocyte/macrophage in resorption of alveolar bone. It is reported that lipopoly- progenitors. OCs require macrophage colony-stimulating saccharide (LPS), a major constituent of Gram-negative factor (M-CSF) for proliferation and receptor activator of bacteria, can stimulate OC formation and bone resorption nuclear factor kappa-B ligand (RANKL) for fusion and through activation of Toll-like receptors (TLRs) [8–10]. maturation [1–5]. OCs act by extruding acid onto the Porphyromonas gingivalis (P. gingivalis) is an anaerobic bone surface and dissolving inorganic components within Gram-negative oral bacterium involved in periodontitis. LPS derived from P. gingivalis is different to that derived from other bacteria and acts as an agonist of both TLR4 * Correspondence: kajiya@college.fdcnet.ac.jp and TLR2 [11, 12]. P. gingivalis aggravates periodontal Department of Physiological Science and Molecular Biology, Fukuoka Dental bone resorption through differential regulation of TLR2 College, Fukuoka 8140193, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 2 of 9 and TLR4 signaling pathways in a RANKL-dependent Methods manner [13]. Although TLR ligands have been shown Cell culture to have stimulatory or inhibitory functions on osteo- All procedures were approved by the Council on Animal clastogenesis in vitro, TLR4 ligand has been shown to Care of Fukuoka Dental College (13013). Mouse BMCs be a potent stimulator of bone loss in vivo [14]. It were obtained from the tibia and femora of 4- to has been reported that P. gingivalis LPS stimulates 5-week-old ddY male mice. All mice were euthanized by periosteal OC formation due to induction of RANKL cervical dislocation under anesthesia by inhalation of in osteoblasts by activation of TLR2 [15]. Of note, al- isoflurane at 0.5–5% with oxygen. BMCs were cultured veolar bone loss is induced by P. gingivalis through in α-Minimum Essential Medium (α-MEM; Invitrogen, TLR2 in mice [13, 16, 17]. Conversely, synthetic Grand Island, NY, USA) containing 10% fetal bovine TLR2 ligand inhibits OC formation in mouse bone serum (FBS; Biowest Nuaille, France) and antibiotics marrow macrophages (BMMs) stimulated with macro- (100 U/ml penicillin G and 0.15 mg/ml streptomycin phage colony-stimulating factor (M-CSF) and RANKL sulfate). After overnight culture to differentiate BMMs, [18, 19]. Although RANKL induces differentiation of non-adherent cells were cultured for 3 days with or with- BMMs to OCs, simultaneous addition of LPS inhibits out Pam3CSK4 (50–100 ng/ml) or lipid A (100 ng/ml) in this process [19, 20]. However, LPS stimulation at the presence of macrophage colony-stimulating factor late-stage osteoclastogenesis enhances the survival and (M-CSF; 20 ng/ml). For the control, phosphate-buffered sa- activation of OCs [20–22]. Although P. gingivalis has line (PBS) was administered instead of the TLR ligands. In known to promote osteoclastogenesis and bone some experiments, BMMs were used as osteoclast precur- resorption which types of TLR receptor, such as sors and further cultured with RANKL (50 ng/ml) in the TLR2 and/or TLR4 promote bone resorption remains presence or absence of TLR ligands for 3 days. To identify unclear. OCs, cells were fixed with 3.7% formaldehyde and stained Lectin-like oxidized low-density lipoprotein receptor-1 with tartrate-resistant acid phosphatase (TRAP) using the (Lox-1) was discovered as a receptor for oxidized Acid Phosphatase Leukocyte Kit (Sigma-Aldrich, St. Louis, low-density lipoprotein (oxLDL) in endothelium and MO, USA), and defined as TRAP-positive multinucleated vascular-rich organs [23]. Lox-1 is a multi-ligand recep- cells (having more than three nuclei). OCs were counted tor that recognizes many ligands, such as activated plate- using a microscope. Each condition was tested in triplicate lets [24], neutrophils [25], apoptotic/aged cells [26], and and all experiments were repeated at least three times. bacteria [27], and is expressed in many cell types [28–30]. Bone-resorbing OCs and bone-forming oste- Reverse transcription (RT)-polymerase chain reaction oblasts have been reported to express Lox-1, and (PCR) and real-time PCR −/− Lox-1 mice have decreased bone mass in the Total RNA was extracted from cells using TRIzol reagent steady state but are resistant to inflammatory bone (Life Technologies Corporation, Rockville, MD, USA). First destruction because of the impairment of osteoblastic strand cDNA was synthesized using 1 mg total RNA with RANKLexpressioninresponse toinflammation [31]. Super Script II RT according to the manufacturer’sinstruc- The pathology of both periodontitis and dyslipidemia tions (Invitrogen, Grand Island, NY, USA). To detect involves OCs, and these lifestyle-related diseases are ex- mRNA expression of Lox-1 and β-actin, RT-PCR was per- acerbated by stimulation with TLRs and Lox-1, respect- formed using the following: Lox-1 sense 5′-AGACTGGCT ively. Consequently, although these disorders are CTGGCATAAAG-3′,antisense 5′-AAGGCCAACATGCT reported to be associated with each other, the mechan- TTACAT-3′; β-actin sense 5′-TGAGAGGGAAATCG ism for this is unclear [32–34]. Some reports have TGCGT-3′,antisense 5′-GCTGGAAGGTGGACAGTGA shown that periodontitis increases the risk of athero- G-3′. PCR was performed under the following conditions: sclerosis in vivo and in vitro [35–38], and that periodon- 1 min denaturation at 95 °C, 1 min annealing at 55 °C, and titis worsens in the apolipoprotein E (ApoE)−/− 1 min extension at 72 °C, using 36 cycles. PCR products hyperlipidemia model [33, 35, 39]. However, there are were subjected to electrophoresis on 2% agarose gel and vi- few reports describing how dyslipidemia exacerbates sualized with ethidium bromide. To examine the effects of periodontitis. MAPK inhibitors on Lox-1 mRNA expression, BMCs in The purpose of this study was to clarify whether osteo- thepresenceofM-CSF (20ng/ml)were culturedwithor clastogenesis is upregulated through Lox-1 by TLR without Pam3CSK4 (100 ng/ml) or Lipid A (100 ng/ml) for stimulation at early- and late-stage. We showed that 3 days after incubation with TLR2/4 inhibitor (25 μM) or osteoclastogenesis was accelerated by activation of TLRs MAPK inhibitors, SP600125 (5 μM) as c-Jun N-terminal through upregulation of Lox-1 expression during bone kinase (JNK) inhibitor or U0126 (10 μM) as MAP/extracel- marrow cell (BMC) differentiation into BMMs, suggesting lular signal-regulated kinase (ERK) kinase (MEK) inhibitor, dyslipidemia increases the risk of periodontitis. respectively, for 1 h. Signals of Lox-1 mRNA were Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 3 of 9 normalized to β-actin mRNA expression levels using Image cells among the total non-adherent BMCs was quantified J software (NIH, Bethesda, MD, USA). using the labeled square. To quantify mRNA levels, cDNA samples were analyzed by quantitative real-time PCR. A total of 1 mg of cDNA Western blot analysis was amplified in a 20 μl volume of Power SYBR Green Cells were lysed in Tris-NaCl-Tween (TNT) buffer contain- PCR Master Mix (Applied Biosystems, Foster City, CA, ing 20 mM tris–HCl (pH 7.5), 200 mM NaCl, 1% Triton USA) in a real-time PCR system (Bio-rad CFX96, Bio-rad X-100, 1 mM dithiothreitol (DTT), and protease inhibitors Technologies, Inc., Santa Clara, CA, USA) and the fluor- (Roche, Basel, Switzerland). The protein content of the sam- escence was monitored at each cycle. Cycle parameters ples was measured using Pierce reagents, following the man- were 95 °C for 30 s to activate Taq followed by 40 cycles ufacturer’s protocol. Protein samples (20 μg) were subjected of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 40 s. For to sodium dodecyl sulfate-polyacrylamide gel electrophor- real-time analysis, two standard curves were constructed esis, and proteins were then transferred to a polyvinylidene from amplicons for both the β-actin and target gene. Tar- difluoride (PVDF) membrane (100 V, 1 h, 4 °C). The mem- get gene cDNA units in each sample were normalized to branes were incubated with anti-FDPS and anti-β-actin anti- β-actin cDNA units. Finally, the relative target gene ex- bodies diluted at 1:1000 in 5% (w/v) skimmed milk solution pression units were expressed as arbitrary units, calculated supplemented with 0.01% (w/v) azide overnight at 4 °C. The according to the following formula: relative target gene ex- blots were washed in Tris- buffered saline with Tween pression units = target gene cDNA units/β-actin cDNA (TTBS) (10 mM tris–HCl, 50 mM NaCl, 0.25% Tween 20) units. To detect mRNA expression of Lox-1, MyD88 and and incubated with an appropriate secondary antibody for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 30 min at room temperature. The immunoreactive proteins real-time PCR was performed using the following sets: were visualized using enhanced chemiluminescence reagents Lox-1 sense 5′-CTGCGAATGACGAGCCTGA-3′,anti- (GE Healthcare, Tokyo, Japan). sense 5′-TCACTGACAACACCAGGCAGAG-3′;MyD88 sense 5′-TACAGGTGGCCAGAGTGGAA-3′,antisense Chemicals 5′-GCAGTAGCAGATAAAGGCATCGAA-3′;GAPDH Recombinant human RANKL and M-CSF were pur- sense 5′-TGTGTCCGTCGTGGATCTGA-3′,antisense chased from Peprotec Co. Ltd. (Minneapolis, MN, USA). 5′-TTGCTGTTGAAGTCGCAGGAG′. All other chemicals were obtained from Sigma-Aldrich. Immunocytochemistry Data analysis Cells were fixed in 4% formaldehyde for 5 min and perme- All data are expressed as mean ± standard error of the abilized with 0.05% Triton-× 100 in phosphate-buffered sa- mean (SEM) of the number of cells (n). Statistical compari- line (PBS) for 5 min. Cells were incubated with goat sons were performed using analysis of variance (ANOVA). polyclonal anti-rabbit Lox-1 antibody (1:100 dilution, A probability (P) of < 0.05 was considered significant. ab60178, Abcam) overnight at 4 °C after blockade of non- specific binding with 3% goat serum for 40 min at room Results temperature. Cells treated with primary antibody were Expression of Lox-1 was upregulated by TLR 2/4 washed with PBS and incubated with Alexa fluor ligands in BMCs 488-conjugated streptavidin (2 μg/ml, Molecular Probes, Eu- To clarify the effect of TLR ligands on the expression of gene, OR) in goat anti-rabbit IgG secondary antibody Lox-1 in BMCs or BMMs, cells were cultured with or (1:1500 dilution, Vector Laboratories, Burlingame, CA) for without Pam3CSK4 (100 ng/ml) or Lipid A (100 ng/ml) in 30 min at room temperature. To label cell nuclei, cells were the presence of M-CSF for 3 days (Fig. 1a). Exposure to rinsed in PBS and covered with encapsulant containing Pam3CSK4 or Lipid A significantly upregulated the ex- 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence was pression of Lox-1 in BMCs (p < 0.05) (upper panel) but observed using fluorescence microscopy (TMD 300, Nikon). suppressed Lox-1 expression in BMMs (lower panel) com- pared with the control. Lox-1 mRNA expression in BMCs FACS analysis peaked on days 1 or 3 after incubation with Pam3CSK4 or BMCs were cultured for 3 days in α-MEM containing 10% Lipid A as assessed by real-time PCR (p < 0.05) (Fig. 1b, FBS and M-CSF with or without Pam3CSK4 (100 ng/ml) or left panel). Furthermore, Lox-1 protein expression peaked Lipid A (100 ng/ml) for 3 days. Cells were washed twice on day 3 after treatment with both TLR ligands (Fig. 1b, with PBS and incubated with anti-Lox-1 and anti-RANK right panel). Similar to the PCR results, protein expression antibodies conjugated with phycoerythrin. The labeled cells of Lox-1 was also upregulated following exposure to were analyzed on a FACS using On Chip (On Chip Bio. Pam3CSK4 or Lipid A in BMCs as assessed by immuno- Tec., Tokyo, Japan). The ratio of Lox-1- or RANK-positive cytochemistry (Fig. 1c). Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 4 of 9 a b Fig. 1 Analysis of LOX-1 in bone marrow cells (BMCs) and bone marrow macrophages (BMMs). (A, upper panel, B, C) BMCs were differentiated into BMMs with or without Pam3CSK4 (TLR2 ligand, 100 ng/ml) or Lipid A (TLR4 ligand, 100 ng/ml) in the presence of macrophage colony-stimulating factor (M-CSF, 20 ng/ml) for 3 days. (A, lower panel) BMMs were cultured with or without Pam3CSK4 (100 ng/ml) or Lipid A (100 ng/ml) in the presence of M-CSF and receptor activator of nuclear factor kappa-B ligand (RANKL, 50 ng/ml) for 3 days. a Polymerase chain reaction (PCR) products of LOX-1, a receptor of oxidative low density lipoprotein (oxLDL), mRNA in BMCs (upper panel) and BMMs (lower panel) were amplified using RT-PCR methods. Following stimulation with Pam3CSK4 or Lipid A, Lox-1 expression was upregulated in BMCs (upper panel) but not in BMMs (lower panel). b Lox-1 mRNA expression peaked on day 1 or 3 as determined by real-time PCR (left panel) and protein expression on day 3 (right panel). c BMCs stimulated with TLR ligands on day 3 were stained with anti-Lox-1 antibody (1:100 dilution) then incubated with Alexa fluor 488-conjugated streptavidin (2 μg/ml) in goat anti-rabbit IgG secondary antibody (1:1500 dilution). Lox-1 expression was upregulated. Data are expressed as mean ± SEM (n =3). *P < 0.05 compared with control TLR2/4 ligands upregulate Lox-1 and RANK in BMCs real-time PCR. Although the addition of Pam3CSK4 or BMC surface expression of Lox-1 and RANK proteins Lipid A significantly increased the expression of Lox-1 was analyzed by FACS. Treatment with M-CSF slightly and MyD88 mRNAs, TLR2 or 4 ligand-induced upregu- increased the cell surface expression of Lox-1 in BMCs lation was reduced in the presence of TLR2/4 and in a time-dependent manner compared with untreated MAPK inhibitor peptides, respectively (Fig. 3a), suggest- cells (day 0; Fig. 2, upper panel). The addition of ing an influence towards Lox-1 expression by activation Pam3CSK4 or Lipid A significantly upregulated the of TLR-MyD88 downstream signaling. Furthermore, the expression of Lox-1 compared with the control cells inhibitors of JNK and MEK significantly inhibited the (incubated with IgG). Similar to the real-time PCR time upregulation of TLR2-induced RANK mRNAs on BMCs course results, Lox-1 expression peaked at day 1 or day (Fig. 3b). These results demonstrated that TLR2/4 3 after stimulation with Pam3CSK4 or Lipid A, respect- ligands activated Lox-1 expression through the MEK ively. In the present experiments, RANKL induced the and JNK activation pathway, including MyD88. expression of RANK, and the addition of Pam3CSK4 or Lipid A upregulated the expression of RANK in BMCs