Messenger RNA (mRNA) expression of nAChR subunits in three different HGEC lines (epi 4, Tfx and E6E7) was assessed using reverse transcription-polymerase chain reaction (RT-PCR). HGECs were stimulated by 1 ¡Á 10? M nicotine in the presence or absence of IL-1¦Â or Porphyromonas gingivalis lipopolysaccharide (LPS). IL-8 production was then examined using real-time PCR and enzyme-linked immunosorbent assay. Nicotine-mediated signalling in the epi 4 cell line was also evaluated by Western blotting.
HGECs expressed several nAChR subunits. Nicotine increased the secretion of IL-8 from HGECs that were cultured in the presence of IL-1¦Â or P. gingivalis LPS and also induced the phosphorylation of extracellular signal-regulated kinase (ERK) in epi 4. Pretreatment with non-selective nAChR antagonist or intracellular calcium chelator reduced the nicotine-induced phosphorylation of ERK. Furthermore, nicotine-induced IL-8 secretion was decreased by pretreatment with non-selective nAChR antagonist, ERK1/2 inhibitor or intracellular calcium chelator.
These findings indicate that nicotine increases IL-8 production in gingival epithelial cells via ERK phosphorylation following Ca2+ signalling after nAChR activation.