Human Buccal Mucosal Fibroblasts Research Articles

Targeting MetaLnc9/miR-143/FSCN1 axis inhibits oxidative stress and myofibroblast transdifferentiation in oral submucous fibrosis

Background/purposePersistent activation of myofibroblasts is attributed to various dysregulated biological events conferring multiple types of fibrosis diseases, including oral submucous fibrosis (OSF). Although the significance of non-coding RNAs (ncRNAs) in the occurrence of fibrosis has been appreciated, the detailed mechanisms still have not been fully elucidated. The aim of this study was to identify key dysregulated ncRNAs and elucidate their pro-fibrotic mechanisms in promoting myofibroblast activation and the pathological development of OSF. Materials and methodsExpression of non-coding RNAs and mRNAs in OSF cohort was determined using RNA sequencing and qRT-PCR. The molecular axis of pro-fibrotic ncRNAs were exploited via luciferase reporter activity assay and RNA expression rescue experiments. Functional assays, including collagen gel contraction, wound healing ability, cell migration, and reactive oxygen species (ROS) production, were conducted to assess the changes in the myofibroblastic phenotypes of primary human buccal mucosal fibroblasts. ResultsHerein, we found that long non-coding RNA MetaLnc9 was upregulated in OSF specimens and positively associated with several fibrosis markers. Silencing of MetaLnc9 diminished the features of activated myofibroblasts and the production of ROS. We not only showed that MetaLnc9 functioned as a competitive endogenous RNA of microRNA (miR)-143, but also demonstrated that the pro-fibrosis effect of MetaLnc9 on myofibroblast activities was mediated by suppression of miR-143. Moreover, our data showed that fascin actin-bundling protein 1 (FSCN1) was a direct target of miR-143 and positively related to MetaLnc9. ConclusionUpregulation of MetaLnc9 may enhance the activation of myofibroblasts by sponging miR-143 and titrating its inhibitory property on FSCN1.

Effects of Vasoactive Intestinal Polypeptide on Proliferation and Migration of Human Buccal Mucosa Fibroblasts

To explore the effects of vasoactive intestinal polypeptide (VIP) on the proliferation and migration of human buccal mucosal fibroblasts (HBMFs). HBMFs were primarily cultured and pretreated with lipopolysaccharide (LPS) as an inflammatory stimulant. The cells in experimental group and control group were transfected with overexpressed pcDNA3.1-VIP and its control pcDNA3.1-VIP-NC, and LPS group was also set. All cells were routinely cultured. Then the cell proliferative activity and migration capacity were detected by MTT assay and wound healing assay, respectively. The mRNA expressions of N-cadherin and proliferating cell nuclear antigen (PCNA) were determined using qRT-PCR, and the expression levels of VIP and macrophage metalloelastase (MME) were detected by Western blotting. Compared with LPS group, the cell proliferative activity and migration capacity, the mRNA expressions of N-cadherin and PCNA, and the expressions of VIP and MME were significantly increased in experimental group (P<0.05). Overexpression of VIP can enhance the proliferation and migration of HBMFs, probably by regulating the expression of MME.

Determination of phenotypic alteration of arecoline-induced buccal mucosal fibroblasts: An in-vitro cell culture study

Introduction: Oral cancer is one of the worldwide health problems accounting as the 6th common of all malignancies. Majority of the oral cancer develop from premalignant conditions of the oral cavity due to the chronic habit of tobacco chewing and smoking. The prominent cells of the oral mucosa are fibroblasts playing a major role in synthesis of extracellular matrix, wound healing, and wound repair. Arecoline, one of main ingredients of tobacco is considered as a risk factor for the development of oral premalignant lesions and cancer. The arecoline is reported to have both genotoxic and morphological alteration of oral fibroblasts leading to Oral submucous fibrosis. Thus in our study a dose dependent effect of arecoline was assessed on the morphology of cultured Human Buccal Mucosal Fibroblasts. Aim: The aim of our experiment is to assess the effect of different concentration of arecoline on the morphological variation of primary cell lines of human buccal mucosal fibroblasts to develop a model of altered fibroblasts. Materials and Methods: The primary cell lines of human buccal mucosa were established in BSRC KAHER Belagavi, and authenticated by STR profiling from DNA forensics Lab New Delhi. The cells were further cultured and assessed after treating with different concentration of arecoline hydrobromide. The treated cells were then observed for the phenotypic changes and recorded. The morphological alterations were compared to the untreated fibroblasts. Results and Conclusion: In our study, a dose-dependent effect of arecoline was assessed on phenotypic or morphological alteration of buccal mucosal fibroblasts. The results justified that concentration of arecoline lower than 125 μg/ml did not show change in the morphology of buccal mucosal fibroblasts, whereas the concentration of arecoline >250 μg/ml showed altered fibroblasts. Hence, it can be concluded that the levels of arecoline in arecanut chewers if it is >250 μg/ml, the mucosal fibroblasts may undergo changes to cause fibrosis of collagen. The future scope of our study is to determine the genotoxic effects of arecoline on buccal mucosal fibroblasts and also to develop the therapeutic effects.

