Lysophosphatidic acid (LPA) is a lipid messenger mediated by G protein-coupled receptors (LPAR1-6). It is involved in the pathogenesis of certain chronic inflammatory and autoimmune diseases. In addition, it controls the self-renewal and differentiation of stem cells. Recent research has demonstrated the close relationship between periodontitis and various diseases in the human body. However, the precise role of LPA in the development of periodontitis has not been studied. We identified that LPAR1 was highly expressed in human periodontal ligament stem cells (PDLSCs). In periodontitis-mimicking conditions with Porphyromonas gingivalis -derived lipopolysaccharide (Pg-LPS) treatment, PDLSCs exhibited a considerable reduction in the cellular viability and osteogenic differentiation potential, in addition to an increase in the inflammatory responses including tumor necrosis factor-α and interleukin-1β expression and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Of the various LPAR antagonists, pre-treatment with AM095, an LPAR1 inhibitor, showed a positive effect on the restoration of cellular viability and osteogenic differentiation, accompanied by a decrease in NF-κB signaling, and action against Pg-LPS. These findings suggest that the modulation of LPAR1 activity will assist in checking the progression of periodontitis and in its treatment.

Human β- defensin-2 (hBD-2) is an antimicrobial peptide which is produced by epithelial cells after stimulation with microorganisms or inflammatory mediators. However, little is known as to whether the LPS and nicotine induces the expression of hBD-2 in periodontal l igament (PDL) cells. T he p urpose o f this s tudy was t o investigate t he r oles o f MAPKs pathway o f nicotine a nd L PS-induced hBD-2 expression in PDL cells. The maximal expression of hBD-2 was observed in nicotine 5 mM and LPS 1 μg/ml treated PDL cells. Nicotine and LPS induced the phosphorylation of p38, c-Jun N-terminal kinase 1 and 2 (JNK1/2), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) MAPK. ERK1/2 inhibitor SB203580, p38 inhibitor PD98059, and JNK inhibitor SP600125, blocked the effects of nicotine and LPS on hBD-2 upregulation in PDL cells. These results collectively suggest that hBD-2 is up-regulated in nicotine and LPS-treated PDL cells through activation of p38, ERK and JNK MAPKs pathway. Our data regarding the up-regulation of hBD-2 may help us to understand the antimicrobial mechanism in periodontal disease

Periodontal ligament (PDL) tissue is a connective tissue that is interposed between the roots of the teeth and the inner wall of the alveolar bone socket. PDL is always exposed to physiologic mechanical force such as masticatory force and PDL cells play important roles during orthodontic tooth movement by synthesizing and secreting different mediators involved in bone remodeling. The Wnt/β-catenin signaling pathway was recently shown to play a significant role in the control of bone formation. In the present study, we applied cyclic tensile stress of 20% elongation to cultured human PDL cells and assessed its impact after six days upon components of the Wnt/β-catenin signaling pathway. RTPCR analysis showed that Wnt1a, Wnt3a, Wnt10b and the Wnt receptor LRP5 were down-regulated, whereas the Wnt inhibitor DKK1 was up-regulated in response to these stress conditions. In contrast, little change was detected in the mRNA expression of Wnt5a, Wnt7b, Fz1, and LRP6. By western blotting we found decreased expression of the β-catenin and p-GSK-3β proteins. Our results thus show that mechanical stress suppresses the canonical Wnt/β-catenin signaling pathway in PDL cells.

Substance P (SP) is known to be expressed in the nerve fibers of dental pulp and periodontal tissues. It was recently reported that SP expression increased in response to orthodontic force. In the present study, we investigated the effect of SP on expression of mineralization markers and heme oxygenase-1 (HO-1) in human immortalized periodontal ligament (IPDL) cells. Cell viability was measured using a 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of mineralization markers, including alkaline phosphatase (ALP), osteonectin (ON) and bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. SP did not significantly change human IPDL cell viability, with the exception of the 24 hour treatment group. Treatment of human IPDL cells with 10-10 to 10-⁴M SP upregulated mineralization marker and HO-1 expression in a time- and concentration-dependent manner. Our results suggest that SP may modulate osteoblastic cell differentiation of human IPDL cells through a mechanism involving HO-1 expression.

