Thrombin Activates Latent TGFß1 via Integrin αvß1 in Gingival Fibroblasts.

Transforming growth factor ß (TGFß) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGFß1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGFß1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFß1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFß-neutralizing antibody, TGFß type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits αv and ß1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFß1 levels; however, similar effects were not observed for integrins αvß3 and αvß5. These results suggest that protease-activated receptor 1-induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvß1-induced latent TGFß1 activation and subsequent TGFß1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFß1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFß1 activation.

NADPH Oxidase 4 Mediates TGFß1-induced CCN2 in Gingival Fibroblasts.

Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFß in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFß. We previously showed that Src, JNK, and Smad3 mediate TGFß1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFß. In this study, we demonstrated that TGFß1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFß1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFß1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFß1-induced NOX4 expression in HGFs.

Curcumin inhibits TGFß1-induced CCN2 via Src, JNK, and Smad3 in gingiva.

Transforming growth factor ß (TGFß) is a key regulator associated with the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) is overexpressed in GO tissues. CCN2 promotes and sustains fibrosis initiated by TGFß. Previous studies have shown that JNK and Smad3 activation is required for TGFß-induced CCN2 expressions in human gingival fibroblasts (HGFs). In this study, we have found that Src is a major signaling mediator for TGFß-induced CCN2 expressions in HGFs. Pre-treatment with 2 Src kinase inhibitors (PP2, Src inhibitor-1) significantly reduced TGFß1-induced CCN2 synthesis and JNK and Smad3 activation in HGFs. These results suggest that Src is an upstream signaling transducer of JNK and Smad3 with respect to TGFß1-stimulated CCN2 expression in HGFs. We further found that curcumin significantly abrogated the TGFß1-induced CCN2 in HGFs by inhibiting the phosphorylations of Src, JNK, and Smad3. Furthermore, curcumin inhibited TGFß1-induced HGF migration and α-SMA expression. Curcumin potentially qualifies as a useful agent for the control of GO.

Arecoline-stimulated placenta growth factor production in gingival epithelial cells: modulation by curcumin.

OBJECTIVE: Placenta growth factor (PlGF) is associated with the progression and prognosis of oral cancer. MATERIALS AND METHODS: This study used ELISA, quantitative polymerase chain reaction, and Western blotting to study the arecoline-stimulated (PlGF) protein or mRNA expression in human gingival epithelial S-G cells. RESULTS: Arecoline, a major areca nut alkaloid and an oral carcinogen, could stimulate PlGF protein synthesis in S-G cells in a dose- and time-dependent manner. The levels of PlGF protein secretion increased about 3.1- and 3.8-fold after 24-h exposure to 0.4 and 0.8 mM arecoline, respectively. Pretreatment with antioxidant N-acetyl-l-cysteine (NAC) and ERK inhibitor PD98059, but not NF-κB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580, and PI3-K inhibitor LY294002, significantly reduced arecoline-induced PlGF protein synthesis. ELISA analyses demonstrated that NAC and PD98059 reduced about 43% and 38% of the arecoline-induced PlGF protein secretion, respectively. However, combined treatment with NAC and PD98059 did not show additive effect. Moreover, 10 µM curcumin and 4 mM NAC significantly inhibited arecoline-induced ERK activation. Furthermore, 10 µM curcumin completely blocked arecoline-induced PlGF mRNA expression. CONCLUSION: Arecoline-induced PlGF synthesis is probably mediated by reactive oxygen species/ERK pathways, and curcumin may be an useful agent in controlling oral carcinogenesis.

Connective tissue growth factor modulates oral squamous cell carcinoma invasion by activating a miR-504/FOXP1 signalling.

