Conditioned Medium of Demineralized Freeze‐Dried Bone Activates Gene Expression in Periodontal Fibroblasts In Vitro

Background: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)‐β signaling in mesenchymal cells. The aim of this study is to determine whether processing of native bone into DBM affects the activity of the conditioned medium. Methods: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid, and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium obtained from DBM (DBCM). Changes in the expression of TGF‐β target genes were determined. Results: DBCM altered the expression of TGF‐β target genes (e.g., adrenomedullin, pentraxin 3, KN motif and ankyrin repeat domains 4, interleukin 11, NADPH oxidase 4, and BTB [POZ] domain containing 11) by at least five‐fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF‐β receptor type I kinase inhibitor SB431542 [4‐(4‐(benzo[d][1,3]dioxol‐5‐yl)‐5‐(pyridin‐2‐yl)‐1H‐imidazol‐2‐yl)benzamide] but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF‐β target genes. Conclusion: The findings suggest that the DBCM can activate TGF‐β signaling in oral fibroblasts.     

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.     

Effects of Resolvin D1 on Cell Survival and Cytokine Expression of Human Gingival Fibroblasts

Background: Tissue breakdown in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis, and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils to initiate resolution. The effects of resolvins on human gingival fibroblasts (HGFs) are unknown. This study examines the effects of resolvin D1 on HGF survival and cytokine expression when treated with or without P. gingivalis supernatant. Methods: Cytotoxicity of resolvin D1 on HGFs with or without a toxic level of P. gingivalis supernatant was measured with lactate dehydrogenase assays. Cytokine arrays were performed on HGF‐conditioned media treated with or without resolvin D1 and with or without P. gingivalis supernatant. Results: Resolvin D1 had no cytotoxic effects on HGFs at concentrations between 1 and 1,000 nM (all P > 0.05). Resolvin D1 (1,000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant on HGFs (P = 0.002). Resolvin D1 significantly reduced the expression of interleukin (IL)‐6 (P = 0.010) and monocyte chemoattractant protein (MCP)‐1 (P = 0.04) in untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the expression levels of granulocyte‐macrophage colony‐stimulating factor (CSF), granulocyte CSF, growth‐regulated oncogene (GRO), IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, MCP‐1, MCP‐2, MCP‐3, and monokine induced by γ‐interferon. Resolvin D1 significantly reduced the expression of GRO (P = 0.04), marginally reduced the levels of MCP‐1 (P = 0.10), and marginally increased the levels of transforming growth factor (TGF)‐β1 (P = 0.07) from HGFs treated with P. gingivalis supernatant. Conclusions: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP‐1, and marginally increased TGF‐β1 from P. gingivalis–treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.     

Aggregatibacter actinomycetemcomitans induces a proatherosclerotic response in human endothelial cells in a three‐dimensional collagen scaffold model

The role of periodontopathogens in inflammatory endothelial dysfunction is not known. This study characterizes a three‐dimensional model with human coronary artery endothelial cells on three‐dimensional (HCAEC‐3D) type I collagen scaffolds to evaluate whether infection with Aggregatibacter actinomycetemcomitans induces a proinflammatory response associated with atherosclerosis. The HCAEC‐3D culture was physicochemically characterized with regard to biocompatibility and barrier function. Then, the culture was infected with A. actinomycetemcomitans strain ATCC 29522 at multiplicities of infection (MOIs) of 1:1, 1:10, and 1:100. Cultures without infection and stimulated with A. actinomycetemcomitans lipopolysaccharide were used as controls. The secretion of soluble factors (IL‐6, IL‐1β, MCP‐1, RANTES, MIP‐1, IL‐8, IL‐1α, and TNF‐α) was evaluated via flow cytometry; TGF‐β1 was evaluated via enzyme‐linked immunosorbent assay (ELISA). The adhesion and migration of fluorescent human THP‐1 monocytes was evaluated. IL‐8, MCP‐1, and IL‐6 secretion increased in a dose‐dependent manner with A. actinomycetemcomitans infection and was significantly greater than that under control treatment. The concentration of TGF‐β1 was significantly higher at MOI 1:100 than in controls. Treatment of the 3D cultures with A. actinomycetemcomitans at different MOIs induced significant differences in the adhesion of monocytes to the endothelium compared to the control without infection. Lastly, conditioned media from 3D cultures treated with A. actinomycetemcomitans induced monocyte migration. The effects of IL‐8, MCP‐1, IL‐6, and TGF‐β1 on the endothelium indicate the ability of A. actinomycetemcomitans to induce an inflammatory response through a mechanism of monocyte adhesion and migration and endothelial dysfunction.     

