The Upregulation of Transglutaminase‐2 by Cyclosporin A in Human Gingival Fibroblasts Is Augmented by Oxidative Stress

Background: Transglutaminase‐2 (TGM‐2) has been implicated in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Hence, the authors hypothesize that cyclosporin A (CsA) may regulate TGM‐2 via ROS, and this regulation may have a role in the pathogenesis of CsA‐induced gingival overgrowth. Methods: Cytotoxicity, 2′,7′‐dichlorodihydrofluorescein diacetate assay, and Western blot were used to investigate the effects of CsA in human gingival fibroblasts (HGFs). In addition, extracellular signal‐regulated kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase inhibitor LY294002, glutathione precursor N‐acetyl‐L‐cysteine (NAC), curcumin, epigallocatechin‐3 gallate (EGCG), and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Concentrations of CsA >500 ng/mL demonstrated cytotoxicity to HGFs (P < 0.05). CsA enhanced the generation of intracellular ROS at concentrations >200 ng/mL (P <0.05). TGM‐2 protein induced by CsA was found in HGFs in a dose‐ and time‐dependent manner (P <0.05). The addition of PD98059, LY294002, NAC, curcumin, EGCG, and SB203580 markedly inhibited TGM‐2 expression induced by CsA (P <0.05). Conclusions: These results demonstrate that CsA significantly upregulates intracellular ROS generation and elevates TGM‐2 expression in HGFs. In addition, TGM‐2 induced by CsA is downregulated by PD98059, LY294002, NAC, curcumin, EGCG, and SB203580.     

Matrix metalloproteinase-3 gene polymorphism in renal transplant patients with gingival overgrowth

Drozdzik A, Kurzawski M, Lener A, Kozak M, Banach J, Drozdzik M. Matrix metalloproteinase‐3 gene polymorphism in renal transplant patients with gingival overgrowth. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01221.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Gingival enlargement frequently occurs in transplant patients receiving immunosuppressive drugs. It was hypothesized that gingival enlargement associated with cyclosporine use results from reduced degradation of extracellular matrix in the gingiva. Matrix metalloproteinase‐3 (MMP‐3) is involved in biodegradation of the extracellular matrix, and its inhibition may contribute to an abnormal accumulation of fibronectin and proteoglycans, which are MMP‐3 substrates. The aim of this study was to investigate whether an association exists between MMP‐3 genotypes and gingival enlargement in kidney transplant patients medicated with cyclosporine A. Material and Methods: Sixty‐four unrelated kidney transplant patients suffering from gingival overgrowth, as well as 111 control transplant patients without gingival overgrowth, were enrolled in the study. Gingival overgrowth was assessed 6 mo after transplantation. During the post‐transplant period all patients were given cyclosporine A as a principal immunosuppressive agent. MMP‐3 polymorphism was determined using a PCR restriction fragment length polymorphism assay. Results: In kidney transplant patients suffering from gingival overgrowth the mean gingival overgrowth score was 1.35 ± 0.57, whereas in control subjects the mean gingival overgrowth score was 0.0. The distribution of MMP‐3‐1178A/*dupA alleles among all kidney transplant patients, as well as in the two study subgroups, did not differ significantly from Hardy–Weinberg equilibrium. The frequency of the MMP‐3‐1171*A/*A genotype (28.1% for gingival overgrowth vs. 26.1% for controls) and of the MMP‐3‐1171*dupA/*dupA genotype (32.8% for gingival overgrowth vs. 22.5% for controls) was similar for both study groups. The risk of gingival overgrowth was lowest among patients carrying the MMP‐3‐1171*A/*dupA genotype (odds ratio 0.52), but this did not differ markedly from the other genotypes. Conclusion: No association between MMP‐3 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A.     

Elevation of collagen type I in fibroblast-keratinocyte cocultures emphasizes the decisive role of fibroblasts in the manifestation of the phenotype of cyclosporin A-induced gingival overgrowth

Background and Objective: Collagen type I elevation in cyclosporin A‐induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo‐like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth‐derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers. Material and methods: Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting. Results: Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture. Conclusion: Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth‐phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth‐derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A‐induced effects under more in vivo‐like conditions.     