Although substance P (SP). a potent pro-inflammatory peptide, is involved in inflammation and immune responses, the effect of SP 011 the expression of macl'ophage inJlammatol'Y protein 3a (MIP-3a. CCL20) in periodontal ligament (PDL) cells a l'e unknown Equally as enigmatic is the link between SP. the stress protein heme oxygenase-l (HO-l) , and CCL20 product ion. We investigated whether SP induces the release of chemokine CCL20 from irrunortalized POL (IPDL) cells. and further claif’y SP mediated pathways . We also exarnined the relationship between HO-l and CCL20 by treating POL cells with SP Incubating IPOL cells with SP incl'eased ex pl'ession of CCL20 mRNA and CCL20 protein in a dose-time dependent manner. Highly selective p38 and ERKl/2 inhibitors abl'ogated SP-induced expression of CCL20 lD IPOL cells SP is also responsible fo l' ini tiating phosphorylation of I/( B‘ degl'adation of IK B. and activation of NF-/( B. SP induced expression of HO-l in both a concentration- and time-dependent manner. and CCL20 refl ected similal' patterns. The inductive effects of SP on HO-l and CCL20 were enhanced by HO- l inducer hemin and the membrane-permea ble cGMP analog 8-bromo-cGMP Conversely, this pathway was inhi bited by the HO-l inhibitor zinc Pl'otoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase‘ 1H- [1. 2. 4]uxad iazole[4, 3-alquinoxal i n- 1-one (ODQ) We report hel'ein the pathway that connects SP a long with other modulators 0 1' neuroimmunoregulationto the induction of HO-1 and the inflanunatol'y mediatol' MIP- 3a /CCL20 in IPDL cel ls. which play an impol'tant role in the development 0 1' pe- I'iodontitis or inflammation during ol'thodontic tooth movement

Al t hough substance P(SP) , a potent pro- inflammatory peptide, is involved in inflammation and immune responses‘ t he eff'ect of SP on t he expression of macrophage inflammatory protein 3a (MIP- 3α CCL20) in periodontal liga ment(PDL) cell s a re unknown, Equally as enigmatic is the link between SP, t he stress protein heme oxygenase- l(HO-l) ‘ and CCL20 procluction, We investigated whether SP induces the release of chemokine CCL20 from immortal ized PDL(IPDL) ceJJ s‘ and fur ther c l a꺼 SP mediated pathways, We also examined the relationship between HO-l a ncl CCL20 by t reating PDL cells with SP, Incubating IPDL cells with SP increased expression of CCL20 mRNA a nd CCL20 protein in a dose-time dependent manner Highly selective p38 and ERKl/2 inhibitors abrogated SP-induced expression of CCL20 in IPDL cell s, SP is a lso responsible for ini t iating phosphorylation of I/C B, degradation of Iκ B‘ ancl activat ion of NF'-/C B, SP induced expression of HO-l in both a concentration- and time-dependent man nel ‘ and CCL20 refl ected s imilar patterns, The inductive effects o[ SP on HO- l and CCL20 wer e enhanced by HO- j inducer hemin and the membrane-permeable cGMP analog 8-bromo-cGMP, Conversely, this pathway was inJübited by t he 1-10난 inhi bitor zinc protoporphyrin IX(ZnPP IX) and the selective inl뼈itor of guanylate cyc1ase‘ lH-[l , 2, 4Joxad iazole[4‘ 3-aJquinoxal in-l-one (ODQ) , We report herein the pathway that connects SP along with other modulators 。f neuroimmunoregulationto the induction of HO-l and t he inflammatory mediator MIP-3a /CCL20 in IPDL cell s‘ which play an important role in the development 01' periodontitis or inflamrnation during orthodontic tooth movem