Endothelin-1 Stimulates Proinflammatory Cytokine Expression in Human Periodontal Ligament Cells via Mitogen-Activated Protein Kinase Pathway

• Localización: Journal of periodontology, ISSN 0022-3492, Nº. 4, 2014, págs. 618-626
• Idioma: inglés
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• Resumen

  • Background: Endothelin-1 (ET-1) is a 21�amino acid peptide with multifunctional regulation. Initial research indicated that ET-1 is related to the inflammatory pathogenesis of periodontitis and involved in the regulation of cytokines, but the mechanisms involved remain unclear. The primary aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression in human periodontal ligament (hPDL) cells.

    Methods: hPDL cells were obtained from both healthy (H)- and periodontitis (P)-affected periodontal tissues. H-hPDL and P-hPDL cells were treated with ET-1 (1, 10, and 100 nM) for 12, 24, and 48 hours. The untreated cells served as a control. To confirm the specificity of the ET-1 effects, 100 nM of the specific endothelin A (ETA) receptor antagonist BQ123 and 100 nM of the specific ETB receptor antagonist BQ788, as negative control, were used. To examine the signaling pathways and molecular mechanisms involved in ET-1�mediated cytokine expression, H-hPDL and P-hPDL cells were pretreated with specific inhibitors for extracellular signal�regulated kinase (ERK1/2) (PD98059), c-Jun N-terminal kinase (SP600125), and p38 kinase (SB203580) for 1 hour before 100 nM ET-1 stimulation. Tumor necrosis factor (TNF)-a, interleukin (IL)-1ß, and IL-6 messenger RNA (mRNA) and protein levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.

    Results: ET-1 dose- and time-dependently induced the production of proinflammatory cytokines TNF-a, IL-1ß, and IL-6 by H-hPDL and P-hPDL cells at both mRNA and protein levels. However, ETA and ETB receptor antagonists inhibited the stimulatory effects of ET-1 on inflammatory cytokine expression in H-hPDL and P-hPDL cells. Furthermore, inhibitors of the mitogen-activated protein kinases (MAPKs) significantly reduced ET-1�stimulated TNF-a, IL-1ß, and IL-6 expression in H-hPDL and P-hPDL cells.

    Conclusion: ET-1 may be involved in the inflammatory process of periodontitis, at least in part, by stimulating proinflammatory cytokine production via the MAPK pathway in hPDL cells.

    Endothelin-1 (ET-1) is a 21�amino acid peptide originally isolated from the culture supernatant of porcine aortic endothelial cell.1,2 ET-1 has multiple biologic actions and is produced and secreted by various tissues and cells.1,3 ET-1 stimulates neutrophils to release elastase, activates mast cells, and stimulates monocytes to produce a variety of cytokines such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-a, which play important roles in the initiation and progression of periodontal disease.4-6 Periodontitis is one of the major infectious diseases, with inflammation that extends deep into the tissues and damages both soft tissues and alveolar bone.7,8 It has recently been found that Porphyromonas gingivalis, one of the major pathogens responsible for causing periodontitis,9 stimulates the expression of ET-1, with upregulation of proinflammatory cytokines and intercellular adhesion molecule 1, in the bronchial epithelial cell line HEp-2 and KB cells.4,10 The expression of ET-1 was strongly enhanced in gingival tissue and endothelial cells during periodontal inflammation.10 Recently, studies found that the ET-1 level in gingival crevicular fluid increased with the progression of periodontal disease,11 and ET-1 was involved in the regulation of IL-1ß expression in gingival tissues.12 Therefore, ET-1 is possibly related to the inflammatory pathogenesis of periodontitis and may be involved in the regulation of cytokines. Nevertheless, little is known about the pathophysiologic role of ET-1 in periodontitis.

    The periodontal ligament (PDL) is a special connective tissue located between the alveolar bone and the tooth cementum. Cells of this ligament play an important role in maintaining the homeostasis of periodontal tissues. Previous studies showed that PDL cells play a role as producers of cytokines and chemokines in periodontal inflammation.13-15 Human PDL (hPDL) cells have been reported to express messenger RNA (mRNA) for endothelins and their receptors.12 However, how ET-1 affects proinflammatory cytokine expression in hPDL cells remains unclear.

    Mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases and play an important role in the intracellular signaling that occurs in response to extracellular stimuli. MAPKs are divided into three subclasses: extracellular signal�regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 kinase.16 ERK1/2 is particularly activated by mitogens and growth factors acting through receptor protein tyrosine kinases17 or Gq protein-coupled receptor agonists such as ET-1.18 JNK and p38 are potently activated by cellular stresses, including oxidative stress and proinflammatory cytokines such as IL-1ß and TNF-a.19-21 The MAPKs control the activity of many transcription factors. ERK1/2 is particularly associated with cell proliferation.22,23 JNK and p38 are associated with the immune response,21 and p38 probably propagates an inflammatory response by, at least in part, increasing production of cytokines, including IL-1ß and TNF-a.24 The authors hypothesized that MAPKs might be involved in ET-1�induced proinflammatory cytokine expression. Therefore, the present study is designed to investigate the effects of ET-1 on the expression of proinflammatory cytokines such as TNF-a, IL-1ß, and IL-6 in hPDL cells and put emphasis on exploring the possible roles of MAPK pathways in ET-1�induced cytokine expression.