Protease‐activated receptor 2 mediates interleukin‐8 and intercellular adhesion molecule‐1 expression in response to Aggregatibacter actinomycetemcomitans

Introduction:  We investigated the mechanisms by which extracts of Aggregatibacter actinomycetemcomitans affect the inflammatory response in gingival epithelial cells. Methods:  Human gingival cells (Ca9‐22) were cultured in bacterial extracts prepared from A. actinomycetemcomitans ATCC 29522. The cells were pretreated with protease inhibitors or transfected with small interfering RNA (siRNA) specific for protease‐activated receptor 2 (PAR‐2). Results:  The pretreatment of cells with serine protease inhibitors significantly inhibited A. actinomycetemcomitans extract‐induced expression of interleukin‐8 (IL‐8) and intercellular adhesion molecule‐1 (ICAM‐1) at both the messenger RNA and protein levels. In addition, A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression was significantly decreased in PAR‐2/siRNA‐transfected cells. Conclusions:  A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression in gingival epithelial cells is mediated by PAR‐2.     

Flutamide inhibits nifedipine‐ and interleukin‐1β‐induced collagen overproduction in gingival fibroblasts

Lu H‐K, Tseng C‐C, Lee Y‐H, Li C‐L, Wang L‐F. Flutamide inhibits nifedipine‐ and interleukin‐1β‐induced collagen overproduction in gingival fibroblasts. J Periodont Res 2010; 45: 451–457. © 2010 John Wiley & Sons A/S Background and Objective: To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin‐1β‐ and nifedipine‐induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. Material and Methods: Gingival fibroblasts from healthy subjects and patients with dihydropyridine‐induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin‐1β or both. The mRNA expression was examined using real‐time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. Results: Interleukin‐1β was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen α1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen α1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin‐1β. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. Conclusion: The data suggest that both nifedipine and interleukin‐1β play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine‐induced overgrowth cells.     

TGF-β1 gene polymorphism in renal transplant patients with and without gingival overgrowth

Oral Diseases (2011) 17 , 414–419 Background: The incidence of gingival overgrowth among renal transplant patients treated with cyclosporine A ranges from 13% to 84.6%, and the overgrowth is not only esthetic but also a medical problem. We studied the determination of association between TGF‐β1 (TGFB1) gene polymorphism and gingival overgrowth in kidney transplant patients medicated with cyclosporin A. Methods: Eighty‐four kidney transplant patients with gingival overgrowth and 140 control transplant patients without overgrowth were enrolled into the case control study. TGFB1 polymorphism was determined using the PCR‐RFLP assay for +869T>C in codon 10 and +915G>C in codon 25 as well as TaqMan real‐time PCR assays for promoter ?800G>A and ?509C>T SNPs. Results: In kidney transplant patients suffering from gingival overgrowth, mean score of gingival overgrowth was 1.38 ± 0.60, whereas in control subjects it was 0.0. The patients with gingival overgrowth were characterized by similar distribution of TGFB1 genotypes and allele in comparison to subjects without gingival overgrowth. Among 16 potentially possible haplotypes of TGFB1 gene, only four were observed in the studied sample of kidney transplant patients: G_C_T_G, G_T_C_G, G_C_C_C, and A_C_T_G, with similar frequency in patients with and without gingival overgrowth. Conclusion: No association between the TGFB1 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A.     