TLRs stimulation with oxLDL accelerates in a time-dependent manner. osteoclastogenesis at early-phase To clarify TLR2-induced Lox-1 upregulation on osteoclas- TLR 2/4 ligands upregulate the expression of Lox-1 in togenesis, we examined the effect of oxLDL (30 μg/ml) on BMCs through activation of MAPK osteoclast differentiation on early-phase (BMC: day 0–3be- To assess TLR2 or 4 activation of Lox-1 expression via fore stimulation with RANKL) and/or on late-phase the downstream pathways of TLR2/4, MyD88, and (BMM: day 0–3 after RANKL stimulation) osteoclastogene- MAPKs, we examined the effects of these inhibitors on sis. The number of TRAP-positive multinucleated cells the expression of Lox-1 and RANK in BMCs using were increased at early phase and decreased at late phase, Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 5 of 9 Fig. 2 BMC surface expression of Lox-1 and RANK proteins. BMCs were cultured with or without Pam3CSK4 (TLR2 ligand, 100 ng/ml) or Lipid A (TLR4 ligand, 100 ng/ml) in the presence of M-CSF (20 ng/ml) for 3 days. Cells were washed with PBS and incubated with anti-Lox-1 or anti-RANK antibodies. Data are expressed as the mean ± SEM (n = 3 experiments) by addition of only oxLDL (Fig. 4a and b each upper panels). [43, 44]. Although atherosclerosis is reported to result Furthermore, Pam3CSK4 (10 ng/ml) had no effect on in inhibited bone formation because oxLDL blocked early-phase, but an inhibition effect at late-phase osteoclas- differentiation of osteoblast progenitor cells [45, 46], togenesis (Fig. 4a and b each middle panels). When we stim- little is known about how dyslipidemia influences ulated BMCs with both Pam3CSK4 (25 ng/ml) and oxLDL bone resorption. In the present study, we found that (15 μg/ml) at early-phase, numbers of TRAP-positive multi- TLRs upregulated the oxLDL receptor, Lox-1, via nucleated cells were significantly increased (Fig. 4a and b MAPK in BMCs, resulting in promotion of osteoclas- each lower panels). togenesis. This finding suggests that periodontitis would worsen with progression of dyslipidemia. Discussion The subcutaneous or intraperitoneal administration of P. gingivalis is known to stimulate TLR2 and/or TLR4 [11, Pam3CSK4 is reported to upregulate bone resorption in 12] via MyD88 to modulate osteoclastogenesis [18, 40–42], vivo, suggesting that Pam3CSK4 directly promotes osteo- resulting in alveolar bone resorption through differential clastogenesisfromBMMsafter RANKLpriming [41, 47]. regulation of TLR2 and TLR4 signaling pathways in a However, synthetic TLR2 ligand inhibits OC formation in RANKL-dependent manner [13]. Although Lipid A, a BMMs treated with M-CSF and RANKL in vitro [18, 19] TLR4 ligand, has been shown to be a potent stimulator of Furthermore, combination of RANKL and LPS inhibited bone loss in vivo [14], little is known about whether other osteoclastogenesis [19, 20]. Our data also showed that TLR ligands have a stimulatory or inhibitory effect on oste- numbers of TRAP-positive cells were decreased when oclastogenesis in vitro. Furthermore, many clinical studies BMMs were stimulated with Pam3CSK, compared with show that osteoporosis is associated with atherosclerosis those treated with M-CSF and RANKL. In contrast, the Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 6 of 9 ab Lox-1 control +Pam3 +Lipid A +Pam3 +TLR2/4 inhibitors control RANK +Lipid A +TLR2/4 inhibitor +Pam +Pam3 +JNK inhibitor +Pam3 +Lipid A 20 +MEK inhibitor +Pam+JNK inhibitor +Lipid A+JNK inhibitor incubation time (day) +Pam+MEK inhibitor control MyD88 +Pam3 +Lipid A +Pam3 +TLR2/4 inhibitors +Lipid A +TLR2/4 inhibitor +Pam3 +JNK inhibitor +Pam3 +MEK inhibitor 01 3 0 1 3 incubation time (day) incubation time (day) Fig. 3 Real time PCR to determine Lox-1, MyD88 and RANK expression in BMCs following addition of inhibitors. BMCs in the presence of M-CSF (20 ng/ml) were cultured with or without Pam3CSK4 (TLR2 ligand, 100 ng/ml) or Lipid A (TLR4 ligand, 100 ng/ml) for 3 days after incubation with TLR2/4 inhibitor (25 μM) or MAPK inhibitors, SP600125 (5 μM) as JNK inhibitor or U0126 (10 μM) as MEK inhibitor, respectively, for 1 h. a Both TLR2/4 inhibitor and MAPK inhibitors significantly suppressed TLR 2 or 4 ligand-induced upregulation of Lox-1 (upper panel) and MyD88 (lower panel) mRNA. b SP600125 significantly suppressed TLR 2, but not TLR 4 ligand-induced upregulation of RANK mRNA, and U0126 upregulated RANK mRNA. Data are expressed as the mean ± SEM (n = 3 experiments) addition of Pam3CSK and oxLDL, a Lox-1 ligand to- kinases, together with the suppression of DNA binding gether at early phase, increased the number of activities of transcriptional factors, such as nuclear TRAP-positive cells. Although the mechanism through factor-kappa B (NF-kappa-B) and nuclear fsctor of acti- which P. gingivalis-induces bone resorption via TLR2 vated T-cells (NFAT) [49]. Our data demonstrated that and TLR4 is not fully understood, our results suggest TLR 2 or 4 ligand-induced Lox-1 upregulation was re- that TLR-stimulation in periodontitis upregulates duced in the presence of TLR2/4 and MAPK inhibitor, alveolar bone resorption under high plasma concentra- respectively, and the inhibitors of JNK and MEK signifi- tion of oxLDL in dyslipidemia. cantly inhibited the upregulation of TLR 2-induced RANK OC progenitors, including BMCs and BMMs, are re- mRNA on BMCs. We also found that TLR-stimulation ported to express abundant levels of LDL receptors, together with oxLDL accelerated early-phase but not such as Lox-1, in a RANKL-independent manner. Ex- late-phase osteoclastogenesis, and only oxLDL stimulation pression levels of Lox-1 are greatly decreased during os- suppressed RANKL-induced osteoclastogenesis, consist- teoclastogenesis, particularly during the fusion of ent with previous studies. In the present study, Pam3CSK4 mononuclear cells to form multinuclear OCs [48]. The was shown to upregulate the expression of RANK and present data showed that Lox-1 expression was upregu- Lox-1 in BMCs. Furthermore, stimulation with both lated by TLR 2 or 4 ligands in BMCs but not in BMMs. Pam3CSK4 and oxLDL was shown to promote progres- −/− Periodontal tissues in ApoE hyperlipidemia model sion of osteoclastogenesis, suggesting that oxLDL-Lox-1 rats are reported to exhibit more TRAP-positive multi- signaling promotes osteoclastogenesis through expression nuclear cells and increased TLR2 and TLR4 expression of Lox-1 by stimulating TLRs. levels [33], suggesting that high oxLDL concentration ef- Similar reports have shown that plasma lipids likely fects osteoclastogenesis via the elevation of TLRs. play a role in maintaining bone mass [48, 50]. A better OxLDL is reported to suppress RANKL-induced osteo- understanding of the coupling between osteoporosis and clastogenesis, TRAP activity, and bone-resorbing activity atherosclerosis is indicated in previous studies whereby derived from human peripheral blood mononuclear cells treatment of one disease may have beneficial effects on in vitro. This suppression is suggested to be caused by another [51–56]. The American Heart Association reported RANKL-induced phosphorylation of ERK, p38, and JNK in 2012 that although periodontal interventions result in a ratio