ABSTRACT
: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.
Subject(s)
Humans , Fibroblasts , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Phosphorylation , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression types of protease-activated receptors(PAR) in human gingival fibroblasts(HGF) and the functions of PAR in periodontitis.</p><p><b>METHODS</b>Primary HGF were cultured.Reverse transcription PCR(RT-PCR) was used to detect the expression of PAR in HGF. Recombinant gingipain R (rRgp) was applied to HGF. The change of PAR expression on the cell surface was analyzed by real-time quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) was used to detect the change of the interleukin (IL)-6 production from HGF. The results of RT-PCR and ELISA were statistically analyzed using the two independent samples t-test of SPSS10.0 software.</p><p><b>RESULTS</b>HGF expressed PAR-1 and PAR-3. The expression of PAR-1 and PAR-3 changed after two rRgp treatment with HGF cells. The relative expression of PAR-1 was decreased from 1.04 ± 0.31 to 0.67 ± 0.11 and 0.31 ± 0.11. The relative expression of PAR-3 was decreased from 1.01 ± 0.44 to 0.79 ± 0.13 and 0.44 ± 0.12(P < 0.05). The level of IL-6 was increased after rRgp treatment for 8 h. The control group was (18.77 ± 4.09) µg/L, the rRgp treatment groups were (179.36 ± 15.81) and (320.56 ± 26.19) µg/L respectively.</p><p><b>CONCLUSIONS</b>HGF expressed PAR-1 and PAR-3 and were involved in periodontal inflammation.</p>
Subject(s)
Humans , Adhesins, Bacterial , Cell Membrane , Cysteine Endopeptidases , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Gingiva , Metabolism , Interleukin-6 , Periodontitis , Metabolism , Receptors, Proteinase-ActivatedABSTRACT
Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been shown to differ from enterobacterial LPS in structure and function; therefore, the Toll-like receptors (TLRs) and the intracellular inflammatory signaling pathways are accordingly different. To elucidate the signal transduction pathway of P. gingivalis, LPS-induced pro-inflammatory cytokine production in the human monocytic cell line THP-1 was measured by ELISA, and the TLRs were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors as well as Phospho-ELISA kits were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. In this study, P. gingivalis LPS showed the ability to induce cytokine production in THP-1 cells and its induction was significantly (P < 0.05) suppressed by anti-TLR2 antibody or JNK inhibitor, and the phosphorylation level of JNK was significantly increased (P < 0.05). These results indicate that TLR2-JNK is the main signaling pathway of P. gingivalis LPS-induced cytokine production, while the cytokine induction by E. coli LPS was mainly via TLR4-NF-kappaB and TLR4-p38MAPK. This suggests that P. gingivalis LPS differs from E. coli LPS in its signaling pathway in THP-1 cells, and that the TLR2-JNK pathway might play a significant role in P. gingivalis LPS-induced chronic inflammatory periodontal disease.
Subject(s)
Interleukin-1beta/metabolism , Interleukin-6/metabolism , Lipopolysaccharides/immunology , Monocytes/immunology , Porphyromonas gingivalis/immunology , Tumor Necrosis Factor-alpha/metabolism , Anthracenes/pharmacology , Cell Line , Enzyme Inhibitors/pharmacology , Escherichia coli/immunology , Humans , Imidazoles/pharmacology , Lipopolysaccharides/isolation & purification , MAP Kinase Kinase 4/antagonists & inhibitors , MAP Kinase Kinase 4/immunology , MAP Kinase Kinase 4/metabolism , Monocytes/drug effects , Monocytes/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/immunology , NF-kappa B/metabolism , Peptides/pharmacology , Pyridines/pharmacology , Toll-Like Receptor 2/immunology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/immunology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Objective To determine the diversity of Porphytomonas gingivalis lipopolysaccharide (LPS)induced IL-1β,TNF-αand IL-6 levels in THP-1 cells and the associated Toll-like receptors(TLR).Methods P.gingivalis strain ATCC33277 LPS (Pg-LPS)was prepared using phenol-water method and then identified by both infrared spectrometry and limulus test.The levels of IL-1β,TNF-α and IL-6 secreted by THP-1 cells under inducement of Pg-LPS were quantitatively detected using commercial ELISA kits.The bloc-king test using TLR2 or TLR4 monoelonal antibody(McAb)plus the ELISA were used to determine the types of Pg-LPS binding TLR on the surface of target cells.In this study.a commercial LPS from E.coli strain O111:B4(E-LPS)was used as the contr01.Results When 1彬ml ofPg.u,s induced THP.1 ceHs for0.5,6 and 6 h.or l∥rnl ofE-LPs induced for 6,24 and 24 h,respectively,tIIe levels ofTNF.a,IL-1B and IL广6 were in.creased obviously(P<0.01).However,the maximal concentration of tlle t11ree cytokines induced by Pg.LP$ were similar to that induced by E-LPS(P>0.05).tLR2 or TLR4 McAb could block the effects of Pg-LPS in-ducing THP-1 cells to secrete IL-1β and IL-6(P<0.05),where as only TLR2 McAb displayed the inhibition of TNF-α secretion(P<0.05).On the contrast,only TLR4 McAb showed the effects blocking the three cytokines secretion in the THP-1 cells under inducement of E-LPS(P<0.05).Conclusion Pg-LPS shows a slight high-er activity inducing THP-1 cells to secrete IL-1β,TNF-α and IL-6 than E-LPS.TLR2,but not TLR4,is the major receptor of Pg-LPS on the target cells to mediate the secretion of the three cytokines.
