Introduction
During the progression of periodontitis, oral bacteria exacerbate immune responses. The continuous inflammatory immune response leads to tissue damage and bone loss due to the increase of osteoclasts and the decrease of osteoblasts [1]. Dental mesenchymal stem cells (MSCs) hold a great potential for dental tissue regeneration [2]. Among different types of dental stem cells isolated from distinct parts of dental tissue, periodontal ligament stem cells (PDLSCs) from a fibrous connective tissue between tooth and alveolar bone is involved in the repair from periodontitis [3]. PDLSCs present multipotent differentiation capacities including osteogenic, adipogenic and chondrogenic lineages [4]. In addition, PDLSCs may mitigate adverse immune responses as MSCs from various other tissues do [5]. Furthermore, PDLSCs can be utilized in a wide range of regenerative medicine as they are an excellent source for the repair of bone or cartilage in other parts of body. During the progression of periodontitis, alveolar bone surrounding the teeth is devastated by strong resorptive bone remodeling by osteoclasts activated by inflammatory immune responses. Modulation of PDLSC activity in the inflammatory condition may provide a novel opportunity in reserving the demolishing process of periodontitis.
Lysophosphatidic acid (LPA) is a signaling phospholipid generated by the enzyme autotaxin. LPA binds to and stimulates a group of G protein-coupled receptors known as LPARs (LPAR1-6) [6]. The binding of LPA to LPARs activates downstream signaling cascade including small GTPase, Ras, Rho, and Rac [7]. LPA signaling modulates a variety of cellular changes in proliferation, migration, adhesion, differentiation, and morphology [8]. The growing evidence suggests that LPA signaling regulates stem cells and progenitors in maintaining stemness and controlling the differentiation toward the intended lineages in adult stem cells and embryonic stem cells [9]. LPA was shown to mildly increase the proliferation of PDLSCs, but a concern regarding the promotion of tumor growth and bone loss remains [10]. Further understanding of LPA signaling with moderation in PDLSCs is necessary for the application to periodontitis.
In this study, we identified that the LPAR1 antagonist (AM095) decreased the expression of pro-inflammatory cytokines and promoted the osteogenic differentiation of PDLSCs in inflammation-mimicking condition induced by Porphyromonas gingivalis (P. gingivalis, Pg). These findings indicate that LPAR1 is a potential therapeutic target in the development of treatment of periodontitis.
Materials and Methods
1. Cell culture and reagents
Human PDLSCs were purchased from Lonza (Basel, Switzerland, Cat No. CC-7049) and prepared according to the manufacturer’s instructions, and cells at passage 3–9 were used for experiments. Cells were maintained in stromal cell growth medium with 5% fetal bovine serum (FBS), 0.1% human fibroblast growth factor, 0.1% insulin, and gentamicin/amphotericin-B (Lonza, Cat No. CC-3205). Bacterial LPS of Pg (InvivoGen, San Diego, CA, USA) was suspended in endotoxin-free water as a stock solution (concentration 1 mg/mL). To determine effect of LPAR inhibition, PDLSCs were incubated with LPAR1 antagonist (AM095), LPAR2 antagonist (Beck35), LPAR 1 and 3 antagonist (Ki16325), LPAR5 antagonists (TC-LPA5-4). LPAR antagonists were kindly provided by Dr. Gabor J. Tigyi (University of Tennessee Health Science Center, Memphis, TN, USA).
2. Multilineage differentiation
The differentiation capacity of the PDLSCs were evaluated using the Human MSC Functional Identification Kit (R&D Systems Inc., Minneapolis, MN, USA, Cat. No. SC006). The cells were incubated in the differentiation media for 2–3 weeks and then evaluated for osteogenesis, adipogenesis, and chondrogensis according to the manufacturer’s instructions.
3. Immunocytochemistry
Osteogenic and adipogenic cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 minutes at room temperature. Chondrogenic pellets were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. Pelleted cells were dehydrated and placed in embedding medium (Tissue- Tek; Sakura Finetek USA Inc., Torrance, CA, USA), and sections were prepared with a cryostat (thickness: 15 μm). Fixed cells or pellets were then incubated for 45 minutes at room temperature in a blocking solution containing 0.3% Triton X-100, 1% bovine serum albumin (BSA), and 10% normal donkey serum in 0.1 M PBS. The samples were then washed and incubated with anti-mFABP4 antibody (adipogenesis), anti- hOsteocalcin (osteogenesis) and anti-hAggrecan (chondrogenesis) overnight at 4℃. For immunofluorescence, samples were incubated with a combination of Alexa Fluor 594-labeled donkey anti-goat (1:2,000, Invitrogen Corp., Carlsbad, CA, USA, Cat No. A11058) and Alexa Fluor 488-labeled donkey anti-mouse secondary antibody (1:2,000, Invitrogen Corp., Cat No. A32766) for 1 hour at room temperature. Images were taken with EVOSTM FL Auto 2 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).