Role of MDR1 gene polymorphisms in gingival overgrowth induced by cyclosporine in transplant patients

Background and Objective: The aim of the present study was to determine the association between genotypes of the MDR1 gene, encoding P‐glycoprotein, and gingival overgrowth in transplant patients treated with cyclosporine, and to evaluate the effect of periodontal treatment in these patients. Material and Methods: Fifty transplant patients receiving therapy with cyclosporine and suffering from gingival overgrowth were subjected to nonsurgical periodontal treatment and received oral hygiene instructions. Hyperplastic index, periodontal probing depths, bleeding and plaque scores were recorded at baseline and after 3 and 6 mo. Patients were dichotomized into two groups: those with a hyperplastic index of < 30% (minimal gingival overgrowth) and those with a hyperplastic index of ≥ 30% (clinically significant gingival overgrowth). MDR1 C3435T and G2677T polymorphisms were evaluated in all patients and in 100 controls. Results: At baseline, 32 patients (64%) had minimal gingival overgrowth and 18 patients (36%) had clinically significant gingival overgrowth. The mutated C3435T genotype was significantly more frequent in the second group (p < 0.019). The significant association between gingival overgrowth and the 3435TT genotype was confirmed by logistic regression analysis (p < 0.031). The differences in hyperplastic index, observed at baseline between patients with the TT genotype and those with the CC/CT genotype disappeared in the second and third evaluation. The mean monthly change of the square root of the gingival overgrowth scores for all patients, assessed using linear models, was significantly different from baseline (?0.17 points per month, p < 0.00001); and this was particularly evident in subjects with renal transplant (?1.62, p < 0.01). Conclusion: Aetiological periodontal and self‐performed maintenance therapy is effective in reducing gingival overgrowth, particularly in subjects with the 3435TT genotype and in patients with renal transplant.     

Interleukin‐6 Family of Cytokines in Crevicular Fluid of Renal Transplant Recipients With and Without Cyclosporine A–Induced Gingival Overgrowth

Background: Interleukin (IL)‐6 family of cytokines, including IL‐6, oncostatin M (OSM), leukemia inhibitory factor (LIF), and IL‐11, have fibrogenic features. The current study determines gingival crevicular fluid (GCF) levels of fibrosis‐related IL‐6–type cytokines in cyclosporine A (CsA)–induced gingival overgrowth (GO). Methods: Eighty non‐smokers were included (40 CsA‐medicated renal transplant patients with GO [GO + ; n = 20] or without GO [GO?; n = 20], 20 individuals with gingivitis, and 20 healthy participants). Probing depth and plaque, papilla bleeding, and hyperplastic index scores were recorded. GCF samples were obtained from the mesio‐buccal aspects of two teeth. GCF IL‐6, IL‐1β, OSM, LIF, and IL‐11 levels were analyzed by enzyme‐linked immunosorbent assay. Results: The GO+ and GO? groups had higher IL‐6 total amounts than the healthy group (P <0.008). IL‐1β total amounts in the GO+ group were significantly higher than in both the healthy and GO? groups (P <0.008). OSM total amount was elevated in the GO+ and GO? groups compared with both the gingivitis and healthy groups (P <0.008). All groups had similar LIF and IL‐11 total amounts (P >0.008). Moderate positive correlations were detected among IL‐6, IL‐1β, OSM, and IL‐11 total amount in GCF and clinical parameters (P <0.05). Conclusions: IL‐6 and OSM increases in GCF as a result of CsA usage or an immunosuppressed state irrespective of the severity of inflammation and the presence of GO. The IL‐6 family of cytokines might not be directly involved in biologic mechanisms associated with CsA‐induced GO. Lack of an association between assessed IL‐6 cytokines and CsA‐induced GO might indicate distinct effects of these cytokines on fibrotic changes of different tissues.     

Human gingival fibroblasts release high‐mobility group box‐1 protein through active and passive pathways

Introduction:  The nuclear protein high‐mobility group box‐1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS‐stimulated HGF. Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time‐dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

Gingival crevicular fluid and serum matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 levels in renal transplant patients undergoing different immunosuppressive therapy