Isolation, culture and characterization of primary cell lines of human buccal mucosal fibroblasts: A combination of explant enzamytic technique.

Background:The cell culture technique has become a routine and a popular method for its wide applications in the field of cell biology and biotechnology and in medical research. Isolation of primary cells over the cancer cells is an essential component of cell culture technology as they are the reliable source to understand normal physiological, morphological and molecular process of human cells. As fibroblasts are the prominent cells of the connective tissue of oral mucosa, many disease entities and histogenesis are linked to fibroblasts. Culture of oral fibroblast cells helps the oral biologists and researchers to study the morphological and molecular process in the oral diseases.Aim:The aim of our experiment is to isolate and culture the human buccal mucosal fibroblast cells from healthy individuals using a combination of explant–enzymatic method and characterization of the cells by short tandem repeat (STR) profiling.Materials and Methods:The tissue samples were collected from healthy individuals undergoing routine impacted third molar extraction. A combination of explant–enzymatic technique was used for the isolation from the tissue samples. The cells were further subcultured, maintained and stored as per the standard protocols. Thus, to confirm the oral fibroblasts of human origin and its uniqueness, they were characterized using STR profiling.Results and Conclusion:Using the combination technique, we were successful in isolating the cells at a faster rate by detachment of cells on day 3 and confluency on day 10. The morphological assessment and STR profiling further confirmed that the isolated cell lines resemble human fibroblast cells.

MiR-200b ameliorates myofibroblast transdifferentiation in precancerous oral submucous fibrosis through targeting ZEB2.

Oral submucous fibrosis (OSF) is a progressive scarring disease. MicroRNA‐200b (miR‐200b) has been reported as a tumour suppressor, but its role in the precancerous OSF remains unknown. In this study, we investigated the impact of miR‐200b on myofibroblastic differentiation activity. Arecoline is a major areca nut alkaloid and has been employed to induce the elevated myofibroblast activity in human buccal mucosal fibroblasts (BMFs). Treatment of arecoline in BMFs dose‐dependently reduced gene expression of miR‐200b, which corresponded with the decreased expression of miR‐200b in fBMFs. The arecoline‐induced myofibroblast activities were abolished by overexpression of miR‐200b in BMFs, and the same results were observed in fBMFs. In addition, α‐SMA was inhibited by an increase in miR‐200b. We further demonstrated that miR‐200b‐mediated decrease in ZEB2 led to down‐regulation of α‐SMA, vimentin. Loss of miR‐200b resulted in enhanced collagen contraction and migration capabilities, and knockdown of ZEB2 reversed these phenomena. Lastly, we showed the expression of miR‐200b was significantly less and ZEB2 was markedly higher in OSF tissues. These results suggested that down‐regulation of miR‐200b may contribute to the pathogenesis of areca quid‐associated OSF through the regulation of ZEB2 and myofibroblast hallmarks.

Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF-β/smad signaling.