4. Flow cytometry analysis
Analysis of fluorescence intensity of the stained cells was performed using a FACSCanto flow cytometry system (BD Biosciences, San Diego, CA, USA). PDLSCs were dissociated with TrypLETM (Thermo Fisher Scientific), and cells were resuspended in PBS with 1% FBS and aliquoted to be 1 × 105 cell/ tube for antibody labeling. Cells were blocked with 1% BSA in PBS and stained with directly-conjugated fluorescent antibodies: CD14-PE, CD34-FITC, CD44-FITC, CD45-PE, CD73- APC, CD90-PE, CD105-APC, CD166-PE (BD Biosciences, Cat No. 555398, 555821, 555478, 555483, 560847, 555596, 562408, 559263), following by counting 10,000 events. 7-AAD (BD Biosciences, Cat No. 559925) was added with 1:100 dilution to distinguish live cells from dead cells. Obtained fluorescence signals were analyzed using the FACSDiva (ver. 6.1.3, BD Biosciences) or the FlowJo (ver. 10, Tree Star Inc., San Carlos, CA, USA).
5. RNA isolation and quantitative reverse transcription polymerase chain reaction (RT-PCR)
To analyze mRNA expression in PDLSCs, RNA was extracted using Sensi-TriJol reagent (Lugen Sci Inc., Bucheon, Korea) according to the manufacturer’s protocol. RNA purity and concentration were determined by NanoDrop ND-1000 Spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA) using absorbance at 260 and 280 nm. cDNA was synthetized using the QIAGEN Whole Transcriptome Kit (QIAGEN, Valencia, CA, USA) and analyzed by real-time PCR using the following primer sets: LPA receptor 1 sense primer, 5’-GCT GCC ATC TCT ACT TCC AT-3’; LPA receptor 1 antisense primer, 5’-CCA TTC TGT GGC AAG ATG CT-3’; LPA receptor 2 sense primer, 3’-CAG CGC ATG GCA GAG CAT GT-5’; LPA receptor 2 antisense primer, 3’-CCA GGA CAT TGC AGG ACT CAC A-5’; LPA receptor 3 sense primer, 3’-AAC GTG AGC GGA TGT TCA CT- 5’; LPA receptor 3 antisense primer, 3’-CCG CGA TGA CCA GAG AAT TA-5’; LPA receptor 4 sense primer, 3’-TTC GAA CTA ATG TGG AGG AA-5’; LPA receptor 4 antisense primer, 3’-TGG AAT TGG AAG TCA ATG AA-5’; LPA receptor 5 sense primer, 3’-CAG AGC AAC ACG GAG CAC AG-5’; LPA receptor 5 antisense primer, 3’-CAC CAG AAT CAT GGC ATG GC- 5’; LPA receptor 6 sense primer, 3’-CTT CAC AAC ACG GAA TTG GC-5’; LPA receptor 6 antisense primer, 3’-AGT TAA CCA CAC GCC AGT GC-5’; actin sense primer, 3’-TCC ATC ATG AAG TGT GAC G-5’; actin antisense primer, 3’-TCA GGA GCA ATG ATC T-5’; interleukin (IL)-1β sense primer, 3’-TCT TCG ACA CAT GGG ATA AC-5’; IL-1β antisense primer, 3’ -CCT TGT ACA AAG GAC ATG GA-5’; tumor necrosis factor-α (TNF-α) sense primer, 3’-CTC TTC TGC CTG CAC TTT G-5’; TNF-α antisense primer, 3’-ATG GGC TAC AGG CTT GTC ACT C-5’. Real-time PCR experiments were performed by Bright- Green 2X qPCR MasterMix-ROX (abm, Vancouver, Canada) using the StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) for ∼ 40 cycles. Data were normalized for gene expression by using GAPDH as an internal control. The 2–ΔΔCT method was used to analyze the relative quantification of gene expression.
6. Proliferation assay
PDLSCs viability was determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The PDLSCs were seeded in 48 well plates (Corning Incorporated, Corning, NY, USA) with a density of 2 × 104 cells/well in a humidified atmosphere with 5% CO2 at 37℃. After the subjection to varying experimental conditions, cells were washed with PBS and incubated with 200 μL of 0.5 mg/mL MTT (Merck KGaA, Darmstadt, Germany) for 2 hours at 37℃. Cells were rinsed with PBS, and the formazan products were dissolved in 200 μL of dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO, USA) for 15 minutes at room temperature with agitation. The absorbance at 540 nm was determined using microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).
