MicroRNA expression profile of human periodontal ligament cells under the influence of Porphyromonas gingivalis LPS

Periodontitis is a chronic inflammatory disease which is caused by bacterial infection and leads to the destruction of periodontal tissues and resorption of alveolar bone. Thus, special attention should be paid to the mechanism under lipopolysaccharide (LPS)‐induced periodontitis because LPS is the major cause of periodontitis. However, to date, miRNA expression in the LPS‐induced periodontitis has not been well characterized. In this study, we investigated miRNA expression patterns in LPS‐treated periodontal ligament cells (PDLCs). Through miRNA array and differential analysis, 22 up‐regulated miRNAs and 28 down‐regulated miRNAs in LPS‐treated PDLCs were identified. Seven randomly selected up‐regulated (miR‐21‐5p, 498, 548a‐5p) and down‐regulated (miR‐495‐3p, 539‐5p, 34c‐3p and 7a‐2‐3p) miRNAs were examined by qRT‐PCR, and the results proved the accuracy of the miRNA array. Moreover, targets of these deregulated miRNAs were analysed using the miRWalk database. Database for Annotation, Visualization and Integration Discovery software were performed to analyse the Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway of differential expression miRNAs, and the results shown that Toll‐like receptor signalling pathway, cAMP signalling pathway, transforming growth factor‐beta signalling pathway, mitogen‐activated protein kinase (MAPK) signalling pathway and other pathways were involved in the molecular mechanisms underlying LPS‐induced periodontitis. In conclusion, this study provides clues for enhancing our understanding of the mechanisms and roles of miRNAs as key regulators of LPS‐induced periodontitis.     

Circ_0062491 alleviates LPS-induced apoptosis and inflammation in periodontitis by regulating miR-498/SOCS6 axis

Periodontitis is a prevalent chronic inflammatory disease. Circular RNAs (circRNAs) have been revealed to play roles in the inflammatory response. Hence, this work aimed to explore the role and mechanism of circ_0062491 in periodontitis progression. Human periodontal ligament cells (PDLCs) were isolated from the periodontal ligament (PDL) of the healthy teeth with orthodontic requirement after tooth extraction. In vitro experiments were conducted by cell counting Kit-8 (CCK-8) assay, flow cytometry, Western blot, and ELISA to determine cell viability, apoptosis, and inflammatory response. The binding between miR-498 and circ_0062491 or SOCS6 was confirmed using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ_0062491 expression was decreased in periodontitis and LPS-induced PDLCs. Restoration of circ_0062491 attenuated LPS-induced apoptosis and inflammation in PDLCs in vitro. Mechanistically, circ_0062491 functioned as a sponge for miR-498, and miR-498 directly targeted SOCS6. Rescue experiments showed that miR-498 up-regulation reversed the protective action of circ_0062491 on PDLCs under LPS treatment. Moreover, silencing of miR-498 protected PDLCs from LPS-induced apoptosis and inflammation, which were abolished by SOCS6 knockdown. Circ_0062491 protected PDLCs from LPS-induced apoptosis and inflammation, suggesting a new target for the amelioration of periodontitis patients.     

The Pro-Apoptotic and Pro-Inflammatory Effects of Calprotectin on Human Periodontal Ligament Cells

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.     

Fibroblast growth factor‐2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011. © 2010 Wiley‐Liss, Inc.     

Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine

Periodontal ligament (PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support the teeth in situ and preserve tissue homeostasis. Recent studies have revealed the existence of stem cells in human dental tissues including periodontal ligament that play an important role, not only in the maintenance of the periodontium but also in promoting periodontal regeneration. In this study, human periodontal ligament cells (hPDLCs) were isolated by outgrowth and enzymatic dissociation methods. Expression of surface markers on PDLCs as human mesenchymal stem cells (MSCs) was identified by flow cytometry. In addition, proliferation and differentiation capacity of cultured cells to osteoblasts, adipocytes were evaluated. As a result, we successfully cultured cells from the human periodontal ligament tissues. PDLCs express mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 and do not express CD34, CD45, and HLA-DR. PDLCs also possess the multipotential to differentiate into various types of cells, such as osteoblast and adipocytes, in vitro. Therefore, these cells have high potential to serve as materials for tissue engineering, especially dental tissue engineering.     

MicroRNA-543-3p down-regulates inflammation and inhibits periodontitis through KLF6

MicroRNA-543-3p (miR-543-3p) has been reported to be involved in many human disease’s progression, but its role in inflammation is still unclear. After bacterial infection, innate immune cells are activated to trigger inflammation by recognizing lipopolysaccharide (LPS) on the bacterial outer membrane. In our research, it showed that miR-543-3p was down-regulated in LPS-treated periodontal ligament cells (PDLCs). And it mediated the apoptosis of PDLC induced by LPS, which may be involved in periodontitis development. Besides, up-regulation of miR-543-3p alleviated the inflammatory damage induced by LPS. Furthermore, our research demonstrated Kruppel-like factor 6 (KLF6) served as a direct downstream target of miR-543-3p to play a vital role in periodontitis. Simply put, these findings suggest that miR-543-3p could down-regulate inflammation and inhibit periodontitis by targeting KLF6, and it provides a new insight into the molecular mechanism of periodontitis, which may be helpful for the early diagnosis and treatment of this disease.     