Oral submucous fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis, and it causes chronic inflammation and persistent activation of myofibroblasts. As yet, existing treatments only provide temporary symptomatic relief and there is a lack of an effective intervention to cure OSF. Therefore, development of approaches to ameliorate myofibroblast activities becomes a crucial objective to prevent the malignant progression of OSF. In this study, we examined the inhibitory effect of glabridin, an isoflavane extracted from licorice root, on the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results showed that myofibroblast activities, including collagen gel contractility, migration, invasion and wound healing abilities were reduced after exposure of glabridin in a dose-dependent manner. Most importantly, we demonstrated that the arecoline-induced myofiroblast activities were abolished by glabridin treatment. Additionally, the expression of the myofibroblast marker α-smooth muscle actin and other fibrogenic marker, type I collagen, in fBMFs were dose-dependently downregulated. Moreover, we showed that the production of TGF-β was suppressed by glabridin in fBMFs and the protein expression of phospho-Smad2 was decreased as well. In summary, our data suggested that glabridin repressed the myofibroblast features in fBMFs via TGF-β/Smad2 signaling pathway. Glabridin also prevented the arecoline-increased myofibroblast activities, and could serve as a natural anti-fibrosis compound for OSF.

Inhibitory effect of GMI, an immunomodulatory protein from Ganoderma microsporum, on myofibroblast activity and proinflammatory cytokines in human fibrotic buccal mucosal fibroblasts.

Oral submucous fibrosis (OSF) has been indicated as one of the oral potentially malignant disorders. Epidemiological studies have attributed this pathological fibrosis to the habit of areca nuts chewing, which causes chronic inflammation and persistent activation of myofibroblasts in the oral cavity. Hence, it is crucial to find an effective intervention to ameliorate inflammation in order to prevent the malignant progression of OSF. In this study, we assessed the anti-inflammatory effect of the immunomodulatory protein, GMI, extracted from Ganoderma microsporum on the expression proinflammatory cytokines and the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results demonstrated that the expression level of interleukin (IL)-6 and IL-8 were decreased after exposure of GMI and the myofibroblast activities, including collagen gel contraction, migration, invasion, and wound healing abilities were inhibited as well. Furthermore, we confirmed these findings in the arecoline-stimulated BMFs. Consistent with the above findings, the expression of the myofibroblast marker α-smooth muscle actin and other fibrogenic markers, such as type I collagen, fibronectin, and vimentin in fBMFs were all reduced in a dose-dependent manner. Collectively, our data suggested that GMI suppressed the proinflammatory cytokines and myofibroblast features in fBMFs, and could serve as a promising and natural antifibrosis agent.

Activation of transforming growth factor-β1 by thrombin via integrins αvβ1, αvβ3, and αvβ5 in buccal fibroblasts: Suppression by epigallocatechin-3-gallate.

Transforming growth factor-beta (TGF-β) plays a central role in the pathogenesis of oral submucous fibrosis (OSF). Thrombin is a key player in tissue repair, inflammation, and fibrosis after injury. Effects of thrombin on activated-TGF-β1 levels, Smad3 phosphorylation, and connective tissue growth factor (CTGF/CCN2) synthesis in primary human buccal mucosal fibroblasts (BMFs) were assessed by enzyme-linked immunosorbent assay or Western blot analysis. Thrombin and protease-activated receptor-1 (PAR-1) agonist induced TGF-β1 activation and Smad3 phosphorylation. Pretreatment with TGF-β-neutralizing antibody completely inhibited thrombin-induced CCN2 synthesis. Neutralizing antibodies to integrin αv, β1, αvβ3, αvβ5, and Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor Y27632 completely blocked thrombin-induced TGF-β1 activation, Smad3 phosphorylation, and CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited thrombin-induced TGF-β1 activation. Thrombin induces αvβ1, αvβ3, and αvβ5 integrins-mediated TGF-β1 activations via ROCK signaling. EGCG inhibits thrombin-induced CCN2 synthesis in BMFs by suppressing latent TGF-β1 activation.

Regulation of hypoxia-inducible factor-1α in human buccal mucosal fibroblasts stimulated with arecoline.

Hypoxia-inducible factor (HIF)-1α is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare HIF-1α expression from fibroblasts derived from human normal buccal mucosa and oral submucous fibrosis (OSF) specimens and further to explore the potential mechanisms that may lead to induce HIF-1α expression. OSF buccal mucosal fibroblasts (BMFs) demonstrated significantly higher HIF-1α mRNA expression than normal BMFs (p<0.005). Arecoline, the major areca nut alkaloid, was also found to elevate HIF-1α mRNA expression in a dose-dependent manner (p<0.05). Moreover, arecoline-induced HIF-1α expression was downregulated by mitogen-activated protein kinase inhibitor U0126, phosphatidylinositol 3-kinase inhibitor LY294002, p38 inhibitor SB203580, cyclooxygenase-2 inhibitor NS-398, and glutathione precursor N-acetyl-L-cysteine (p<0.05). Taken together, hypoxia plays an important role in the pathogenesis of areca quid chewing-associated OSF. These pharmacological agents may be further used as chemoprevention agents for OSF.

