Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts

Background and Objective: In our previous study, we found that flutamide [an androgen receptor (AR) antagonist] inhibited the up-regulation of collagen induced by interleukin (IL)-1β and/or nifedipine in gingival fibroblasts. The present study attempted to verify the role of nitric oxide (NO) in the IL-1β/nifedipine-AR pathway in gingival overgrowth. Material and Methods: Confluent gingival fibroblasts derived from healthy individuals (n=4) and those with dihydropyridine-induced gingival overgrowth (DIGO) (n=6) were stimulated for 48h with IL-1β (10ng/mL), nifedipine (0.34μm) or IL-1β + nifedipine. Gene and protein expression were analyzed with real-time RT-PCR and western blot analyses, respectively. Meanwhile, Sircol dye-binding and the Griess reagent were, respectively, used to detect the concentrations of total soluble collagen and nitrite in the medium. Results: IL-1β and nifedipine simultaneously up-regulated the expression of the AR and type-I collagen α1 [Colα1(I)] genes and the total collagen concentration in DIGO cells (p<0.05). IL-1β strongly increased the expression of inducible nitric oxide synthase (iNOS) mRNA and the nitrite concentration in both healthy and DIGO cells (p<0.05). However, co-administration of IL-1β and nifedipine largely abrogated the expression of iNOS mRNA and the nitrite concentration with the same treatment. Spearman's correlation coefficients revealed a positive correlation between the AR and total collagen (p<0.001), but they both showed a negative correlation with iNOS expression and the NO concentration (p<0.001). The iNOS inhibitor, 1400W, enhanced IL-1β-induced AR expression; furthermore, the NO donor, NONOate, diminished the expression of the AR to a similar extent in gingival fibroblasts derived from both healthy patients and DIGO patients (p<0.05). Conclusion: IL-1β-induced NO attenuated AR-mediated collagen production in human gingival fibroblasts. The iNOS/NO system down-regulated the axis of AR/Colα1(I) mRNA expression and the production of AR/total collagen proteins by DIGO cells.