Connective tissue growth factor (CTGF) is a multi-functional secreted protein, and it has been shown either to promote or suppress tumor progression among different kinds of cancers. Here, we investigated the role of CTGF in oral squamous cell carcinoma (OSCC) invasion and metastasis. In five OSCC cell lines, endogenous CTGF negatively correlated with invasiveness. Exogenous CTGF protein or forced expression of CTGF gene in the oral cancer cell line SAS significantly decreased their invasive and migratory abilities. MicroRNA (miRNA) microarray analysis was performed in CTGF-overexpressed SAS cells (SAS/CTGF-M3) versus control cells to investigate the mechanism of CTGF-mediated inhibition of OSCC invasion. Among the miRNAs regulated by CTGF, miR-504 and miR-346 were the top two miRNAs downregulated in CTGF transfectants, and the result was confirmed by quantitative reverse transcriptase-PCR. Ectopic miR-504 increased migration and invasion in SAS/CTGF-M3, however, miR-346 did not have such impact on migration/invasion. Furthermore, we identified FOXP1, a member of forkhead transcription factors, as a target gene that takes part in the miR-504-induced cellular invasion. Knockdown of FOXP1 increased invasiveness in SAS/CTGF-M3, confirming the signal axis of CTGF/miR-504/FOXP1 in OSCC. Animal experiments showed that SAS/CTGF-M3-formed orthotopic tumors were associated with a lesser invasive phenotype than control cells. Expression of miR-504 in SAS/CTGF-M3 increased lymph node metastasis, and co-expression of FOXP1 in miR-504-transfected SAS/CTGF-M3 alleviated miR-504-induced metastasis. In OSCC samples, high CTGF was associated with a lower clinical stage and a better outcome. A reverse correlation between CTGF and miR-504, miR-504 and FOXP1, and a positive correlation between CTGF and FOXP1 were shown. Our study discovers a novel signal pathway involving the regulation of miRNA machinery by a secreted cytokine, which will be beneficial for developing therapeutic strategy against advanced OSCC.

Nitric oxide promotes the progression of periapical lesion via inducing macrophage and osteoblast apoptosis.

This study aimed to elucidate the modulation by nitric oxide (NO) of the apoptosis of macrophages and osteoblasts, the essential cellular components in the development of periapical lesions. Lipopolysaccharide (LPS) induced prominent nitrite synthesis in J774 mouse macrophage cell lines. Exposure to LPS induced obvious apoptosis in J774 cells, whereas transient transfection with murine inducible nitric oxide synthase (iNOS), small interfering RNA (siRNA) diminished this effect. Tumor necrosis factor-alpha (TNF-alpha) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) (a NO donor) triggered apoptosis in UMR-106 rat osteoblastic cell lines and a synergistic effect was noted when TNF-alpha and SNAP were added to the medium together. Administration of siRNAs for c-Fos and c-Jun: components of activator protein-1 (AP-1) and transforming growth factor-beta1 attenuated the combined effect markedly. Terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) stain in a rat model of induced periapical lesion showed positive apoptotic signals in macrophages and osteoblasts. Administration of N(G)-monomethyl-l-arginine markedly diminished the extent of bone loss and the amounts of apoptotic macrophages and osteoblasts. In conclusion, NO mediates LPS-stimulated apoptosis of macrophages. It also induces osteoblast apoptosis and augments the pro-apoptotic effect of cytokines. Inhibition of NO synthesis in vivo attenuates apoptosis and the size of periapical lesions. Taken together, these results suggest that NO may promote the progression of periapical lesion by inducing the apoptosis of macrophages and osteoblasts.

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis.

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.

Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes.

Norcantharidin (NCTD) is the demethylated analogue of cantharidin. In this study, multi-parameter assessments of morphological alterations, clonogenic efficiency, cell growth curves, DNA synthesis, and DNA strand break were employed to determine and compare the cytotoxic effects of NCTD on oral cancer KB cell line and normal buccal keratinocytes. The results showed NCTD induced significant cytotoxicity in KB cells after 24 h of exposure. Normal buccal keratinocytes were more resistant to NCTD induced cytotoxicity. The IC(50) of 24 h NCTD treatment for KB and keratinocytes were 15.06 and 216.29 microg/ml, respectively with a keratinocyte/KB selective index of 14.36. Anoikis and membrane blebbing, morphological characterization of apoptosis, were observed in about 90% of KB cells after exposure to 100 microg/ml of NCTD for 24 h compared to about 30% in keratinocytes. In addition, inhibition of colony formation was noted in KB cells even when exposed to low concentration of drug (5 microg/ml) for a short period of time (6 h). NCTD inhibited subsequent cell proliferation in KB but growth of normal keratinocytes was retarded only temporarily. NCTD inhibited DNA synthesis in both KB and normal keratinocytes. However, keratinocytes were more sensitive to DNA synthesis inhibition by low dose of NCTD. Significant DNA strand break was noted in KB cells only after cell viability was reduced to less than 60% of the control. In comparison, normal keratinocytes were resistant to NCTD induced DNA strand break. These results indicated KB cells were more sensitive to NCTD induced cytotoxicity compared to normal keratinocytes. NCTD may be of value in treating oral cancers. The underlying mechanisms of the differential actions of NCTD on these two cell types are worthy of further investigations.

High incidence of autoantibodies in Taiwanese patients with oral submucous fibrosis.