Local delivery of simvastatin maintains tooth anchorage during mechanical tooth moving via anti-inflammation property and AMPK/MAPK/NF-kB inhibition

Simvastatin (SMV) could increase tooth anchorage during orthodontic tooth movement (OTM). However, previous studies on its bone-specific anabolic and anti-inflammation properties were based on static in vitro and in vivo conditions. AMPK is a stress-activated kinase that protects tissue against serious damage from overloading inflammation. Rat periodontal ligament cells (PDLCs) were subjected to a serial of SMV concentrations to investigate the optimization that promoted osteogenic differentiation. The PDLCs in static and/or tensile culturing conditions then received the proper concentration SMV. Related factors expression was measured by the protein array, real-time PCR and Western blot. The 0.05UM SMV triggered osteogenic differentiation of PDLCs. The inhibition of AMPK activation through a pharmacological approach (Compound C) caused dramatic decrease in osteogenic/angiogenic gene expression and significant increase in inflammatory NF-κB phosphorylation. In contrast, pharmacological activation of AMPK by AICAR significantly inhibited inflammatory factors expression and activated ERK1/2, P38 MAPK phosphorylation. Moreover, AMPK activation induced by SMV delivery significantly attenuated the osteoclastogenesis and decreased the expression of pro-inflammatory TNF-α and NF-κB in a rodent model of OTM. The current studies suggested that SMV could intrigue intrinsic activation of AMPK in PDLCs that promote attenuate the inflammation which occurred under tensile irritation through AMPK/MAPK/NF-kB Inhibition.     

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase‐activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF‐kB, and their inhibitors diminished both proMMP9‐overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2‐Ets1, p38/HSP27, and PAR2/NF‐kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.     

Analysis of histone deacetylase inhibitor-induced responses in human periodontal ligament fibroblasts

Periodontal ligament (PDL) fibroblasts play critical roles in the regeneration of periodontal tissues damaged by periodontitis. Histone deacetylase inhibitors (HDIs) have been suggested to be potential tools in tissue engineering. The feasibility of using the HDI, sodium butyrate (NaB) for periodontal regeneration was examined by evaluating its effect on the osteogenic differentiation of human PDL fibroblasts and its modulation of the inflammatory responses to lipopolysaccharide (LPS). NaB did not cause significant cell death at 100 μM but promoted the expression of the osteoblast phenotype (Runx2, osterix, osteocalcin, and bone sialoprotein). NaB significantly inhibited the LPS-induced production of reactive oxygen species and the expression of pro-inflammatory cytokines (IL-1β and TNF-α). These results suggest that HDIs can offer a potential therapeutic agent for periodontal regeneration.     

Sodium hydrogen sulfide inhibits nicotine and lipopolysaccharide‐induced osteoclastic differentiation and reversed osteoblastic differentiation in human periodontal ligament cells

Although previous studies have demonstrated that hydrogen sulfide (H₂S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H₂S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H₂S on bone metabolism, we investigated the in vitro effects of H₂S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine‐stimulated human periodontal ligament cells (hPDLCs). The H₂S donor, NaHS, protected hPDLCs from nicotine and LPS‐induced cytotoxicity and recovered nicotine‐ and LPS‐downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in mouse bone marrow cells and blocked nicotine‐ and LPS‐induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M‐CSF, MMP‐9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS‐induced activation of p38, ERK, MKP‐1, PI3K, PKC, and PKC isoenzymes, and NF‐κB. The effects of H₂S on nicotine‐ and LPS‐induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP‐1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H₂S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine‐ and periodontopathogen‐stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases. J. Cell. Biochem. 114: 1183–1193, 2013. © 2012 Wiley Periodicals, Inc.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Tyrosine‐protein phosphatase non‐receptor type 2 inhibits alveolar bone resorption in diabetic periodontitis via dephosphorylating CSF1 receptor

Tyrosine‐protein phosphatase non‐receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25‐Hydroxyvitamin D₃ (25(OH)2D₃) on diabetic periodontitis. 25(OH)2D₃ photo‐affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D₃, which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony‐stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss‐related diseases.     

Nicotinamide‐induced silencing of SIRT1 by miR‐22‐3p increases periodontal ligament stem cell proliferation and differentiation

Dental stem cell proliferation and osteoblast differentiation are key cellular processes involved in periodontitis diseases. Researchers have found that SIRT1 (sirtuin 1, silent mating type information regulation 2 homolog 1) and microRNAs play a pivotal role in the process, but a clear underlying mechanism has not been determined. In this study, the has‐miR‐22‐3p that target SIRT1 was predicted by TargetScan. Luciferase reporter assay was used to confirm that SIRT1 is the direct target of miR‐22‐3p. Importantly, miR‐22‐3p was revealed to control SIRT1 in periodontal ligament stem cell (PDLSC) and to regulate the proliferation and differentiation of PDLSC by SIRT1 silencing. Furthermore, we detected the induction of miR‐22‐3p expression by nicotinamide treatment on PDLSC. Induction of PDLSC proliferation and differentiation by nicotinamide treatment was blocked by miR‐22‐3p knockdown. These results suggested that the effect of nicotinamide on PDLSC is through miR‐22‐3p. In addition, miR‐22‐3p also upregulated the expression levels of the inflammatory cytokines tumor necrosis factor‐α, interleukin‐1β (IL‐1β), and IL‐8 in PDLSC through SIRT1 pathway and downregulated the expression of TLR‐2 and TLR‐4. miR‐22‐3p is a new target either for the treatment of periodontitis or the improvement of inflammation caused by orthodontics.