of Ct ratio of Ct ratio of Ct Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 7 of 9 N.S. control EL E & L oxLDL N.S. Pam 3 (-) 100 µm control L E & L oxLDL N.S. EL control E & L Pam oxLDL (-) 100 µm Pam control E L E & L EL control E & L oxLDL E Pam (+) N.S. 100 µm oxLDL control E L E & L Fig. 4 Effect of stimulation to TLRs and/or Lox-1 to TRAP-positive multinucleated cells. The effect of stimulation to Lox-1 with oxLDL and/or to TLR 2 with Pam3CSK4 was examined using TRAP staining. In each panel, E indicates stimulation at early-phase (BMC: day 0–3 before stimulation with RANKL) and L indicates stimulation at late-phase (BMMs: day 4–6 after stimulation with RANKL). (a and b upper) BMCs in the presence of M- CSF or BMMs in the presence of M-CSF and RANKL were cultured with oxLDL (30 μg/ml) at indicated phase. (a and b middle) BMCs in the presence of M-CSF or BMMs in the presence of M-CSF and RANKL were cultured with Pam3CSK4 (10 ng/ml) at indicated phase. (a and b lower) BMCs in the presence of M-CSF and Pam3CSK4 (25 ng/ml) were stimulated with oxLDL (15 μg/ml) at early- or late-phase. Data are expressed as the mean ± SEM (n = 3 experiments). *P < 0.05 compared with control reduction in systemic inflammation and endothelial periodontitis at least partially worsens by the compli- dysfunction in short-term studies, there is no evi- cation of dyslipidemia. dencethattheyprevent atherosclerotic cardiovascular disease (ASVD) or modify its outcomes [57]. Al- Conclusion though our data could not support that ASVD devel- In conclusion, the activation of TLR 2 and/or 4 upregu- oped due to periodontitis worsening dyslipidemia, it lated the expression of Lox-1 through activation of did show that osteoclastogenesis was upregulated by MAPK in BMCs but not in osteoblasts, suggesting the TLR-stimulation, like bacterial infection, in periodontitis promotion of osteoclastogenesis during dyslipidemia. with dyslipidemia. Abbreviations In this study, we focused on elucidating the correl- ApoE: Apolipoprotein E; ASVD: Atherosclerotic cardiovascular disease; ation between dyslipidemia and progression of peri- BMC: Bone marrow cell; BMM: Bone marrow macrophage; ERK: Extracellular odontitis. Our results indicated that stimulation of signal-regulated kinase; FACS: Fluorescence activated cell sorting; JNK: c-Jun N-terminal kinase; Lox-1: Lectin-like oxidized low-density-lipoprotein BMCs with TLRs, representative of a bacterial infec- receptor 1; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein tion, upregulated Lox-1 expression resulting in pro- kinase;M-CSF:Macrophagecolony-stimulating factor; MEK: MAPK/ERK motion of osteoclastogenesis in the presence of kinase; MyD88: Myeloid differentiation factor 88; NFAT: Nuclear factor of activated T-cells; NF-kappa-b: Nuclear factor-kappa B; OC: Osteoclast; oxLDL: oxidized oxLDL in dyslipidemia. Although the present data low-density lipoprotein; P. gingivalis: Porphyromonas gingivalis; PCR: Polymerase did not clarify the direct relationships between chain reaction; RANKL: Receptor activator of nuclear factor kappa-B ligand; periodontitis and atherosclerotic vascular disease, TLR: Toll-like receptor; TRAP: Tartrate-resistant acid phosphatase TRAP+ MNCs TRAP+ MNCs TRAP+ MNCs Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 8 of 9 Acknowledgements contains multiple lipid a species that functionally interact with both toll-like We thank Elizabeth Finnie, PhD, from Edanz Group (http://www.edanzediting.com/ac) receptors 2 and 4. Infect Immun. 2004;72(9):5041–51. for editing a draft of this manuscript. 13. Lin J, Bi L, Yu X, Kawai T, Taubman MA, Shen B, Han X. Porphyromonas gingivalis exacerbates ligature-induced, RANKL-dependent alveolar bone Funding resorption via differential regulation of toll-like receptor 2 (TLR2) and TLR4. This work was supported by a Grants-in-Aid for Scientific Research from the Infect Immun. 2014;82(10):4127–34. Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 14. Sakuma Y, Tanaka K, Suda M, Komatsu Y, Yasoda A, Miura M, Ozasa A, 15 K11405) and Private University Research Branding Project. The authors Narumiya S, Sugimoto Y, Ichikawa A, Ushikubi F, Nakao K. Impaired bone report no conflicts of interest related to this study. resorption by lipopolysaccharide in vivo in mice deficient in the prostaglandin E receptor EP4 subtype. Infect Immun. 2000;68(12):6819–25. Authors’ contributions 15. Kassem A, Henning P, Lundberg P, Souza PP, Lindholm C, Lerner UH. KO (Kimiko Ohgi): Conceived, designed and performed the experiments and Porphyromonas gingivalis stimulates bone resorption by enhancing RANKL wrote the paper; HK: Conceived, designed and performed the experiments (receptor activator of NF-κB ligand) through activation of toll-like receptor 2 and wrote the paper; RS: Conceived and designed the experiments; KO: in osteoblasts. J Biol Chem. 2015;290(33):20147–58. Performed the experiments; KGT: Analyzed the data; FO: Analyzed the data; 16. Burns E, Bachrach G, Shapira L, Nussbaum G. Cutting edge: TLR2 is required YY: Analyzed the data. All authors read and approved the final manuscript. for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone Ethics approval and consent to participate resorption. J Immuno. 2006;177(12):8296–300. Not applicable. 17. Papadopoulos G, Weinberg EO, Massari P, Gibson FC 3rd, Wetzler LM, Morgan EF, Genco CA, et al. 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Toll-like receptor 2 activation primes and upregulates osteoclastogenesis via lox-1

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Abstract

Background: Lectin-like oxidized low-density-lipoprotein receptor 1 (Lox-1) is the receptor for oxidized low-density lipoprotein (oxLDL), a mediator in dyslipidemia. Toll-like receptor (TLR)-2 and − 4 are receptors of lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major pathogen of chronic periodontitis. Although some reports have demonstrated that periodontitis has an adverse effect on dyslipidemia, little is clear that the mechanism is explained the effects of dyslipidemia on osteoclastogenesis. We have hypothesized that osteoclast oxLDL has directly effect on osteoclasts (OCs), and therefore alveolar bone loss on periodontitis may be increased by dyslipidemia. The present study aimed to elucidate the effect of Lox-1 on osteoclastogenesis associated with TLRs in vitro. Methods: Mouse bone marrow cells (BMCs) were stimulated with macrophage colony-stimulating factor into bone marrow macrophages (BMMs). The cells were also stimulated with synthetic ligands for TLR2 (Pam3CSK4) or TLR4 (Lipid A), with or without receptor activator of nuclear factor kappa-B ligand (RANKL), and assessed for osteoclastogenesis by tartrate-resistant acid phosphatase (TRAP) staining, immunostaining, western blotting, flow activated cell sorting (FACS) analysis, real-time polymerase chain reaction (PCR), and reverse transcription PCR. Results: Lox-1 expression was significantly upregulated by Pam3CSK4 and Lipid A in BMCs (p < 0.05), but not in BMMs. FACS analysis identified that Pam3CSK4 upregulated RANK and Lox-1 expression in BMCs. TRAP-positive cells were not increased by stimulation with Pam3CSK4 alone, but were increased by stimulation