Signal transduction pathways interrupted by nicotine may lead to delayed wound healing

Abstract

Smokers have delayed healing response subsequent to periodontal therapy. Cell migration and myofibroblast differentiation are necessary for wound contraction. The small G protein, Rac is necessary for cell migration, and TGFβ 1 induces myofibroblast differentiation. We asked if nicotine had effects on signal transduction pathways involved in myofibroblast differentiation and cell migration in human gingival fibroblasts (HGFs). Primary HGFs were cultured to confluence, serum starved for 24 hrs, treated with nicotine (0 or 0.5μM) and wounded in vitro. Live cell assays were used to assess migration rates. Rac activity, p21-activated kinase 1/2 (PAK1/2) and activated p44/42 MAP kinase were assessed in monolayer wound cultures (+/− nicotine). Myofibroblast differentiation was determined in HGFs pretreated with nicotine (0, 0.01, 0.1, and 1 mM) for 2 hours, and then incubated with TGF-β 1 (0, 0.25, 0.5, and 1 ng/ml) (+/−) nicotine for 30 hrs. Alpha-smooth muscle actin (α-SMA) was analyzed by Western blots, immunocytochemistry, and RT- PCR. MAP kinase (p38 MAPK) activity was analyzed by Western blots. Our results demonstrated that nicotine decreased cell migration rates 50% compared to controls and altered the activation patterns of Rac and PAK 1/2 and up-regulated p44/42 MAPK (erk). TGFβ 1 induced an increase of α-SMA protein and mRNA expression, while nicotine (1mM) inhibited the TGFβ 1-induced expression of α-SMA. Nicotine also down-regulated TGFβ 1-induced p38 MAPK activity. In conclusion, nicotine decreased fibroblast cell migration and inhibited myofibroblast differentiation in HGFs in vitro.