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts

J Oral Pathol Med (2010) 39 : 250–256 Background: Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL‐4, TGF‐β, tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Methods: Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme‐linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. Results: TNF‐α and IFN‐γ were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL‐4 was reactive in 24% of cysts, and TGF‐β was positive in all samples. Patients with tenderness showed significantly higher levels of IFN‐γ and IL‐4 (P < 0.05). Swelling was associated with high levels of TNF‐α, IFN‐γ, and IL‐4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). Conclusions: Periapical granulomas display a regulatory environment characterized by high TGF‐β and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN‐γ, TNF‐α, and IL‐4.     

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.     

Immunoexpression of TNF-α and TGF-β in central and peripheral giant cell lesions of the jaws

J Oral Pathol Med (2012) 41 : 194–199 Background: Peripheral giant cell lesion (PGCL) is a reactive process associated with a local irritating factor that shows low recurrence after treatment, especially if the irritating factor is eliminated. On the other hand, central giant cell lesion (CGCL) presents a variable clinical behavior ranging from slow and asymptomatic growth without recurrence to rapid, painful and recurrent growth. Our aim was to compare the immunoexpression of tumor necrosis factor‐alpha (TNF‐α) and transforming growth factor‐beta (TGF‐β) in CGCL and PGCL. Methods: Twenty CGCL and 20 PGCL were selected for analysis of the immunoexpression of TNF‐α and TGF‐β in multinucleated giant cells (MGC) and mononucleated cells (MC). Results: The PGCL showed lightly higher expression of TNF‐α than CGCL. In comparison with PGCL, the CGCL showed higher expression of TGF‐β in MC and MGC (P < 0.05) and in total cells (P < 0.05). Significant positive correlation was found between expressions of TGF‐β and TNF‐α in CGCL (P < 0.05). Conclusions: Our results suggest that, in CGCL, coordinated interactions between TGF‐β and TNF‐α may be important for osteoclastogenesis and bone resorption. PGCL occasionally cause bone resorption but to a lower extent, a fact that might be explained by the lower expression of TGF‐β in these lesions.     

Whole‐Blood Cultures From Patients With Chronic Periodontitis Respond Differently to Porphyromonas gingivalis but not Escherichia coli Lipopolysaccharide

Background: Porphyromonas gingivalis lipid A heterogeneity modulates cytokine expression in human cells. This study investigates the effects of two lipid A isoforms of P. gingivalis, lipopolysaccharide (LPS)_1435/1449 and LPS₁₆₉₀, on the secretion of proinflammatory and regulatory cytokines in total blood cultures from patients with and without chronic periodontitis (CP). Methods: A cross‐sectional study was conducted in 38 systemically healthy individuals divided in two groups: 1) the CP group (n = 19), in which patients were diagnosed with CP; and 2) the no periodontitis (NP) group (n = 19), which included control patients without CP. Blood samples were collected from all patients, and whole‐blood cell cultures (WBCCs) were stimulated for 48 hours with P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ and Escherichia coli LPS. Unstimulated WBCCs served as negative controls. The secretion of interferon‐γ (IFN‐γ), interleukin‐10 (IL‐10), and transforming growth factor‐β (TGF‐β) was detected in WBCC supernatants by enzyme‐linked immunosorbent assays. Results: E. coli LPS significantly increased the expression of all cytokines in WBCCs from both the NP and CP groups when compared to non‐stimulated cells (control treatment). P. gingivalis LPS preparations increased IFN‐γ levels in the CP group but not in the NP group when compared with controls (P <0.05). P. gingivalis LPS preparations also increased IL‐10 and TGF‐β levels in both CP and NP groups, but P. gingivalis LPS₁₆₉₀ showed a three‐fold increase on IL‐10 production in the NP group (P <0.05) when compared to P. gingivalis LPS_1435/144. Conclusions: These data demonstrate that WBCC cell populations obtained from healthy individuals and patients with CP may differ in the cytokine response to P. gingivalis but not E. coli LPS. This is consistent with the notion that CP alters the systemic WBCC response and that this can be detected by the different P. gingivalis LPS structures.     

