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

Abstract

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. 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)-α, interleukin (IL)-1β, and IL-6 messenger RNA (mRNA) and protein levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. ET-1 dose- and time-dependently induced the production of proinflammatory cytokines TNF-α, IL-1β, 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-α, IL-1β, and IL-6 expression in H-hPDL and P-hPDL cells. 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.