with combination combined Pam3CSK and oxLDL. Expression of both Lox-1 and myeloid differentiation factor 88 (MyD88), an essential adaptor protein in the TLR signaling pathway, were suppressed by inhibitors of TLR2, TLR4 and mitogen-activated protein kinase (MAPK). Conclusions: This study supports that osteoclastogenesis is promoted under the coexistence of oxLDL by TLR2-induced upregulation of Lox-1 in BMCs. This indicates that periodontitis could worsen with progression of dyslipidemia. Keywords: Dyslipidemia, Osteoclastogenesis, Toll-like receptor, Lectin-like oxidized low-density-lipoprotein receptor 1 Background the bone matrix [5]. Periodontitis is a chronic disease Osteoclasts (OCs) are multinucleated, bone-adhering cells, caused by bacterial infection in the gingiva [6, 7], resulting formed by fusion of mononuclear monocyte/macrophage in resorption of alveolar bone. It is reported that lipopoly- progenitors. OCs require macrophage colony-stimulating saccharide (LPS), a major constituent of Gram-negative factor (M-CSF) for proliferation and receptor activator of bacteria, can stimulate OC formation and bone resorption nuclear factor kappa-B ligand (RANKL) for fusion and through activation of Toll-like receptors (TLRs) [8–10]. maturation [1–5]. OCs act by extruding acid onto the Porphyromonas gingivalis (P. gingivalis) is an anaerobic bone surface and dissolving inorganic components within Gram-negative oral bacterium involved in periodontitis. LPS derived from P. gingivalis is different to that derived from other bacteria and acts as an agonist of both TLR4 * Correspondence: kajiya@college.fdcnet.ac.jp and TLR2 [11, 12]. P. gingivalis aggravates periodontal Department of Physiological Science and Molecular Biology, Fukuoka Dental bone resorption through differential regulation of TLR2 College, Fukuoka 8140193, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 2 of 9 and TLR4 signaling pathways in a RANKL-dependent Methods manner [13]. Although TLR ligands have been shown Cell culture to have stimulatory or inhibitory functions on osteo- All procedures were approved by the Council on Animal clastogenesis in vitro, TLR4 ligand has been shown to Care of Fukuoka Dental College (13013). Mouse BMCs be a potent stimulator of bone loss in vivo [14]. It were obtained from the tibia and femora of 4- to has been reported that P. gingivalis LPS stimulates 5-week-old ddY male mice. All mice were euthanized by periosteal OC formation due to induction of RANKL cervical dislocation under anesthesia by inhalation of in osteoblasts by activation of TLR2 [15]. Of note, al- isoflurane at 0.5–5% with oxygen. BMCs were cultured veolar bone loss is induced by P. gingivalis through in α-Minimum Essential Medium (α-MEM; Invitrogen, TLR2 in mice [13, 16, 17]. Conversely, synthetic Grand Island, NY, USA) containing 10% fetal bovine TLR2 ligand inhibits OC formation in mouse bone serum (FBS; Biowest Nuaille, France) and antibiotics marrow macrophages (BMMs) stimulated with macro- (100 U/ml penicillin G and 0.15 mg/ml streptomycin phage colony-stimulating factor (M-CSF) and RANKL sulfate). After overnight culture to differentiate BMMs, [18, 19]. Although RANKL induces differentiation of non-adherent cells were cultured for 3 days with or with- BMMs to OCs, simultaneous addition of LPS inhibits out Pam3CSK4 (50–100 ng/ml) or lipid A (100 ng/ml) in this process [19, 20]. However, LPS stimulation at the presence of macrophage colony-stimulating factor late-stage osteoclastogenesis enhances the survival and (M-CSF; 20 ng/ml). For the control, phosphate-buffered sa- activation of OCs [20–22]. Although P. gingivalis has line (PBS) was administered instead of the TLR ligands. In known to promote osteoclastogenesis and bone some experiments, BMMs were used as osteoclast precur- resorption which types of TLR receptor, such as sors and further cultured with RANKL (50 ng/ml) in the TLR2 and/or TLR4 promote bone resorption remains presence or absence of TLR ligands for 3 days. To identify unclear. OCs, cells were fixed with 3.7% formaldehyde and stained Lectin-like oxidized low-density lipoprotein receptor-1 with tartrate-resistant acid phosphatase (TRAP) using the (Lox-1) was discovered as a receptor for oxidized Acid Phosphatase Leukocyte Kit (Sigma-Aldrich, St. Louis, low-density lipoprotein (oxLDL) in endothelium and MO, USA), and defined as TRAP-positive multinucleated vascular-rich organs [23]. Lox-1 is a multi-ligand recep- cells (having more than three nuclei). OCs were counted tor that recognizes many ligands, such as activated plate- using a microscope. Each condition was tested in triplicate lets [24], neutrophils [25], apoptotic/aged cells [26], and and all experiments were repeated at least three times. bacteria [27], and is expressed in many cell types [28–30]. Bone-resorbing OCs and bone-forming oste- Reverse transcription (RT)-polymerase chain reaction oblasts have been reported to express Lox-1, and (PCR) and real-time PCR −/− Lox-1 mice have decreased bone mass in the Total RNA was extracted from cells using TRIzol reagent steady state but are resistant to inflammatory bone (Life Technologies Corporation, Rockville, MD, USA). First destruction because of the impairment of osteoblastic strand cDNA was synthesized using 1 mg total RNA with RANKLexpressioninresponse toinflammation [31]. Super Script II RT according to the manufacturer’sinstruc- The pathology of both periodontitis and dyslipidemia tions (Invitrogen, Grand Island, NY, USA). To detect involves OCs, and these lifestyle-related diseases are ex- mRNA expression of Lox-1 and β-actin, RT-PCR was per- acerbated by stimulation with TLRs and Lox-1, respect- formed using the following: Lox-1 sense 5′-AGACTGGCT ively. Consequently, although these disorders are CTGGCATAAAG-3′,antisense 5′-AAGGCCAACATGCT reported to be associated with each other, the mechan- TTACAT-3′; β-actin sense 5′-TGAGAGGGAAATCG ism for this is unclear [32–34]. Some reports have TGCGT-3′,antisense 5′-GCTGGAAGGTGGACAGTGA shown that periodontitis increases the risk of athero- G-3′. PCR was performed under the following conditions: sclerosis in vivo and in vitro [35–38], and that periodon- 1 min denaturation at 95 °C, 1 min annealing at 55 °C, and titis worsens in the apolipoprotein E (ApoE)−/− 1 min extension at 72 °C, using 36 cycles. PCR products hyperlipidemia model [33, 35, 39]. However, there are were subjected to electrophoresis on 2% agarose gel and vi- few reports describing how dyslipidemia exacerbates sualized with ethidium bromide. To examine the effects of periodontitis. MAPK inhibitors on Lox-1 mRNA expression, BMCs in The purpose of this study was to clarify whether osteo- thepresenceofM-CSF (20ng/ml)were culturedwithor clastogenesis is upregulated through Lox-1 by TLR without Pam3CSK4 (100 ng/ml) or Lipid A (100 ng/ml) for stimulation at early- and late-stage. We showed that 3 days after incubation with TLR2/4 inhibitor (25 μM) or osteoclastogenesis was accelerated by activation of TLRs MAPK inhibitors, SP600125 (5 μM) as c-Jun N-terminal through upregulation of Lox-1 expression during bone kinase (JNK) inhibitor or U0126 (10 μM) as MAP/extracel- marrow cell (BMC) differentiation into