Folic acid rescue of ATRA‐induced cleft palate by restoring the TGF‐β signal and inhibiting apoptosis

J Oral Pathol Med (2010) 40 : 433–439 Background: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF‐β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all‐trans retinoic acid (ATRA)‐induced cleft palate in organ culture were tested. Methods: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. Results: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase‐9 in MEPM cells were detected in the ATRA group than in the control or the ATRA + FA group. Compared with the control or the ATRA + FA group, ATRA had little effect on TGF‐β3 in MEPM cells but significantly inhibited TGF‐β receptor II. Conclusions: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA‐induced cleft palate.     

Effect of cytokine and antigen stimulation on peripheral blood lymphocyte syndecan‐1 expression

Introduction: Cytokines are not only produced by activated lymphocytes but also interact with a number of cell‐surface molecules on the same cells. Syndecan‐1 is one such cell‐surface molecule, which has the capacity to bind a variety of growth factors as well as cytokines. The aim of this study was to examine the effects of transforming growth factor β (TGF‐β), interleukin‐1 (IL‐1), IL‐2, IL‐4, lipopolysaccharide (LPS) from Porphyromonas gingivalis and tetanus toxoid on syndecan‐1 expression by B and T lymphocytes. Methods: B and T lymphocytes were obtained from the peripheral blood of healthy donors. Following exposure to the above growth factors, cytokines and antigens, syndecan‐1 expression was determined by flow cytometry. Results: Subjects could be categorized as high or low expressors of syndecan‐1. In the high‐responder group TGF‐β1 alone resulted in a significant increase in syndecan‐1 expression by both B and T cells. None of the other cytokines and antigens produced a significant response. When analysed in combination, TGF‐β1 in combination with IL‐2, IL‐4, P. gingivalis LPS and tetanus toxoid all produced significant increases in syndecan‐1 expression by B cells. For T cells, combinations of TGF‐β1 with IL‐2 and tetanus toxoid resulted in increased syndecan‐1 expression. Conclusions: Both B and T lymphocytes synthesize the cell‐surface proteoglycan syndecan‐1 and its expression can be modulated by TGF‐β1, either alone or in combination with IL‐2, IL‐4 and LPS from P. gingivalis and tetanus toxoid. While these may reflect general responses under inflammatory conditions their biological significance requires further investigation.     

Retinoic acid‐inducible gene‐I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin‐1β, ‐6 and ‐8 expression

Retinoic acid‐inducible gene‐I (RIG‐I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1β (IL‐1β) induced RIG‐I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG‐I expression induced by lipopolysaccharide (LPS) or double‐stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG‐I in the expression of IL‐1β, IL‐6 and IL‐8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG‐I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG‐I induction by these stimuli was examined. The expression of IL‐1β, IL‐6 and IL‐8 in gingival fibroblasts transfected with RIG‐I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG‐I in concentration‐ and time‐dependent manners. We also examined the localization of RIG‐I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA‐induced RIG‐I expression. Introduction of RIG‐I cDNA into gingival fibroblasts resulted in enhanced expression of IL‐1β, IL‐6 and IL‐8; moreover, overexpression of RIG‐I stimulated with LPS or dsRNA synergistically increased expression of IL‐1β, IL‐6 and IL‐8. RIG‐I may have important roles in the innate immune response in the regulation of IL‐1β, IL‐6 and IL‐8 expression in gingival fibroblasts in response to LPS and dsRNA.     

Effects of tobacco smoke on the secretion of interleukin‐1β, tumor necrosis factor‐α, and transforming growth factor‐β from peripheral blood mononuclear cells