Effect of isobutyl methacrylate and methacrylic acid eluted from chairside denture hard reliners on enzymatic cellular antioxidants: An in vitro study in human primary buccal mucosal fibroblasts.

Aim:This study was conducted with the objective to evaluate the cytotoxicity of monomers isobutyl methacrylate (IBMA) and methacrylic acid (MA) in human buccal mucosal fibroblast primary cell culture and to study their effect on cellular enzymatic antioxidants-glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT).Materials and Methods:The tissue for fibroblast cell culture was harvested from oral buccal mucosa of a healthy donor. Fibroblast cells were plated at a density of 1 × 104 cells per well in 96-well tissue culture plates. Cells were exposed to various concentrations of IBMA and MA. The cell viability and various enzyme activities were evaluated 24 h after exposure to the above treatments. All tests were done in triplicate. Cell viability was assessed by trypan blue dye exclusion assay and all enzyme activities were done using assay kits from Cayman Chemicals, Ann Arbor, USA.Results:At all concentrations tested a statistically significant decrease in viability was observed in IBMA- and MA-treated cells. Around 42% cells were viable at the highest test concentration of IBMA (80 μmol/L) and only 20% cells were viable at the highest dose (144 μmol/L) of MA exposure (P < 0.05). Dose-dependent decrease in the GPx and SOD activities was observed in cells treated with IBMA and MA (P < 0.05). CAT activity was not detectable in the controls. However, a fall in CAT activity was detected in cells exposed to IBMA and MA at all concentrations tested (P < 0.05).Conclusion:IBMA and MA leaching out from the chairside denture hard reliners are cytotoxic on human buccal fibroblast primary cell cultures. This could be due to the oxidative stress caused by the generation of reactive oxygen species which is evidenced by the fall in activities of antioxidant enzymes (GPx, SOD, and CAT) and cytotoxicity.

Aberrant SSEA-4 upregulation mediates myofibroblast activity to promote pre-cancerous oral submucous fibrosis.

Oral submucous fibrosis (OSF), regarded as a precancerous condition, is characterized by juxta-epithelial inflammatory reaction followed by fibro-elastic change in the lamina properia and epithelial atrophy. The pathologic mechanisms of OSF still need to be further clarified. In the study, we investigated the functional expression of SSEA-4, which is a well-known stemness marker, in myofibroblast activity and the clinical significance in OSF tissues. The expression of SSEA-4 in OSF was evaluated by immunohistochemical staining. Functional analysis of SSEA-4 on myofibroblast activity of OSF was achieved by lentiviral silencing ST3GAL2. Immunohisitochemistry demonstrated that SSEA-4 expression was significantly higher expression in areca quid chewing-associated OSF tissues than those of normal oral mucosa tissues. From flow cytometry analysis, arecoline dose-dependently activated SSEA-4 expression in primary human normal buccal mucosal fibroblasts (BMFs). Sorted SSEA-4-positive cells from fibrotic BMFs (fBMFs) have higher colony-forming unit, collagen gel contraction, and α-smooth muscle actin (α-SMA) expression than SSEA-4-negative subset. Knockdown of ST3GAL2 in fBMFs suppressed SSEA-4 expression, collagen contraction, migration, invasiveness, and wound healing capability. Consistently, silencing ST3GAL2 was found to repress arecoline-induced myofibroblast activity in BMFs. The study highlights SSEA-4 as a critical marker for therapeutic intervention to mediate myofibroblast transdifferentiation in areca quid chewing-associated OSF.

Resveratrol suppresses myofibroblast activity of human buccal mucosal fibroblasts through the epigenetic inhibition of ZEB1 expression.