7. Immunoblotting
Proteins from PDLSCs were homogenized in M-PER lysis buffer (Pierce Chemical Co., Rockford, IL, USA) with a protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). The extracted proteins (20 μg) were resuspended in sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS), 1% 2-mecaptoethanol, 0.02% bromophenol blue and separated by SDS-polyacrylamide gel electrophoresis. Proteins on the gel were transferred onto a PVDF membrane (Merck KGaA) by an electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in blocking buffer and incubated with anti-phospho-IKK (Cell Signaling Technology, Danvers, MA, USA, Cat No. 2697), anti- IKK (Cell Signaling Technology, Cat No. 2694), anti-phospho- IκB (Cell Signaling Technology, Cat No. 2859), anti-IκB (Cell Signaling Technology, Cat No. 9242), anti-phospho-NF-κB p65 (Cell Signaling Technology, Cat No. 9242), anti-NF-κB p65 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, Cat No. sc-8008), anti-beta actin (Sigma Aldrich, Cat No. A5316) and anti-Histone H2B (Santa Cruz Biotechnology, Inc., Cat No. sc-8650) antibodies. Primary antibodies were detected using horseradish peroxidase-conjugated anti-rabbit (Santa Cruz Biotechnology, Inc., Cat No. sc-2004) or ant-mouse (Santa Cruz Biotechnology, Inc., Cat No. 2005) antibodies. Immunoactivity was detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech., Buckinghamshire, UK).
8. Induction of osteogenic differentiation
PDLSCs were grown in mineralization inducing medium containing 10% FBS, 50 μM ascorbic acid, 10 mM β-glycerophosphate and 0.1 μM dexamethasone in α-MEM. To detect mineralization, cells were fixed with 4% paraformaldehyde and stained with Alizarin red S (Sigma, St. Louis, MO, USA) solution of 2% w/v diluted by deionized water at pH 4.3 for 20 minutes. To quantify the calcium deposition, the stained samples were destained and dissolved in 10% cetylpyridinium chloride solution in 10 mM PBS (pH 7.0). The absorbance at 562 nm was determined using microplate spectrophotometer (BioTek Instruments).
9. Statistical analysis
Data are presented as the mean ± standard deviation of results from three independent experiments. The Student’s ttest was used to determine the significance of difference between two groups.
Results
1. Characterization of PDLSCs in MSC properties and LPAR expression
To evaluate the characteristics of PDLSCs as MSCs, PDLSCs were subjected to the tri-lineage differentiation and evaluated for the surface marker expression. Multipotent differentiation potential of PDLSCs at the 4–6 passage was evaluated by osteogenic, adipogenic and chondrogenic differentiation. After 21 days of differentiation-inducing culture, osteogenesis, adipogenesis and chondrogenesis were analyzed by the immunostaining using FABP4 (adipogenesis), Osteocalcin (osteogenesis) and Aggrecan (chondrogenesis) antibodies (Fig. 1A). Flow cytometry analysis showed that PDLSCs were positive for the expression of MSC surface markers, such as CD44, CD73, and CD90, but negative for hematopoietic stem cell markers, such as CD14, CD34, and CD45 (Fig. 1B). To evaluate the expression of LPARs, PDLSCs in the maintenance culture were subjected to the quantitative real-time PCR analysis (Fig. 1C). Among LPAR1-6, LPAR1 mRNA expression was significantly higher in comparison with other LPA receptors (LPA 2-6).
2. Effect of LPAR antagonists on the viability of PDLSCs
To evaluate the effect of LPAR signaling on the maintenance of PDLSC viability, various LPAR antagonists were treated on PDLSCs, followed by MTT assay. PDLSCs were treated with increasing doses (0, 0.5, 5 μM) of inhibitors for LPAR1 (AM095), LPAR2 (Beck35), LAPR 1and 3 (Ki16425) and LPAR5 (TCLPA5- 4) individually, and cells were subjected to MTT assay at different time pointes (24, 48, 72 hours). As shown in Fig. 2, most of LPAR antagonists, expect for Beck35 at 5 μM and Ki16425 at 72 hours, showed little effect on the viability of PDLSCs.
