Smad7 blocks transforming growth factor-β1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor

Background: Transforming growth factor‐β1 (TGF‐β1), its downstream signaling mediators (Smad proteins), and specific targets, including connective tissue growth factor (CTGF), play important roles in tissue remodeling and fibrosis via myofibroblast activation. We investigated the effect of overexpression of Smad7, a TGF‐β1 signaling inhibitor, on transition of gingival fibroblast to myofibroblast. Moreover, we analyzed the participation of CTGF on TGF‐β1–mediated myofibroblast transformation. Methods: To study the inhibitory effect of Smad7 on TGF‐β1/CTGF‐mediating gingival fibroblast transition into myofibroblasts, we stably overexpressed Smad7 in normal gingival fibroblasts and in myofibroblasts from hereditary gingival fibromatosis (HGF). Myofibroblasts were characterized by the expression of the specific marker isoform α of the smooth muscle actin (α‐SMA) by Western blot, flow cytometry, and immunofluorescence. Enzyme‐linked immunosorbent assay for type I collagen was performed to measure myofibroblast activity. CTGF's role on myofibroblast transformation was examined by enzyme‐linked immunosorbent assay and small interference RNA. Results: TGF‐β1 induced the expression of α‐SMA and CTGF, and small interference RNA–mediating CTGF silencing prevented fibroblast‐myofibroblast switch induced by TGF‐β1. In Smad7‐overexpressing fibroblasts, ablation of TGF‐β1–induced Smad2 phosphorylation marked decreased α‐SMA, CTGF, and type I collagen expression. Similarly, HGF transfectants overexpressing Smad7 demonstrated low levels of α‐SMA and phospho‐Smad2 and significant reduction on CTGF and type I collagen production. Conclusions: CTGF is critical for TGF‐β1–induced gingival fibroblast‐myofibroblast transition, and Smad7 overexpression is effective in the blockage of myofibroblast transformation and activation, suggesting that treatments targeting myofibroblasts by Smad7 overexpression may be clinically effective in gingival fibrotic diseases, such as HGF.     

Tumor Necrosis Factor‐α Inhibits Transforming Growth Factor‐β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor‐β1 (TGF‐β1) and tumor necrosis factor‐α (TNF‐α) modulate myofibroblastic differentiation and the production of ECM components. Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF‐β1 and TNF‐α. Protein levels of α‐smooth muscle actin (α‐SMA), type I collagen, heat shock protein‐47 (HSP‐47), fibronectin (FN), ED‐A‐FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α‐SMA and actin fibers were identified by immunofluorescence. TGF‐β1, TNF‐α, and α‐SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF‐β1 activity was evaluated using a plasminogen activator inhibitor‐1 (PAI‐1) reporter transfected in HGFs. Results: TGF‐β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α‐SMA and ED‐A‐FN. Moreover, TGF‐β1 induced the production of type I collagen, HSP‐47, FN, and periostin. Costimulation with TNF‐α and TGF‐β1 significantly reduced the expression of all the above‐mentioned proteins. TNF‐α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI‐1 reporter. Conclusions: TNF‐α inhibits several cell responses induced by TGF‐β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.     

Role of Transforming Growth Factor‐beta1 in Cyclosporine‐Induced Epithelial‐to‐Mesenchymal Transition in Gingival Epithelium