Oral submucous fibrosis (OSF) is a precancerous condition of the oral mucosa without specific therapeutic drugs. We previously demonstrated that the zinc finger E-box binding homeobox 1 (ZEB1) plays a pathogenic role in the induction of the myofibroblast activity of buccal mucosal fibroblasts (BMFs) and contributes to the pathogenesis of OSF. Resveratrol is a natural polyphenolic flavonoid with anti-fibrosis activity in various tissues and has the capability to inhibit ZEB1 in oral cancer cells. We examined the effect of resveratrol on the myofibroblast activity of human primary fibrotic BMFs (fBMFs) derived from OSF tissues. With the collagen contraction assay, resveratrol displayed anti-myofibroblast activity in three fBMF lines. Resveratrol also inhibited the expression of fibrogenic genes at the mRNA and protein levels in a dose- and time-dependent manner. The downregulation of ZEB1 in fBMFs by resveratrol was mediated by epigenetic mechanisms, such as the upregulated expression of miR-200c and the enhancer of zeste homolog 2 (EZH2), as well as the trimethylated lysine 27 of histone H3 (H3K27me3). Resveratrol also increased the binding of H3K27me3 to the ZEB1 promoter. The knockdown of EZH2 in fBMFs caused the upregulation of ZEB1 and suppressed the inhibitory effect of resveratrol. Furthermore, the reversed expression pattern between EZH2 and ZEB1 was observed in 6/8 OSF tissues with twofold upregulation of ZEB1 expression compared with the adjacent normal mucosa. In conclusion, our data suggest that resveratrol epigenetically inhibits ZEB1 expression to suppress the myofibroblast activity of fBMFs and may serve as a dietary supplement for OSF patients.

Epigallocatechin-3-gallate inhibits transforming-growth-factor-β1-induced collagen synthesis by suppressing early growth response-1 in human buccal mucosal fibroblasts

Transforming growth factor (TGF)-β is a key regulator in the pathogenesis of oral submucous fibrosis (OSF). Early growth response (Egr)-1 is essential for fibrotic responses to TGF-β. Because TGF-β signaling is cell-type- and context-dependent, we investigated the signaling involved in TGF-β-induced Egr-1 in primary human buccal mucosal fibroblasts (BMFs). TGF-β-induced Egr-1 and its signaling were assessed by western blotting in BMFs. Egr-1 small interfering RNA was used to define the role of Egr-1 on TGF-β-induced mRNAs of the α1- and α2-chains of type I collagen (COL1A1 and COL1A2) and acid-soluble collagen production (via Sircol collagen assay). The effects of epigallocatechin-3-gallate (EGCG) on TGF-β-induced Egr-1 protein and acid-soluble collagen were also evaluated. TGF-β1 stimulated Egr-1 production in BMFs. Pretreatment with PD98059, SP600125, SB431542, and SIS3, but not SB203580, significantly reduced TGF-β1-induced Egr-1 protein expression. Genetic targeting of Egr-1 completely inhibited TGF-β1-induced type I collagen mRNAs and collagen protein expression. EGCG fully inhibited TGF-β1-induced Egr-1 and TGF-β1-stimulated production of acid-soluble collagens. We conclude that activin receptor-like kinase (ALK)5, Smad3, extracellular signal-regulated kinase, and c-Jun N-terminal kinase are involved in the TGF-β1-induced Egr-1 protein production in BMFs. Egr-1 mediates TGF-β1-induced COL1A1 and COL1A2 mRNA expression and acid-soluble collagen production in BMFs. EGCG can block TGF-β1-induced collagen production by attenuating Egr-1 expression in BMFs. Egr-1 is a key mediator in TGF-β1-induced pathogenesis of OSF. EGCG may be useful in the prevention or treatment of OSF.

Elevation of Twist expression by arecoline contributes to the pathogenesis of oral submucous fibrosis.

Oral submucous fibrosis (OSF), a chronic progressive scarring disease, has been considered as a precancerous condition of oral mucosa. In this study, we investigated the functional role of Twist, an epithelial-mesenchymal transition (EMT) transcriptional factor, in myofibroblastic differentiation activity of OSF. Arecoline, a major areca nut alkaloid, was used to explore whether expression of Twist could be changed dose-dependently in human primary buccal mucosal fibroblasts (BMFs). Collagen gel contraction and migration capability in arecoline-stimulated BMFs and primary oral submucous fibrosis-derived fibroblasts (OSFs) with Twist knockdown was presented. We observed that the treatment of arecoline dose-dependently increased Twist expression transcript and protein levels in BMFs. The myofibroblast activity including collagen gel contraction and migration capability also induced by arecoline, while knockdown of Twist reversed these phenomena. Importantly, inhibition of Twist led to the suppression collagen contraction and wound healing capability of primary cultivated OSFs. Clinically, Twist transcript and protein expression was higher in areca quid chewing-associated OSF tissues than in normal oral mucosa tissues. This evidence suggests that upregulation of Twist might be involved in the pathogenesis of areca quid-associated OSF through dysregulation of myofibroblast activity.