Aim: We investigated gingival crevicular fluid (GCF) and serum matrix metalloproteinase‐8 (MMP‐8) and tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) levels from renal transplant patients receiving cyclosporine‐A (CsA) and having gingival overgrowth (GO), from patients receiving CsA therapy and having no GO and patients receiving tacrolimus therapy. Material and Methods: GCF samples were collected from sites with GO (GO+) and without GO (GO?) in CsA patients having GO; and GO? sites in CsA patients having no GO; sites from tacrolimus, gingivitis and healthy subjects. GCF and serum MMP‐8 and TIMP‐1 levels were determined by a time‐resolved immunofluorometric assay (IFMA) and enzyme‐linked immunosorbent assay. Results: GO+ sites in CsA patients having GO had elevated GCF MMP‐8 levels compared with those of CsA patients having no GO, tacrolimus and healthy subjects (p<0.005), but these levels were similar to those of gingivitis. The GCF MMP‐8 level was higher in GO+ compared with GO? sites in CsA patients having GO (p<0.05). GCF TIMP‐1 levels were similar between groups. Tacrolimus patients had lower GCF MMP‐8 levels than gingivitis (p<0.005), but levels similar to the healthy group. Conclusion: These results show that CsA and tacrolimus therapy has no significant effect on GCF MMP‐8 levels, and gingival inflammation seems to be the main reason for their elevations.     

Molecular events associated with ciclosporin A-induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts

Sobral LM, Aseredo F, Agostini M, Bufalino A, Pereira MCC, Graner E, Coletta RD. Molecular events associated with ciclosporin A‐induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts. J Periodont Res 2012; 47: 149–158. © 2011 John Wiley & Sons A/S Background and Objective: Ciclosporin A (CsA)‐induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor‐β1 (TGF‐β1). Herein, the in vitro investigation of effects of overexpression of Smad7, a TGF‐β1 signaling inhibitor, in the events associated with CsA‐induced extracellular matrix accumulation was performed. Material and Methods: The effects of Smad7 were assessed by stable overexpression of Smad7 in fibroblasts from normal gingiva. Smad7‐overexpressing cells and control cells were incubated with CsA, and synthesis of type I collagen, production and activity of MMP‐2 and cellular proliferation were evaluated by ELISA, zymography, growth curve, bromodeoxyuridine incorporation assay and cell cycle analysis. The effects of CsA on cell viability and apoptosis of fibroblasts from normal gingiva were also evaluated. Western blot and immunofluorescence for phospho‐Smad2 were performed to measure the activation of TGF‐β1 signaling. Results: Although the treatment with CsA stimulated TGF‐β1 production in both control and Smad7‐overexpressing fibroblasts, its signaling was markedly inhibited in Smad7‐overexpressing cells, as revealed by low levels of phospho‐Smad2. In Smad7‐overexpressing cells, the effects of CsA on proliferation, synthesis of type I collagen and the production and activity of MMP‐2 were significantly blocked. Smad7 overexpression blocked CsA‐induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. Conclusion: The data presented here confirm that TGF‐β1 expression is related to the molecular events associated with CsA‐induced gingival overgrowth and suggest that Smad7 overexpression is effective in blocking these events, including proliferation, type I collagen synthesis and MMP‐2 activity.     

Stimulatory effect of Aggregatibacter actinomycetemcomitans DNA on proinflammatory cytokine expression by human gingival fibroblasts

ObjectiveWhile different virulence factors have been reported of Aggregatibacter actinomycetemcomitans (Aa), there is little information about the stimulatory effect of its DNA. The main purpose of this study was to assess the inflammatory response of human gingival fibroblasts (HGFs) stimulated with A. actinomycetemcomitans DNA.DesignCytokine levels of IL-6, IL-1α and TNF-α were measured on the supernatant of HGFs activated with 10, 25, 50 and 100 μg/ml DNA of Aa during 24 h. Primary cultures of HGFs were infected with Aa and its DNA at different times and concentrations to compare its cytotoxic effect. Cell damage and adhesion of Aa to HGFs were evaluated under light microscopy and Scanning electron microscopy respectively.ResultsThere was a statistical difference (p < 0.05) in cytokine expression in HGFs activated by bacterial DNA with a dose dependent on IL-6 expression and a significantly elevated expression of IL-1α and TNF-α compared to Human DNA negative control. Substantial morphological alterations were observed after infection of A. actinomycetemcomitans in HGFs but not with bDNA exposure. Aggregatibacter actinomycetemcomitans showed a high rate of adhesion and cell damage to HGFs after 30 min.ConclusionsGenomic DNA of A. actinomycetemcomitans could be a factor in the pathogenesis of periodontitis that might play a major role in the inflammatory response.     