Alterations of the host response caused by short‐term exposure to high levels of smoke during the act of smoking (acute smoke exposure) as well as long‐term exposure to lower levels of tobacco substances in the bloodstream of smokers (chronic smoke exposure) may play a role in the pathogenesis of periodontal diseases in smokers. In this study, we examined the secretion of three cytokines [interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, and transforming growth factor (TGF)‐β] from mononuclear blood cells from current smokers and non‐smokers exposed to in vitro tobacco smoke (which may be comparable to in vivo acute smoke exposure) and mononuclear blood cells from current smokers not exposed to further in vitro smoke (which may be comparable to chronic smoke exposure). Peripheral blood mononuclear cells were isolated from eight healthy current smokers and eight healthy non‐smokers, plated in culture wells, exposed in vitro for 1–5 min to cigarette smoke in a smoke box system or not exposed (baseline controls), and then incubated without further smoke exposure for another 24 h. Supernatants from each well were then collected and assayed for the concentrations of the three cytokines by enzyme‐linked immunosorbent assay (ELISA). At baseline, mean IL‐1β levels were higher in smokers than in non‐smokers (mean: 10.6 vs. 5.9 pg/ml, anova : P < 0.05). In both smokers and non‐smokers, secreted levels of IL‐1β increased from 0 to 5 min of in vitro smoke exposure (mean: 5.9–9.9 pg/ml, t‐test: P < 0.05 for non‐smokers only) with levels in smokers higher than in non‐smokers (P > 0.05). Mean TNF‐α levels increased from 0 to 2 min of smoke exposure and decreased from 2 to 5 min in smokers and non‐smokers, with higher levels in non‐smokers than smokers at all time‐points (P > 0.05). Mean TGF‐β levels were higher in smokers than in non‐smokers at all time‐points (mean: 180.5 vs. 132.0 pg/ml, P < 0.05 at 5 min only) with no significant alteration of the pattern of secretion with cigarette smoke exposure. These observed alterations in the secretion of cytokines from mononuclear blood cells in smokers, relative to non‐smokers, and with in vitro smoke exposure may play a role in the pathogenesis of periodontal diseases in smokers.     

Redefining the induction of periodontal tissue regeneration in primates by the osteogenic proteins of the transforming growth factor‐β supergene family

The molecular bases of periodontal tissue induction and regeneration are the osteogenic proteins of the transforming growth factor‐β (TGF‐β) supergene family. These morphogens act as soluble mediators for the induction of tissues morphogenesis sculpting the multicellular mineralized structures of the periodontal tissues with functionally oriented ligament fibers into newly formed cementum. Human TGF‐β₃ (hTGF‐β₃) in growth factor‐reduced Matrigel^® matrix induces cementogenesis when implanted in class II mandibular furcation defects surgically prepared in the non‐human primate Chacma baboon, Papio ursinus. The newly formed periodontal ligament space is characterized by running fibers tightly attached to the cementoid surface penetrating as mineralized constructs within the newly formed cementum assembling and initiating within the mineralized dentine. Angiogenesis heralds the newly formed periodontal ligament space, and newly sprouting capillaries are lined by cellular elements with condensed chromatin interpreted as angioblasts responsible for the rapid and sustained induction of angiogenesis. The inductive activity of hTGF‐β₃ in Matrigel^® matrix is enhanced by the addition of autogenous morcellated fragments of the rectus abdominis muscle potentially providing myoblastic, pericytic/perivascular stem cells for continuous tissue induction and morphogenesis. The striated rectus abdominis muscle is endowed with stem cell niches in para/perivascular location, which can be dominant, thus imposing stem cell features or stemness to the surrounding cells. This capacity to impose stemness is morphologically shown by greater alveolar bone induction and cementogenesis when hTGF‐β₃ in Matrigel^® matrix is combined with morcellated fragments of autogenous rectus abdominis muscle. The induction of periodontal tissue morphogenesis develops as a mosaic structure in which the osteogenic proteins of the TGF‐β supergene family singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. In primates, the presence of several homologous yet molecularly different isoforms with osteogenic activity highlights the biological significance of this apparent redundancy and indicates multiple interactions during embryonic development and bone regeneration in postnatal life. Molecular redundancy with associated different biological functionalities in primate tissues may simply represent the fine‐tuning of speciation‐related molecular evolution in anthropoid apes at the early Pliocene boundary, which resulted in finer tuning of the bone induction cascade.     

Transforming growth factor‐β‐regulated fractalkine as a marker of erosive bone invasion in oral squamous cell carcinoma

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF‐β), as a novel biomarker for correct prediction and early detection of OSCC‐associated bone invasion. TGF‐β knockdown and treatment with a TGF‐β‐neutralizing antibody decreased the level of fractalkine in the culture media of HSC‐2 and YD10B OSCC cells. Treatment with a fractalkine‐neutralizing antibody reduced TGF‐β‐stimulated invasion by HSC‐2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild‐type or TGF‐β knockdown OSCC cells in mouse calvaria. TGF‐β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor‐bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF‐β and mediates TGF‐β‐induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC‐induced bone destruction.     

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.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.