Endothelin Regulates Porphyromonas gingivalis-Induced Production of Inflammatory Cytokines

Abstract

Periodontitis is a very common oral inflammatory disease that results in the destruction of supporting connective and osseous tissues of the teeth. Although the exact etiology is still unclear, Gram-negative bacteria, especially Porphyromonas gingivalis in subgingival pockets are thought to be one of the major etiologic agents of periodontitis. Endothelin (ET) is a family of three 21-amino acid peptides, ET-1, -2, and -3, that activate G protein-coupled receptors, ETA and ETB. Endothelin is involved in the occurrence and progression of various inflammatory diseases. Previous reports have shown that ET-1 and its receptors, ETA and ETB are expressed in the periodontal tissues and, that ET-1 levels in gingival crevicular fluid are increased in periodontitis patients. Moreover, P. gingivalis infection has been shown to induce the production of ET-1 along with other inflammatory cytokines. Despite these studies, however, the functional significance of endothelin in periodontitis is still largely unknown. In this study, we explored the cellular and molecular mechanisms of ET-1 action in periodontitis using human gingival epithelial cells (HGECs). ET-1 and ETA, but not ETB, were abundantly expressed in HGECs. Stimulation of HGECs with P. gingivalis or P. gingivalis lipopolysaccharide increased the expression of ET-1 and ETA suggesting the activation of the endothelin signaling pathway. Production of inflammatory cytokines, IL-1β, TNFα, and IL-6, was significantly enhanced by exogenous ET-1 treatment, and this effect depended on the mitogen-activated protein kinases via intracellular Ca2+ increase, which resulted from the activation of the phospholipase C/inositol 1,4,5-trisphosphate pathway. The inhibition of the endothelin receptor-mediated signaling pathway with the dual receptor inhibitor, bosentan, partially ameliorated alveolar bone loss and immune cell infiltration. These results suggest that endothelin plays an important role in P. gingivalis-mediated periodontitis. Thus, endothelin antagonism may be a potential therapeutic approach for periodontitis treatment.

Fig 1. Endothelin expression in human gingival epithelial cells (HGECs) following P. gingivalis infection.

(A and B) Expression of ET-1 mRNA (A), and ET_A and ET_B protein (B) in HGECs was analyzed by RT-PCR and western blot, respectively. (C and D) Induction of ET-1 peptide and ET_A protein by P. gingivalis and P. gingivalis LPS. After 18h infection with P. gingivalis (100 MOI) and 24h treatment with P. gingivalis LPS (1 μg/ml), ET-1 peptide and ET_A protein levels were analyzed by ELISA and western blot, respectively. ET-1 levels were measured in culture supernatants from five samples (sample #1–5). A representative blot from sample #1 is shown. *P<0.05; **P<0.01. (E and F) Involvement of TLR in ET-1 production. After 24h treatment with HKLM (10⁸ cells/ml), or Pam3 (50 ng/ml) (E), or Pg LPS-ulp (1 μg/ml) or E. coli LPS (50 ng/ml) (F), ET-1 production was evaluated by ELISA in culture supernatants from five samples (sample #1–5). *P<0.05; **P<0.01.

Fig 2. Effect of endothelin inhibitors on the production of inflammatory cytokines in HGECs.

(A to C) Reduced production of IL-1β (A), TNFα (B), and IL-6 (C) after treatment with endothelin receptor antagonists and P. gingivalis. After treatment with BQ123 (100 nM), BQ788 (100 nM), or bosentan (100 nM) for 1h, cells were infected with P. gingivalis (100 MOI) for 24h. (D to F) Reduced production of IL-1β (D), TNFα (E), and IL-6 (F) after treatment with endothelin receptor antagonists and P. gingivalis LPS (1 μg/ml). After treatment with BQ123 (100 nM), BQ788 (100 nM), or bosentan (100 nM) for 1h, cells were treated with P. gingivalis LPS (1 μg/ml) for 24h. (G to I) Reduced production of IL-1β (G), TNFα (H), and IL-6 (I) after treatment with endothelin receptor antagonists and ET-1. After pre-treatment with BQ123 (100 nM), BQ788 (100 nM), and bosentan (100 nM) for 1h, cells were treated with ET-1 (100 nM) for 24h. Secretion of cytokines or ET-1 was examined by ELISA in the culture supernatants from five samples (sample #1–5). *P<0.05; **P<0.01; ***P<0.001.

Fig 3. Involvement of the Ca²⁺/PLC/IP₃ pathway in endothelin-mediated inflammatory cytokine production.

(A and B) ET-1-induced increase in intracellular Ca²⁺ concentration [Ca²⁺]_i. The fura-2/AM- stained cells were treated with ET-1 and BQ123 (100 nM) (A) or bosentan (100 nM) (B). Change of [Ca²⁺]_i was measured through fluorescence intensity at excitation wavelengths of 340 and 380 nm. (C to E) Effect of BAPTA-AM on ET-1-mediated production of inflammatory cytokines. After pre-treatment with BAPTA-AM (5 μM) for 20 min, HGECs were treated with ET-1 (100 nM) for 24h and production of IL-1β (F), TNFα (G), and IL-6 (H) was measured by ELISA in culture supernatants from five samples (sample #1–5). *P<0.05; **P<0.01. (F to H) Involvement of the PLC/IP₃ pathway in the ET-1-induced production of inflammatory cytokines. After pre-treatment with U73122 (10 μM), U73342 (10 μM), or 2-APB (75 μM) for 30 min, HGECs were treated with ET-1 (100 nM) for 24h and production of IL-1β (C), TNFα (D), and IL-6 (E) was measured by ELISA in culture supernatants from five samples (sample #1–5). *P<0.05; **P<0.01.

Fig 4. Involvement of MAPK in endothelin-mediated inflammatory cytokine production.

(A to C) Effect of MAPK inhibitors on the ET-1-mediated mRNA expression of inflammatory cytokines. After pre-treatment with PD98059 (10 μM), SP600125 (10 μM), and SB203580 (10 μM) for 20 min, HGECs were treated with ET-1 (100 nM) for 24h and mRNA expression of IL-1β (F), TNFα (G), and IL-6 (H) was measured by quantitative RT-PCR. *P<0.05; **P<0.01; **P<0.001. (D to F) Effect of MAPK inhibitors on the ET-1-mediated production of inflammatory cytokines. After pre-treatment with PD98059 (10 μM), SP600125 (10 μM) and SB203580 (10 μM) for 20 min, HGECs were treated with ET-1 (100 nM) for 24h and production of IL-1β (F), TNFα (G), and IL-6 (H) was measured by ELISA in culture supernatants from five samples (sample #1–5). *P<0.05; **P<0.01; ns: non-significant.

Fig 5. Effect of endothelin inhibition on ligature-induced periodontitis.

(A) Experimental design and time schedule. Ligature was placed on day 0, and bosentan was treated 1 day before ligature ligation (-1 day). On days 3 and 9 after ligature ligation, the mandibles were dissected and histomorphometric and immunohistochemical analyses were performed. (B and C) Effect of endothelin inhibition on alveolar bone loss on days 3 and 9. ABC-CEJ distances measured by histomorphometric analysis (B) are shown along with representative image (C). *P<0.05; ***P<0.001; ns: non-significant. (D and E) Effect of endothelin inhibition on the number of inflammatory cells in the gingiva on days 3 and 9. Number of inflammatory cells (D) measured by immunohistochemical analysis are shown along with the representative image (E). **P<0.01; ***P<0.001. Scale bar = 100 μm; original magnification ×20.