3. Effect of LPAR antagonists on Pg-LPS-induced viability decrease and inflammatory response
To simulate the inflammatory condition, PDLSCs were treated with the increasing doses of Pg-LPS (0, 1, 10 μg) for varying time points (24, 48, 72 hours). PDLSCs viability was significantly decreased with a high concentration of Pg-LPS treatment (10 μg) (Fig. 3A). To demonstrate the effect of LPAR antagonist on Pg-LPS-induced decrease of viability, various LPAR inhibitors were pre-treated for 3 hours prior to Pg-LPS treatment. As shown in Fig. 3B-3E, LPAR1, 2, and 3 antagonists (AM095, Beck35, Ki16425) showed a significant recov-ery from Pg-LPS-induced inhibition. Among them, AM095 showed little adversary effect at the higher dose (5 μM). LPAR5 antagonist (TC-LPA5-4), however, showed little effect. To evaluate the effect of AM095 on the expression of the proinflammatory cytokines, IL-1β and TNF-α mRNA expression was measured by qRT-PCR. As shown in Fig. 3F and 3G, Pg- LPS treatment increased the expression of IL-1β and TNF-α in PDLSCs, and 3 hours pre-treatment of AM095 reduced Pg- LPS-induced expression. To determine the activation of NF-κB signaling in PDLSCs, immunoblotting was performed by probing related signaling molecules. As shown in Fig. 3H, 1 hour Pg-LPS treatment significantly increased the phosphorylation of IKK, IκB and p65 of NF-κB, all of which showed the reduction in the phosphorylation with pre-treatment of AM095.
4. Effect of LPAR1 antagonist on Pg-LPS-induced decrease of osteogenic differentiation
To evaluate the effect of Pg-LPS and LPAR1 inhibitor, AM095, on the osteogenic differentiation of PDLSCs, cells were subjected to osteogenic culture in combination of Pg- LPS and AM095 treatment. Following osteogenic induction for 4, 7, and 14 days, PDLSCs exhibited gradual increase in the mineral formation, which was analyzed by Alizarin red S staining (Fig. 4). Pg-LPS treatment significantly decreased osteogenic differentiation at all time points. AM095 treatment, however, restored the osteogenic differentiation potential in the presence of Pg-LPS. These results suggest that LPAR1 blockade promoted the osteogenic differentiation of PDLSCs in periodontitis-mimicking inflammatory condition.
Discussion
The involvement of LPAR1 has in several diseases, including lung inflammation, diabetic nephropathy and neuropathic pain, has been reported [11-13]. In this study, we investigated the effect of LPAR modulation on PDLSCs. Among different LPAR modulators, LPAR1 inhibitor, AM095, rescued PDLSCs from Pg-LPS-induced decrease of cellular viability. AM095 itself showed little effect, neither increase nor decrease, on the viability of PDLSCs. These PDLSCs exhibited the excellent cellular morphology, the surface expression of stem cell markers such as CD44, CD73, and CD90, and the differentiation potential toward osteogenic, adipogenic, and chondrogenic lineages. Among LPAR1-6, PDLSCs showed the higher expression of LPAR1 in comparison with other LPARs. Pg-LPS treatment on PDLSCs, which represents periodontitis condition, activated NF-κB pathway and decreased osteogenic differentiation of PDLSCs. Pre-treatment of AM095 along with Pg-LPS inhibited the activation of NF-κB signaling by reducing the phosphorylation of IKK and NF-κB and restored the osteogenic differentiation potential of PDLSCs. These results indicate that LPAR1 takes an important part in the progression of periodontitis.
PDLSCs can be obtained from the periodontal tissue on the root surface of extracted teeth and present a potential source for stem cell therapy as they possess the characteristics of MSC [14,15]. PDLSCs have been suggested as a key contributor for the regeneration of dental tissue damaged by periodontal diseases. In LPS-induced inflammatory environment, the low level of bone formation is observed, but the cause-andeffect relationship between inflammation-PDLSC-bone loss remains yet to be understood. Previous studies have reported that NF-κB signaling is important in regulating the osteogenic differentiation of MSCs in inflammatory microenvironments [16,17]. The osteogenic differentiation of PDLSCs in the inflammatory condition was also reported to be affected by NF-κB signaling [18]. In addition, LPAR1 is involved in the osteogenic progression in the aortic valve through RhoA/ROCKNF- κB pathway [19]. In present study, we identified that the NF-κB pathway was suppressed in LPAR1 antagonist treatment in PDLSCs at Pg-LPS-induced periodontitis-mimicking condition. These results indicate that the modulation of NF-κB signaling in PDLSCs weighs significantly in the protection from periodontitis.
In summary, our findings revealed the novel contribution of LPAR1 in the progression of Pg-LPS-induced periodontitis and suggest the targets for therapeutic intervention at the receptor level along with intracellular signaling clues, which may lead to the development of the original treatment for periodontitis.