BMMs, suggesting lular signal-regulated kinase (ERK) kinase (MEK) inhibitor, dyslipidemia increases the risk of periodontitis. respectively, for 1 h. Signals of Lox-1 mRNA were Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 3 of 9 normalized to β-actin mRNA expression levels using Image cells among the total non-adherent BMCs was quantified J software (NIH, Bethesda, MD, USA). using the labeled square. To quantify mRNA levels, cDNA samples were analyzed by quantitative real-time PCR. A total of 1 mg of cDNA Western blot analysis was amplified in a 20 μl volume of Power SYBR Green Cells were lysed in Tris-NaCl-Tween (TNT) buffer contain- PCR Master Mix (Applied Biosystems, Foster City, CA, ing 20 mM tris–HCl (pH 7.5), 200 mM NaCl, 1% Triton USA) in a real-time PCR system (Bio-rad CFX96, Bio-rad X-100, 1 mM dithiothreitol (DTT), and protease inhibitors Technologies, Inc., Santa Clara, CA, USA) and the fluor- (Roche, Basel, Switzerland). The protein content of the sam- escence was monitored at each cycle. Cycle parameters ples was measured using Pierce reagents, following the man- were 95 °C for 30 s to activate Taq followed by 40 cycles ufacturer’s protocol. Protein samples (20 μg) were subjected of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 40 s. For to sodium dodecyl sulfate-polyacrylamide gel electrophor- real-time analysis, two standard curves were constructed esis, and proteins were then transferred to a polyvinylidene from amplicons for both the β-actin and target gene. Tar- difluoride (PVDF) membrane (100 V, 1 h, 4 °C). The mem- get gene cDNA units in each sample were normalized to branes were incubated with anti-FDPS and anti-β-actin anti- β-actin cDNA units. Finally, the relative target gene ex- bodies diluted at 1:1000 in 5% (w/v) skimmed milk solution pression units were expressed as arbitrary units, calculated supplemented with 0.01% (w/v) azide overnight at 4 °C. The according to the following formula: relative target gene ex- blots were washed in Tris- buffered saline with Tween pression units = target gene cDNA units/β-actin cDNA (TTBS) (10 mM tris–HCl, 50 mM NaCl, 0.25% Tween 20) units. To detect mRNA expression of Lox-1, MyD88 and and incubated with an appropriate secondary antibody for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 30 min at room temperature. The immunoreactive proteins real-time PCR was performed using the following sets: were visualized using enhanced chemiluminescence reagents Lox-1 sense 5′-CTGCGAATGACGAGCCTGA-3′,anti- (GE Healthcare, Tokyo, Japan). sense 5′-TCACTGACAACACCAGGCAGAG-3′;MyD88 sense 5′-TACAGGTGGCCAGAGTGGAA-3′,antisense Chemicals 5′-GCAGTAGCAGATAAAGGCATCGAA-3′;GAPDH Recombinant human RANKL and M-CSF were pur- sense 5′-TGTGTCCGTCGTGGATCTGA-3′,antisense chased from Peprotec Co. Ltd. (Minneapolis, MN, USA). 5′-TTGCTGTTGAAGTCGCAGGAG′. All other chemicals were obtained from Sigma-Aldrich. Immunocytochemistry Data analysis Cells were fixed in 4% formaldehyde for 5 min and perme- All data are expressed as mean ± standard error of the abilized with 0.05% Triton-× 100 in phosphate-buffered sa- mean (SEM) of the number of cells (n). Statistical compari- line (PBS) for 5 min. Cells were incubated with goat sons were performed using analysis of variance (ANOVA). polyclonal anti-rabbit Lox-1 antibody (1:100 dilution, A probability (P) of < 0.05 was considered significant. ab60178, Abcam) overnight at 4 °C after blockade of non- specific binding with 3% goat serum for 40 min at room Results temperature. Cells treated with primary antibody were Expression of Lox-1 was upregulated by TLR 2/4 washed with PBS and incubated with Alexa fluor ligands in BMCs 488-conjugated streptavidin (2 μg/ml, Molecular Probes, Eu- To clarify the effect of TLR ligands on the expression of gene, OR) in goat anti-rabbit IgG secondary antibody Lox-1 in BMCs or BMMs, cells were cultured with or (1:1500 dilution, Vector Laboratories, Burlingame, CA) for without Pam3CSK4 (100 ng/ml) or Lipid A (100 ng/ml) in 30 min at room temperature. To label cell nuclei, cells were the presence of M-CSF for 3 days (Fig. 1a). Exposure to rinsed in PBS and covered with encapsulant containing Pam3CSK4 or Lipid A significantly upregulated the ex- 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence was pression of Lox-1 in BMCs (p < 0.05) (upper panel) but observed using fluorescence microscopy (TMD 300, Nikon). suppressed Lox-1 expression in BMMs (lower panel) com- pared with the control. Lox-1 mRNA expression in BMCs FACS analysis peaked on days 1 or 3 after incubation with Pam3CSK4 or BMCs were cultured for 3 days in α-MEM containing 10% Lipid A as assessed by real-time PCR (p < 0.05) (Fig. 1b, FBS and M-CSF with or without Pam3CSK4 (100 ng/ml) or left panel). Furthermore, Lox-1 protein expression peaked Lipid A (100 ng/ml) for 3 days. Cells were washed twice on day 3 after treatment with both TLR ligands (Fig. 1b, with PBS and incubated with anti-Lox-1 and anti-RANK right panel). Similar to the PCR results, protein expression antibodies conjugated with phycoerythrin. The labeled cells of Lox-1 was also upregulated following exposure to were analyzed on a FACS using On Chip (On Chip Bio. Pam3CSK4 or Lipid A in BMCs as assessed by immuno- Tec., Tokyo, Japan). The ratio of Lox-1- or RANK-positive cytochemistry (Fig. 1c). Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 4 of 9 a b Fig. 1 Analysis of LOX-1 in bone marrow cells (BMCs) and bone marrow macrophages (BMMs). (A, upper panel, B, C) BMCs were differentiated into BMMs with or without Pam3CSK4 (TLR2 ligand, 100 ng/ml) or Lipid A (TLR4 ligand, 100 ng/ml) in the presence of macrophage colony-stimulating factor (M-CSF, 20 ng/ml) for 3 days. (A, lower panel) BMMs were cultured with or without Pam3CSK4 (100 ng/ml) or Lipid A (100 ng/ml) in the presence of M-CSF and receptor activator of nuclear factor kappa-B ligand (RANKL, 50 ng/ml) for 3 days. a Polymerase chain reaction (PCR) products of LOX-1, a receptor of oxidative low density lipoprotein (oxLDL), mRNA in BMCs (upper panel) and BMMs (lower panel) were amplified using RT-PCR methods. Following stimulation with Pam3CSK4 or Lipid A, Lox-1 expression was upregulated in BMCs (upper panel) but not in BMMs (lower panel). b Lox-1 mRNA expression peaked on day 1 or 3 as determined by real-time PCR (left panel) and protein expression on day 3 (right panel). c BMCs stimulated with TLR ligands on day 3 were stained with anti-Lox-1 antibody (1:100 dilution) then incubated with Alexa fluor 488-conjugated streptavidin (2 μg/ml) in goat anti-rabbit IgG secondary antibody (1:1500 dilution). Lox-1 expression was upregulated. Data are expressed as mean ± SEM (n =3). *P < 0.05 compared with control TLR2/4 ligands upregulate Lox-1 and RANK in BMCs real-time PCR. Although the addition of Pam3CSK4 or BMC surface expression of Lox-1 and RANK proteins Lipid A significantly increased the expression of Lox-1 was analyzed by FACS. Treatment with M-CSF slightly and MyD88 mRNAs, TLR2 or 4 ligand-induced upregu- increased the cell surface expression of Lox-1 in BMCs lation was reduced in the presence of TLR2/4 and in a time-dependent manner compared with untreated MAPK inhibitor peptides, respectively (Fig. 