Background: It has been proposed that cyclosporin A (CsA) may induce epithelial‐to‐mesenchymal transition (EMT) in gingiva. The aims of the present study are to confirm the notion that EMT occurs in human gingival epithelial (hGE) cells after CsA treatment and to investigate the role of transforming growth factor beta1 (TGF‐β1) on this CsA‐induced EMT. Methods: The effects of CsA, with and without TGF‐β1 inhibitor, on the morphologic changes of primary culture of hGE cells were examined in vitro. The changes of protein and messenger RNA (mRNA) expressions of two EMT markers (E‐cadherin and alpha‐smooth muscle actin) in the hGE cells after CsA treatment with and without TGF‐β1 inhibitor were evaluated with immunocytochemistry and real‐time polymerase chain reaction. Results: The epithelial cells became spindle‐like, elongated, and disassociated from neighboring cells and lost their original cobblestone monolayer pattern when CsA was added. However, the epithelial cells stayed in their original cobblestone morphology with treatment of TGF‐β1 inhibitor on top of the CsA treatment. When CsA was given, the protein and mRNA expressions of E‐cadherin and α‐SMA were significantly altered, and these alterations were significantly reversed with pretreatment of TGF‐β1 inhibitor. Conclusions: CsA could induce Type 2 EMT in gingiva by changing the morphology of epithelial cells and altering the EMT markers/effectors. The CsA‐induced gingival EMT is dependent or at least partially dependent on TGF‐β1.     

Gingival fibroblast response to cyclosporin A and transforming growth factor β1

This study investigates a potential role for TGFβ₁ in the pathogenesis of cyclosporin A-induced gingival overgrowth (CsA-OG). TGFβ₁ was localized immunohistochemically in the connective tissue of both normal gingiva and CsA-OG. Intense staining for TGFβ₁ was detected at the tips of the dermal papillae of the overgrown gingiva. In addition, fibroblasts derived from healthy gingiva and fibroblasts derived from CsA-OG were cultured both as monolayers or embedded in a 3D-collagen gel. Fibroblast activity was monitored in terms of protein and collagen production in the presence of (i) 1 ng/ml TGFβ₁ (ii) 500 ng/ml CsA, or (iii) 500 ng/ml CsA and 1 ng/ml TGFβ₁. In monolayer culture TGFβ₁ significantly increased protein and collagen production in all cell strains (p<0.05); however, there was no difference in response between fibroblasts from overgrown and healthy tissue. The production of both protein and collagen was significantly lower in the presence of the combination of CsA and TGFβ₁ when compared with the maximal stimulation produced by TGFβ₁ alone. In gel, TGFβ₁ significantly elevated matrix production by all overgrown cell strains (p<0.05) but had little or no effect on the normal cell strains. The combination of CsA and TGFβ₁ in gel cultures reduced protein and collagen production by overgrown cell strains compared with TGFβ¹ alone. It is concluded that the cellular activity of gingival fibroblasts is dependant on culture conditions and that fibroblasts derived from overgrown gingival tissue are more responsive to TGFβ₁ than normal gingival fibroblasts when cultured in type I collagen gel.     

姜黄素对环孢素A诱导激活的大鼠牙龈成纤维细胞TGF-β1/Smad3通路的抑制作用

目的：体外实验研究姜黄素（curcumin,Cur）对环孢素A（cyclosporine A,CsA）作用大鼠牙龈成纤维细胞TGF-β1/Smad3通路的影响,为进一步探讨Cur抑制CsA所致药物性牙龈增生的发生机制提供理论依据。方法：使用CCK-8法观察0、5、10、20、30 μmol/L Cur对大鼠牙龈成纤维细胞增殖的影响, 20 μmol/L Cur+200 ng/mL CsA共同作用对大鼠牙龈成纤维细胞增殖的影响。实时荧光定量PCR检测20 μmol/L Cur+200 ng/mL CsA共同作用下,牙龈成纤维细胞中转化生长因子TGF-β1、Smad3、平滑肌肌动蛋白α-SMA和I型胶原蛋白COL-I的mRNA表达变化;蛋白免疫印迹实验检测TGF-β1、Smad3、p-Smad3、α-SMA和COL-I的蛋白表达变化。细胞划痕实验观察20 μmol/L Cur+200 ng/mL CsA 对牙龈成纤维细胞迁移能力的影响。采用SPSS 23.0 软件包对数据进行统计学分析。结果：大鼠牙龈成纤维细胞在20 μmol/L Cur+200 ng/mL CsA共同作用下,细胞增殖和迁移能力明显降低;20 μmol/L Cur显著下调了牙龈成纤维细胞中TGF-β1、α-SMA 和COL-I的mRNA 表达,蛋白免疫印迹实验提示,TGF-β1、p-Smad3、α-SMA 和COL-I的表达同样显著下调。结论：Cur可能通过抑制CsA激活的牙龈成纤维细胞TGF-β1/Smad3 信号通路,从而降低牙龈成纤维细胞增殖、迁移、平滑肌肌动蛋白和胶原分泌,改善牙龈增生。     