Levels of Aggregatibacter actinomycetemcomitans,Porphyromonas gingivalis,inflammatory cytokines and species‐specific immunoglobulin G in generalized aggressive and chronic periodontitis

Casarin RCV, Del Peloso Ribeiro É, Mariano FS, Nociti FH Jr, Casati MZ, Gonçalves RB. Levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, inflammatory cytokines and species‐specific immunoglobulin G in generalized aggressive and chronic periodontitis. J Periodont Res 2010; 45: 635–642. © 2010 John Wiley & Sons A/S Background and Objective: Aggressive periodontitis pathogenesis still is not completely understood in the literature regarding the relationship between microbial and inflammatory aspects. So this study aimed to compare microbial and inflammatory patterns in the gingival crevicular fluid of generalized aggressive and chronic periodontitis patients. Material and Methods:  Forty aggressive and 28 chronic periodontitis patients were selected. Biofilm and gingival crevicular fluid were collected from a deep pocket (periodontal probing depth >7 mm) and a moderate pocket (periodontal probing depth = 5 mm) of each patient, and microbiological and immunoenzymatic assays were performed. Real‐time PCR was used to determine quantities of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Enzyme‐linked immunosorbent assay (ELISA) was employed to determine gingival crevicular fluid levels of interleukin‐1β, interferon‐γ, prostaglandin E₂ and interleukin‐10. In addition, immunoglobulin G (IgG) levels against A. actinomycetemcomitans and P. gingivalis lipopolysaccharide were also determined by ELISA. Analysis of variance/Tukey test, Mann–Whitney U‐test and the Pearson correlation test were used to determine differences and correlations between variables analysed (α = 5%). Results:  Patients suffering from generalized aggressive periodontitis had their mouth colonized by higher amounts of A. actinomycetemcomitans and P. gingivalis than chronic periodontitis patients. Conversely, the gingival crevicular fluid levels of IgG against both pathogens were statistically inferior in aggressive periodontitis patients (p < 0.05). With regard to gingival crevicular fluid levels of cytokines, aggressive periodontitis patients presented reduced levels of interleukin‐10 (p < 0.05). Conclusion:  In comparison to chronic periodontitis, generalized aggressive periodontitis patients have an imbalance in the host response, with reduced levels of interleukin‐10 and IgG, and increased periodontal pathogens.     

The pharmacokinetics of phenytoin in gingival crevicular fluid and plasma in relation to gingival overgrowth

Abstract The aim of this study was to determine whether phenytoin (PHT) could be detected in gingival crevicular fluid (GCF), and to relate its concentration to both plasma level and degree of gingival overgrowth. 23 patients medicated with phenytoin for at least 6 months were clinically examined for signs of periodontal disease and gingival overgrowth. 12 patients out of these demonstrated clinically significant overgrowth and their plaque scores and gingival inflammation were greater than for the non-overgrowth group (p<0.001). Phenytoin concentrations were determined by high performance liquid chromatography, and was detected in GCF. There was a significant correlation between the GCF and plasma phenytoin concentrations (p<0.05), but it was not related to the extent of gingival overgrowth. Inflammation increased the GCF volume, but was not a determinant of GCF phenytoin concentration. It is concluded that effusion of phenytoin into GCF is regulated by the plasma levels of the drug, but its concentration in GCF is not related to the incidence of gingival overgrowth.     

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

Lin S‐J, Lu H‐K, Lee H‐W, Chen Y‐C, Li C‐L, Wang L‐F. Nitric oxide inhibits androgen receptor‐mediated collagen production in human gingival fibroblasts. J Periodont Res 2012; 47: 701–710. © 2012 John Wiley & Sons A/S 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 48 h with IL‐1β (10 ng/mL), nifedipine (0.34 μm ) 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.