3a), suggest- cells (day 0; Fig. 2, upper panel). The addition of ing an influence towards Lox-1 expression by activation Pam3CSK4 or Lipid A significantly upregulated the of TLR-MyD88 downstream signaling. Furthermore, the expression of Lox-1 compared with the control cells inhibitors of JNK and MEK significantly inhibited the (incubated with IgG). Similar to the real-time PCR time upregulation of TLR2-induced RANK mRNAs on BMCs course results, Lox-1 expression peaked at day 1 or day (Fig. 3b). These results demonstrated that TLR2/4 3 after stimulation with Pam3CSK4 or Lipid A, respect- ligands activated Lox-1 expression through the MEK ively. In the present experiments, RANKL induced the and JNK activation pathway, including MyD88. expression of RANK, and the addition of Pam3CSK4 or Lipid A upregulated the expression of RANK in BMCs TLRs stimulation with oxLDL accelerates in a time-dependent manner. osteoclastogenesis at early-phase To clarify TLR2-induced Lox-1 upregulation on osteoclas- TLR 2/4 ligands upregulate the expression of Lox-1 in togenesis, we examined the effect of oxLDL (30 μg/ml) on BMCs through activation of MAPK osteoclast differentiation on early-phase (BMC: day 0–3be- To assess TLR2 or 4 activation of Lox-1 expression via fore stimulation with RANKL) and/or on late-phase the downstream pathways of TLR2/4, MyD88, and (BMM: day 0–3 after RANKL stimulation) osteoclastogene- MAPKs, we examined the effects of these inhibitors on sis. The number of TRAP-positive multinucleated cells the expression of Lox-1 and RANK in BMCs using were increased at early phase and decreased at late phase, Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 5 of 9 Fig. 2 BMC surface expression of Lox-1 and RANK proteins. BMCs were cultured with or without Pam3CSK4 (TLR2 ligand, 100 ng/ml) or Lipid A (TLR4 ligand, 100 ng/ml) in the presence of M-CSF (20 ng/ml) for 3 days. Cells were washed with PBS and incubated with anti-Lox-1 or anti-RANK antibodies. Data are expressed as the mean ± SEM (n = 3 experiments) by addition of only oxLDL (Fig. 4a and b each upper panels). [43, 44]. Although atherosclerosis is reported to result Furthermore, Pam3CSK4 (10 ng/ml) had no effect on in inhibited bone formation because oxLDL blocked early-phase, but an inhibition effect at late-phase osteoclas- differentiation of osteoblast progenitor cells [45, 46], togenesis (Fig. 4a and b each middle panels). When we stim- little is known about how dyslipidemia influences ulated BMCs with both Pam3CSK4 (25 ng/ml) and oxLDL bone resorption. In the present study, we found that (15 μg/ml) at early-phase, numbers of TRAP-positive multi- TLRs upregulated the oxLDL receptor, Lox-1, via nucleated cells were significantly increased (Fig. 4a and b MAPK in BMCs, resulting in promotion of osteoclas- each lower panels). togenesis. This finding suggests that periodontitis would worsen with progression of dyslipidemia. Discussion The subcutaneous or intraperitoneal administration of P. gingivalis is known to stimulate TLR2 and/or TLR4 [11, Pam3CSK4 is reported to upregulate bone resorption in 12] via MyD88 to modulate osteoclastogenesis [18, 40–42], vivo, suggesting that Pam3CSK4 directly promotes osteo- resulting in alveolar bone resorption through differential clastogenesisfromBMMsafter RANKLpriming [41, 47]. regulation of TLR2 and TLR4 signaling pathways in a However, synthetic TLR2 ligand inhibits OC formation in RANKL-dependent manner [13]. Although Lipid A, a BMMs treated with M-CSF and RANKL in vitro [18, 19] TLR4 ligand, has been shown to be a potent stimulator of Furthermore, combination of RANKL and LPS inhibited bone loss in vivo [14], little is known about whether other osteoclastogenesis [19, 20]. Our data also showed that TLR ligands have a stimulatory or inhibitory effect on oste- numbers of TRAP-positive cells were decreased when oclastogenesis in vitro. Furthermore, many clinical studies BMMs were stimulated with Pam3CSK, compared with show that osteoporosis is associated with atherosclerosis those treated with M-CSF and RANKL. In contrast, the Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 6 of 9 ab Lox-1 control +Pam3 +Lipid A +Pam3 +TLR2/4 inhibitors control RANK +Lipid A +TLR2/4 inhibitor +Pam +Pam3 +JNK inhibitor +Pam3 +Lipid A 20 +MEK inhibitor +Pam+JNK inhibitor +Lipid A+JNK inhibitor incubation time (day) +Pam+MEK inhibitor control MyD88 +Pam3 +Lipid A +Pam3 +TLR2/4 inhibitors +Lipid A +TLR2/4 inhibitor +Pam3 +JNK inhibitor +Pam3 +MEK inhibitor 01 3 0 1 3 incubation time (day) incubation time (day) Fig. 3 Real time PCR to determine Lox-1, MyD88 and RANK expression in BMCs following addition of inhibitors. BMCs in the presence of M-CSF (20 ng/ml) were cultured with or without Pam3CSK4 (TLR2 ligand, 100 ng/ml) or Lipid A (TLR4 ligand, 100 ng/ml) for 3 days after incubation with TLR2/4 inhibitor (25 μM) or MAPK inhibitors, SP600125 (5 μM) as JNK inhibitor or U0126 (10 μM) as MEK inhibitor, respectively, for 1 h. a Both TLR2/4 inhibitor and MAPK inhibitors significantly suppressed TLR 2 or 4 ligand-induced upregulation of Lox-1 (upper panel) and MyD88 (lower panel) mRNA. b SP600125 significantly suppressed TLR 2, but not TLR 4 ligand-induced upregulation of RANK mRNA, and U0126 upregulated RANK mRNA. Data are expressed as the mean ± SEM (n = 3 experiments) addition of Pam3CSK and oxLDL, a Lox-1 ligand to- kinases, together with the suppression of DNA binding gether at early phase, increased the number of activities of transcriptional factors, such as nuclear TRAP-positive cells. Although the mechanism through factor-kappa B (NF-kappa-B) and nuclear fsctor of acti- which P. gingivalis-induces bone resorption via TLR2 vated T-cells (NFAT) [49]. Our data demonstrated that and TLR4 is not fully understood, our results suggest TLR 2 or 4 ligand-induced Lox-1 upregulation was re- that TLR-stimulation in periodontitis upregulates duced in the presence of TLR2/4 and MAPK inhibitor, alveolar bone resorption under high plasma concentra- respectively, and the inhibitors of JNK and MEK signifi- tion of oxLDL in dyslipidemia. cantly inhibited the upregulation of TLR 2-induced RANK OC progenitors, including BMCs and BMMs, are re- mRNA on BMCs. We also found that TLR-stimulation ported to express abundant levels of LDL receptors, together with oxLDL accelerated early-phase but not such as Lox-1, in a RANKL-independent manner. Ex- late-phase osteoclastogenesis, and only oxLDL stimulation pression levels of Lox-1 are greatly decreased during os- suppressed RANKL-induced osteoclastogenesis, consist- teoclastogenesis, particularly during the fusion of ent with previous studies. In the present study, Pam3CSK4 mononuclear cells to form multinuclear OCs [48]. The was shown to upregulate the expression of RANK and present data showed that Lox-1 expression was upregu- Lox-1 in BMCs. Furthermore, stimulation with both lated by TLR 2 or 4 ligands in BMCs but not in BMMs. Pam3CSK4 and oxLDL was shown to promote progres- −/− Periodontal tissues in ApoE hyperlipidemia model sion of osteoclastogenesis, suggesting that oxLDL-Lox-1 rats are reported to exhibit more TRAP-positive multi- signaling promotes osteoclastogenesis through expression nuclear cells and increased