Cyclosporine-A inhibits MMP-2 and -9 activities in the presence of Porphyromonas gingivalis lipopolysaccharide: an experiment in human gingival fibroblast and U937 macrophage co-culture

Kuo PJ, Tu HP, Chin YT, Lu SH, Chiang CY, Chen RY, Fu E. Cyclosporine‐A inhibits MMP‐2 and ‐9 activities in the presence of Porphyromonas gingivalis lipopolysaccharide: an experiment in human gingival fibroblast and U937 macrophage co‐culture. J Periodont Res 2012; 47: 431–438. © 2012 John Wiley & Sons A/S Background and Objective:  Studies have shown that bacterial plaque and the associated gingival inflammation increase the severity of gingival overgrowth induced by cyclosporine‐A (CsA). This in vitro study aimed to evaluate the effect of CsA on the activities of MMPs from the co‐culture of human gingival fibroblasts and U937 macrophages in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS). Material and Methods:  Activities of pro‐MMP‐2, MMP‐2 and pro‐MMP‐9 in the supernatants of independent cultures and co‐cultures were examined by zymography. RT‐PCR was selected to evaluate the expression of mRNA for membrane type‐1 (MT1) MMP in the co‐cultures. Results:  Activities of MMPs in the co‐cultures were significantly greater when compared with any of the independent cultures. Lipopolysaccharide significantly increased the MMP activities in a dose‐dependent manner in the co‐cultures, whereas CsA inhibited these activities. In the presence of both CsA and LPS, the MMP activities inhibited by CsA could still be observed in the co‐cultures. In the individual cultures, in contrast, the CsA‐inhibited MMP activities, in the presence of LPS, were minimally detected. The mRNA expression of MT1‐MMP was significantly enhanced after LPS treatment; however, this enhancement was inhibited by CsA. Conclusion:  This study demonstrated that, in co‐cultures of human gingival fibroblasts and U937 macrophages, CsA could inhibit MMP activities in the presence of P. gingivalis LPS. It might be part of the underlying reason for the persistent overgrowth of gingiva seen when bacterial plaque and local inflammation are present during CsA therapy.     

Mechanism of cyclosporine-induced overgrowth in gingiva

Cyclosporine A (CsA) is a widely used immunosuppressant but with significant side-effects, such as gingival overgrowth. This study investigates how CsA induces gingival proliferation and shows the effects of the CsA-associated signaling messengers, IL-6 and TGF-beta1, on gingival proliferation. CsA increased both IL-6 and TGF-beta1 levels. In addition to CsA, an IL-6 or TGF-beta1 treatment also induced gingival fibroblast proliferation. Inhibiting the cytokine resulted in the suppression of CsA-induced proliferation. MAPKs and PI3K are known to be involved in cell proliferation. Therefore, the effect of CsA on the kinase activities was examined. The results showed that both p38 MAPK and PI3K are essential for gingival fibroblast proliferation. TGF-beta1 and IL-6 and their associated signaling transduction may be novel bona fide molecular targets for the prevention of gingival overgrowth in CsA-treated patients. (Abbreviations: MAPK, mitogen-activated protein kinase; P13K, phosphatidylinositol 3-kinase.)     