MiR-203 inhibits arecoline-induced epithelial-mesenchymal transition by regulating secreted frizzled-related protein 4 and transmembrane-4 L six family member 1 in oral submucous fibrosis

Oral submucous fibrosis (OSF) is a potentially malignant disease predominantly found in Asian people. The areca nut has been implicated in this disease. Arecoline, one of the areca alkaloids, induces epithelial-mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts. Yet, the mechanisms of the underlying arecoline-induced EMT in OSF remain unknown. In the present study, we aimed to investigate the role of microRNAs (miRNAs) in arecoline-induced EMT in HaCaT cells. We found that miR-203 was significantly downregulated in OSF tissues compared to that in normal buccal mucosa tissues, and that miR-203 negatively regulated secreted frizzled-related protein 4 (SFRP4) and positively regulated transmembrane-4 L six family member 1 (TM4SF1). We observed that upregulation of miR-203 significantly decreased the cell proliferation of HaCaT cells, and significantly upregulated the expression of cytokeratin 19 (CK19) and E-cadherin proteins, whereas it significantly downregulated the expression of N-cadherin and vimentin compared to these levels in the vehicle control cells. Thus, we provide evidence to illustrate that miR-203 plays a role in the pathogenesis of OSF, which may be a target for OSF management.

Arecoline stimulated early growth response‐1 production in human buccal fibroblasts: Suppression by epigallocatechin‐3‐gallate

Early growth response-1 (Egr-1) protein plays an important role in many human fibrotic diseases. Areca nut chewing is the most important risk factor of oral submucous fibrosis (OSF). Egr-1 protein expression in OSF was examined using antibody to Egr-1. Arecoline-induced Egr-1 expression and its signaling pathways were assessed by Western blot analyses in human buccal mucosal fibroblasts (BMFs). Elevated Egr-1 staining was observed in epithelial cells, fibroblast, and inflammatory cells in 7 of 10 OSF cases. Arecoline, a main alkaloid found in the areca nut, stimulated Egr-1 synthesis in BMFs. Pretreatment with antioxidant N-acetyl-L-cysteine, c-Jun NH2-terminal kinase inhibitor SP600125, and extracellular signal-regulated kinase inhibitor PD98059 significantly reduced arecoline-induced Egr-1 synthesis. Epigallocatechin-3-gallate (EGCG) inhibited arecoline-induced Egr-1 synthesis and collagen gel contraction in a dose-responsive manner. Constitutive Egr-1 expression during areca nut chewing may play a role in the pathogenesis of OSF. EGCG could be a good candidate for prevention or treatment of OSF.

Arecoline‐induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1

Oral submucous fibrosis (OSF) is considered as a pre-cancerous condition of the oral mucosa and is highly associated with habitual areca quid chewing. Arecoline is the major alkaloid in areca quid and is thought to be involved in the pathogenesis of OSF. Our previous studies have demonstrated that arecoline could induce epithelial–mesenchymal transition (EMT)-related factors in primary human buccal mucosal fibroblasts (BMFs). Therefore, we investigated the expression of zinc finger E-box binding homeobox 1 (ZEB1), which is a well-known transcriptional factor in EMT, in OSF tissues and its role in arecoline-induced myofibroblast transdifferentiation from BMFs. The expression of ZEB1, as well as the myofibroblast marker α-smooth muscle actin (α-SMA), was significantly increased in OSF tissues, respectively. With immunofluorescence analysis, arecoline induced the formation of α-SMA-positive stress fibres in BMFs expressing nuclear ZEB1. Arecoline also induced collagen contraction of BMFs in vitro. By chromatin immunoprecipitation, the binding of ZEB1 to the α-SMA promoter in BMFs was increased by arecoline. The promoter activity of α-SMA in BMFs was also induced by arecoline, while knockdown of ZEB1 abolished arecoline-induced α-SMA promoter activity and collagen contraction of BMFs. Long-term exposure of BMFs to arecoline induced the expression of fibrogenic genes and ZEB1. Silencing of ZEB1 in fibrotic BMFs from an OSF patient also suppressed the expression of α-SMA and myofibroblast activity. Inhibition of insulin-like growth factor receptor-1 could suppress arecoline-induced ZEB1 activation in BMFs. Our data suggest that ZEB1 may participate in the pathogenesis of areca quid–associated OSF by activating the α-SMA promoter and inducing myofibroblast transdifferentiation from BMFs.

EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in buccal fibroblasts

Transforming growth factor β (TGFβ) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) and cyclooxygenase-2 (COX-2) were found to overexpress in OSF. The aim of this study was to investigate the molecular mechanism underlying the TGFβ-induced CCN2 expressions in human buccal mucosal fibroblasts (BMFs) to identify the potential targets for drug intervention or chemoprevention of OSF. TGFβ-induced CCN2 expression and its signaling pathways were assessed by Western blot analyses in BMFs. TGFβ1 stimulated CCN2 synthesis in BMFs. Pretreatment with c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and activin receptor-like kinase 5 (ALK5) inhibitor SB431542 significantly reduced TGFβ1-induced CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) completely blocked TGFβ1-induced CCN2 synthesis by inhibiting the phosphorylation of JNK and p38 MAPK. Prostaglandin E(2) (PGE(2)) inhibited the TGFβ1-induced CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs. The TGFβ1-induced CCN2 synthesis in BMFs could be mediated by the ALK5, JNK, and p38 MAPK pathways. EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in BMFs. The exceptional signal transduction pathways of TGFβ1-induced CCN2 production in BMFs contribute to the resistance of PGE(2) downregulation of CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. EGCG may serve as a useful agent in controlling OSF.

Thrombin‐stimulated connective tissue growth factor (CTGF/CCN2) production in human buccal mucosal fibroblasts: Inhibition by epigallocatechin‐3‐gallate

Connective tissue growth factor (CTGF/CCN2) is associated with many human fibrotic disorders and was found to overexpress in oral submucous fibrosis (OSF). OSF is the result of persistent chemical irritation and microtrauma to oral mucosa from areca nut. Microtrauma could lead to the release of thrombin. Thrombin-induced CCN2 expression and its signaling pathways were assessed by Western blot analyses in human buccal mucosal fibroblasts. Thrombin stimulated CCN2 synthesis in buccal mucosal fibroblasts via activation of protease-activated receptor-1. Pretreatment with antioxidant N-acetyl-L-cysteine, apoptosis signal-regulating kinase 1 inhibitor thioredoxin, and c-Jun NH(2) -terminal kinase inhibitor SP600125 significantly reduced thrombin-induced CCN2 synthesis. Epigallocatechin-3-gallate completely inhibited thrombin-induced CCN2 synthesis. Thrombin produced by microtrauma may contribute to the pathogenesis of OSF by up-regulating CCN2 expression. This effect could be mediated by protease-activated receptor-1, reactive oxygen species, apoptosis signal-regulating kinase 1, and c-Jun NH(2) -terminal kinase pathways and prevented by epigallocatechin-3-gallate.

Arecoline-stimulated connective tissue growth factor production in human buccal mucosal fibroblasts: Modulation by curcumin

Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). We immunohistochemically examined the expression of CTGF protein in 20 cases of OSF and found positive CTGF staining in fibroblasts and endothelial cells in all cases. Western blot analysis showed that arecoline, a main alkaloid found in AN, stimulated CTGF synthesis in a dose- and time-dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of CTGF during AN chewing may enhance the fibrotic activity in OSF and play a role in the pathogenesis of OSF. Pretreatment with NF-kappaB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580 and antioxidant N-acetyl-l-cysteine, but not ERK inhibitor PD98059, significantly reduced arecoline-induced CTGF synthesis. Furthermore, curcumin completely inhibited arecoline-induced CTGF synthesis and the inhibition is dose-dependent. These results indicated that arecoline-induced CTGF synthesis was mediated by ROS, NF-kappaB, JNK, P38 MAPK pathways and curcumin could be a useful agent in controlling OSF.