TLR2 and TLR4 expression of Lox-1 by stimulating TLRs. levels [33], suggesting that high oxLDL concentration ef- Similar reports have shown that plasma lipids likely fects osteoclastogenesis via the elevation of TLRs. play a role in maintaining bone mass [48, 50]. A better OxLDL is reported to suppress RANKL-induced osteo- understanding of the coupling between osteoporosis and clastogenesis, TRAP activity, and bone-resorbing activity atherosclerosis is indicated in previous studies whereby derived from human peripheral blood mononuclear cells treatment of one disease may have beneficial effects on in vitro. This suppression is suggested to be caused by another [51–56]. The American Heart Association reported RANKL-induced phosphorylation of ERK, p38, and JNK in 2012 that although periodontal interventions result in a ratio of Ct ratio of Ct ratio of Ct Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 7 of 9 N.S. control EL E & L oxLDL N.S. Pam 3 (-) 100 µm control L E & L oxLDL N.S. EL control E & L Pam oxLDL (-) 100 µm Pam control E L E & L EL control E & L oxLDL E Pam (+) N.S. 100 µm oxLDL control E L E & L Fig. 4 Effect of stimulation to TLRs and/or Lox-1 to TRAP-positive multinucleated cells. The effect of stimulation to Lox-1 with oxLDL and/or to TLR 2 with Pam3CSK4 was examined using TRAP staining. In each panel, E indicates stimulation at early-phase (BMC: day 0–3 before stimulation with RANKL) and L indicates stimulation at late-phase (BMMs: day 4–6 after stimulation with RANKL). (a and b upper) BMCs in the presence of M- CSF or BMMs in the presence of M-CSF and RANKL were cultured with oxLDL (30 μg/ml) at indicated phase. (a and b middle) BMCs in the presence of M-CSF or BMMs in the presence of M-CSF and RANKL were cultured with Pam3CSK4 (10 ng/ml) at indicated phase. (a and b lower) BMCs in the presence of M-CSF and Pam3CSK4 (25 ng/ml) were stimulated with oxLDL (15 μg/ml) at early- or late-phase. Data are expressed as the mean ± SEM (n = 3 experiments). *P < 0.05 compared with control reduction in systemic inflammation and endothelial periodontitis at least partially worsens by the compli- dysfunction in short-term studies, there is no evi- cation of dyslipidemia. dencethattheyprevent atherosclerotic cardiovascular disease (ASVD) or modify its outcomes [57]. Al- Conclusion though our data could not support that ASVD devel- In conclusion, the activation of TLR 2 and/or 4 upregu- oped due to periodontitis worsening dyslipidemia, it lated the expression of Lox-1 through activation of did show that osteoclastogenesis was upregulated by MAPK in BMCs but not in osteoblasts, suggesting the TLR-stimulation, like bacterial infection, in periodontitis promotion of osteoclastogenesis during dyslipidemia. with dyslipidemia. Abbreviations In this study, we focused on elucidating the correl- ApoE: Apolipoprotein E; ASVD: Atherosclerotic cardiovascular disease; ation between dyslipidemia and progression of peri- BMC: Bone marrow cell; BMM: Bone marrow macrophage; ERK: Extracellular odontitis. Our results indicated that stimulation of signal-regulated kinase; FACS: Fluorescence activated cell sorting; JNK: c-Jun N-terminal kinase; Lox-1: Lectin-like oxidized low-density-lipoprotein BMCs with TLRs, representative of a bacterial infec- receptor 1; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein tion, upregulated Lox-1 expression resulting in pro- kinase;M-CSF:Macrophagecolony-stimulating factor; MEK: MAPK/ERK motion of osteoclastogenesis in the presence of kinase; MyD88: Myeloid differentiation factor 88; NFAT: Nuclear factor of activated T-cells; NF-kappa-b: Nuclear factor-kappa B; OC: Osteoclast; oxLDL: oxidized oxLDL in dyslipidemia. Although the present data low-density lipoprotein; P. gingivalis: Porphyromonas gingivalis; PCR: Polymerase did not clarify the direct relationships between chain reaction; RANKL: Receptor activator of nuclear factor kappa-B ligand; periodontitis and atherosclerotic vascular disease, TLR: Toll-like receptor; TRAP: Tartrate-resistant acid phosphatase TRAP+ MNCs TRAP+ MNCs TRAP+ MNCs Ohgi et al. Lipids in Health and Disease (2018) 17:132 Page 8 of 9 Acknowledgements contains multiple lipid a species that functionally interact with both toll-like We thank Elizabeth Finnie, PhD, from Edanz Group (http://www.edanzediting.com/ac) receptors 2 and 4. Infect Immun. 2004;72(9):5041–51. for editing a draft of this manuscript. 13. Lin J, Bi L, Yu X, Kawai T, Taubman MA, Shen B, Han X. Porphyromonas gingivalis exacerbates ligature-induced, RANKL-dependent alveolar bone Funding resorption via differential regulation of toll-like receptor 2 (TLR2) and TLR4. This work was supported by a Grants-in-Aid for Scientific Research from the Infect Immun. 2014;82(10):4127–34. Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 14. Sakuma Y, Tanaka K, Suda M, Komatsu Y, Yasoda A, Miura M, Ozasa A, 15 K11405) and Private University Research Branding Project. The authors Narumiya S, Sugimoto Y, Ichikawa A, Ushikubi F, Nakao K. Impaired bone report no conflicts of interest related to this study. resorption by lipopolysaccharide in vivo in mice deficient in the prostaglandin E receptor EP4 subtype. Infect Immun. 2000;68(12):6819–25. Authors’ contributions 15. Kassem A, Henning P, Lundberg P, Souza PP, Lindholm C, Lerner UH. KO (Kimiko Ohgi): Conceived, designed and performed the experiments and Porphyromonas gingivalis stimulates bone resorption by enhancing RANKL wrote the paper; HK: Conceived, designed and performed the experiments (receptor activator of NF-κB ligand) through activation of toll-like receptor 2 and wrote the paper; RS: Conceived and designed the experiments; KO: in osteoblasts. J Biol Chem. 2015;290(33):20147–58. Performed the experiments; KGT: Analyzed the data; FO: Analyzed the data; 16. Burns E, Bachrach G, Shapira L, Nussbaum G. Cutting edge: TLR2 is required YY: Analyzed the data. All authors read and approved the final manuscript. for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone Ethics approval and consent to participate resorption. J Immuno. 2006;177(12):8296–300. Not applicable. 17. Papadopoulos G, Weinberg EO, Massari P, Gibson FC 3rd, Wetzler LM, Morgan EF, Genco CA, et al. Macrophage-specific TLR2 signaling mediates Competing interests pathogen-induced TNF-dependent inflammatory oral bone loss. J Immunol. The authors declare that they have no competing interests. 2013;190(3):1148–57. 18. Zhang P, Liu J, Xu Q, Harber G, Feng X, Michalek SM, Katz J. TLR2- dependent modulation of osteoclastogenesis by Porphyromonas gingivalis Publisher’sNote through differential induction of NFATc1 and NF-kappaB. J Biol Chem. 2011; Springer Nature remains neutral with regard to jurisdictional claims in 286(27):24159–69. published maps and institutional affiliations. 19. Yang J, Ryu YH, Yun CH, Han SH. Impaired osteoclastogenesis by staphylococcal lipoteichoic acid through toll-like receptor 2 with partial Author details involvement of MyD88. J Leukoc Biol. 2009;86(4):823–31. Department of Odontology, Fukuoka Dental College, Fukuoka 8140193, 20. Takami M, Kim N, Rho J, Choi Y. Stimulation by toll-like receptors inhibits Japan. 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