Mechanism of azithromycin treatment on gingival overgrowth

Azithromycin is effective for the remission of cyclosporine A-induced gingival overgrowth (CIGO) in persons who have undergone renal transplant. To explain its mechanism in alleviating the clinical symptoms of these individuals, we examined the effect of azithromycin on cell proliferation and collagen turnover modified by cyclosporin A in human gingival fibroblasts from healthy persons and from persons who had undergone renal transplant. Cyclosporin A-induced proliferation of renal transplant fibroblasts and normal fibroblasts was inhibited by azithromycin. Azithromycin elevated the reduced metalloproteinase (MMP)-1 and MMP-2 activities in cyclosporine A-treated renal transplant fibroblasts and normal fibroblasts. In cyclosporine A-treated renal transplant fibroblasts, azithromycin blocked the accumulation of total collagen in culture media and the increase in type I collagen mRNA level, but recovered the reduced MMP-2 mRNA level to the control. These results suggest that azithromycin may improve CIGO by blocking cyclosporine A-induced cell proliferation and collagen synthesis, and by activating MMP-2 in gingival fibroblasts of persons with cyclosporine A-induced gingival overgrowth.     

Involvement of MT1‐MMP and TIMP‐2 in human periodontal disease

Oral Diseases (2010) 16 , 388–395 Objectives: Periodontal disease is characterized by an increased collagen metabolism. Although membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined. Methods: MT1‐MMP and TIMP‐2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis‐affected human subjects. MT1‐MMP and TIMP‐2 expression were assessed through Western‐blot of tissue homogenates. The main cell types involved in MT1‐MMP and TIMP‐2 production were evaluated by means of immunohistochemistry. Results: Both MT1‐MMP and TIMP‐2 were significantly increased in periodontitis‐affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1‐MMP and its inhibitor TIMP‐2 was altered in periodontitis‐affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1‐MMP in fibroblasts and macrophages in gingival tissues. MT1‐MMP was detected in cells in close association with the gingival collagen matrix. TIMP‐2 expression was identified in fibroblasts, macrophages and epithelial cells. Conclusions: Our observations show an increased expression of MT1‐MMP and TIMP‐2 in periodontitis‐affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.     

Effects of long-term cyclosporin therapy on gingiva of rats--analysis by stereological and biochemical estimation

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth, which remains a significant problem. The risk factors appraised include the duration of treatment. However, there are no stereological and biochemical studies exploring the effects of long-term CsA therapy on gingival tissue. The purpose of the present study was to investigate the level of TGF-beta1 in saliva and describe the densities of fibroblasts and collagen fibers in the gingival tissue of rats treated with CsA for long periods. Rats were treated for 60, 120, 180 and 240 days with a daily subcutaneous injection of 10 mg/kg of body weight of CsA. At the end of the experimental periods, saliva was collected for the determination of TGF-beta1 levels. After histological processing, the oral epithelium and the connective tissue area were measured as well as the volume densities of fibroblasts (Vf) and collagen fibers (Vcf). After 60 and 120 days of CsA treatment, there was a significant increase in Vf and Vcf as well as a significant increase in TGF-beta1 levels. After 180 and 240 days, reduction in the gingival overgrowth associated with significant decreases in the level of TGF-beta1, and also decreased Vf and Vcf, were observed. The data presented here suggest that after long-term therapy, a decrease in TGF-beta1 levels occurs, which might contribute to an increase in the proteolytic activity of fibroblasts in the gingiva, favoring the normality of extracellular matrix synthesis.     

Lack of influence of cyclosporin A on levels of gingival procollagen types I and III mRNAs in rats of different ages

Previous studies showed that gingival overgrowth following cyclosporin A (CsA) administration is not associated with an increase in interstitial collagen. It also was shown that CsA causes a significant decrease in collagen content within the gingival stroma. In order to determine whether this decrease is caused by down-regulation of collagen mRNA, the procollagen mRNA level in gingiva of young and old rats was measured and correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of ³²P-labelled cDNA probes for procollagen types I and III with total RNA extracted from the molar gingiva showed that administration of CsA did not change the steady-state levels of mRNAs for both procollagens in the gingiva of either young or old rats. The ratio of gingival interstitial collagen to DNA was significantly reduced in the CsA-treated animals (4.2 ± 0.85) relative to the controls (7.8 ± 1.6). It is concluded that the reduction in interstitial collagen following CsA treatment is not age-related, and is most probably caused by increased degradation rather by decreased biosynthesis.