BACKGROUND: Previous study has shown a high incidence of autoantibodies including antinuclear (ANA), antismooth muscle (SMA), antigastric parietal cell (GPCA), antithyroid microsomal (TMA), and antireticulin antibodies in a small group of 26 patients with oral submucous fibrosis (OSF). The reasons why some of the OSF patients have high titers of autoantibodies in serum have not been completely explained and no further study on autoantibodies in OSF patients has been done in a large group of patients. METHODS: In this study, we determined the serum levels of ANA, SMA, GPCA, and TMA in a large group of 109 male Taiwanese patients with OSF by an indirect immunofluorescence technique (for ANA, SMA, and GPCA), and by a semiquantitative microtiter particle agglutination test (for TMA). The presence of serum autoantibodies in OSF patients was further correlated with patients' oral habits and the severity of OSF measured by maximum mouth opening (MMO) and sites of involvement. RESULTS: We found that the frequencies of presence of serum ANA (23.9%), SMA (23.9%), and GPCA (14.7%) in OSF patients were significantly higher than those (9.2, 7.3, and 5.5%, respectively) in healthy control subjects (P < 0.01, P < 0.005, and P < 0.05, respectively). Although the frequency of presence of TMA (5.5%) in OSF patients was also greater than that (2.8%) in healthy control subjects, the difference was not significant (P > 0.05). The presence of serum GPCA in OSF patients was significantly associated with daily areca quid (AQ) consumption (P < 0.05). The presence of serum ANA in OSF patients associated with daily AQ consumption was of borderline statistical significance (P = 0.066). However, no significant correlations were demonstrated between the presence of serum autoantibodies in OSF patients and other variables of oral habits, MMO, and sites of involvement. CONCLUSION: In this study, all the 109 OSF patients had AQ chewing habit and 73.4% of the OSF patients swallowed the 'juice' of AQ during the chewing process. The presence of serum GPCA and ANA in OSF patients was associated with daily consumption of AQs. AQ chewing caused mucosal microtrauma, and ulcerations facilitated the diffusion of genotoxic and cytotoxic AQ ingredients into the oral and gastric tissues. Altered autoantigens released from AQ ingredients-damaged cells may induce autoantibody production. Higher frequencies of specific HLA-DR antigens in OSF patients may also help autoantibody production. Therefore, we conclude that the high incidence of autoantibodies in OSF patients may be due to AQ chewing habit, toxic AQ ingredients, and genetic susceptibility of the OSF patients.

Quantitative analysis of immunocompetent cells in oral submucous fibrosis in Taiwan.

Previous studies have shown that the local and systemic upregulation of inflammatory and fibrogenic cytokines and downregulation of antifibrotic cytokines are central to the pathogenesis of oral submucous fibrosis (OSF). The immunocompetent cells, especially the macrophages and lymphocytes, are likely the main source of cytokine synthesis. Therefore, this study used an immunohistochemical method to quantify the T lymphocyte, B lymphocyte and macrophage densities in the epithelium and subepithelial connective tissue of 50 specimens of moderately advanced and advanced OSF and 10 specimens of normal oral mucosa (NOM). The mean T lymphocyte, B lymphocyte and macrophage densities in OSF specimens were 555.2+/-417.4, 63.4+/-44.3 and 66.9+/-76.4 cells/mm(2) in the subepithelial connective tissue and 308.1+/-261.1, 1.4+/-3.5 and 6.6+/-11.9 cells/mm(2) in the epithelium, respectively. These findings suggest that T lymphocytes were the major immunocompetent cells in both the subepithelial connective tissue and epithelium of OSF specimens. Macrophages and B lymphocytes are the minor immunocompetent cells in the subepithelial connective tissue and are only occasionally found in the epithelium of OSF specimens. Similar distribution of immunocompetent cells was also found in NOM specimens. However, the mean T lymphocyte, B lymphocyte and macrophage densities in the subepithelial connective tissue (271.2+/-107.0, 13.3+/-18.4 and 17.3+/-19.1 cells/mm(2), respectively) and the mean T lymphocyte density in the epithelium (97.7+/-51.4) of NOM specimens were significantly lower than the corresponding mean cell densities in OSF specimens. Using frozen tissue sections, we further quantified the CD4+ and CD8+ lymphocyte numbers in eight moderately advanced or advanced OSF specimens. It was found that the CD4+ and CD8+ lymphocyte densities were 213.3+/-140.7 and 101.5+/-72.8 cells/mm(2) in the subepithelial connective tissue of OSF specimens, respectively. The CD4+ to CD8+ lymphocyte ratio was 2.1:1. Our results showed a significant increase in the number of T lymphocytes and macrophages and a predominance of CD4+ lymphocytes over CD8+ lymphocytes in the subepithelial connective tissue of OSF specimens. We conclude that the cellular immune response may play an important role in the pathogenesis of OSF.