TGF-ß1 stimulates cyclooxygenase-2 expression and PGE2 production of human dental pulp cells: Role of ALK5/Smad2 and MEK/ERK signal transduction pathways.

BACKGROUND/PURPOSES: TGF-ß1 is an important growth factor that may influence the odontoblast differentiation and matrix deposition in the reactionary/reparative dentinogenesis to dental caries or other tooth injuries. TGF-ß1 exerts its effects through various signaling pathways, such as Smads and MAPKs. Cyclooxygenase-2 (COX-2) is a membrane-associated enzyme that produces prostaglandin E2 (PGE2) at sites of pulpal injury and inflammation, which leads to tissue swelling, redness and pain. The purposes of this study were to investigate the differential signal transduction pathways of TGF-ß1 that mediate COX-2 stimulation and PGE2 production in dental pulp cells. METHODS: Pulp cells were exposed to TGF-ß1 with/without SB431542 (an ALK5/Smad2 inhibitor) and U0126 (a MEK/ERK inhibitor). MTT assay was used to estimate cell viability. Enzyme-linked immunosorbent assay (ELISA) was used for measurement of PGE2 levels. RT-PCR and western blot were used to determined COX-2 mRNA and protein, respectively. RESULTS: Exposure to TGF-ß1 (1-10 ng/ml) increased the COX-2 mRNA and protein level of cultured pulp cells. Exposure to TGF-ß1 (0.1-10 ng/mL) significantly stimulated PGE2 production of dental pulp cells. Under the pretreatment of SB431542, the stimulatory effect of TGF-ß1 on COX-2 level of pulp cells was inhibited. Similarly, U0126 also partly inhibited the TGF-ß1-induced COX-2 expression. CONCLUSION: TGF-ß1 increased the COX-2 and PGE2 level of cultured pulp cells. The effect of TGF-ß1 on COX-2 protein expression was associated with ALK5/Smad2/3 and MEK/ERK pathways. These events are important in the early inflammation, repair and regeneration of dental pulp in response to injury.

Transforming growth factor beta 1 increases collagen content, and stimulates procollagen I and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: Role of MEK/ERK and activin receptor-like kinase-5/Smad signaling.

BACKGROUND/PURPOSE: In order to clarify the role of transforming growth factor beta 1 (TGF-ß1) in pulp repair/regeneration responses, we investigated the differential signaling pathways responsible for the effects of TGF-ß1 on collagen turnover, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human dental pulp cells. METHODS: Pulp cells were exposed to TGF-ß1 with/without pretreatment and coincubation by 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene (U0126; a mitogen-activated protein kinase kinase [MEK]/extracellular signal-regulated kinase [ERK] inhibitor) and 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H- imidazol-2-yl)-benzamide hydrate (SB431542; an activin receptor-like kinase-5/Smad signaling inhibitor). Sircol collagen assay was used to measure cellular collagen content. Culture medium procollagen I, TIMP-1, and MMP-3 levels were determined by enzyme-linked immunosorbent assay. RESULTS: TGF-ß1 increased the collagen content, procollagen I, and TIMP-1 production, but slightly decreased MMP-3 production of pulp cells. SB431542 and U0126 prevented the TGF-ß1-induced increase of collagen content and TIMP-1 production of dental pulp cells. CONCLUSION: These results indicate that TGF-ß1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with activin receptor-like kinase-5/Smad2/3 and MEK/ERK signaling.

Signaling pathways for induction of platelet aggregation by SAS tongue cancer cells--a mechanism of hematogenous metastasis.

BACKGROUND: Tongue cancer metastasis is mainly through blood stream and possibly associated with tumor cell-induced platelet aggregation (TCIPA). METHODS: Platelet aggregation was induced by different amounts of SAS tongue cancer cells with/without inhibitors and the latent period for induction of platelet aggregation was recorded. Gene expression was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: SAS cells (4 x 10(4) to 1 x 10(6) cells/ml) induced platelet aggregation in a cell density-dependent manner. The latent period for induction of platelet aggregation reduced from 11.3 min (2 x 10(5) cells/ml) to 0.9 min (5 x 10(5) cells/ml). The extent of platelet aggregation increased from 39% to 76% by 2 x 10(5) and 5 x 10(5) SAS cells. Pre-treatment of SAS cells with aspirin showed little effect on its induction of platelet aggregation. SAS cells expressed tissue factor (TF) mRNA and the SAS cells-induced TCIPA was inhibited by TF neutralization antibody (5-20 microg/ml), heparin (5-10 U/ml), Hirudin fragment 54-65 (50 microg/ml) and D-Phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. But areca nut (AN, a betel quid component known to generate reactive oxygen species (ROS)) extract showed little effect on TF expression in SAS cells. Pre-treatment with U73122 and 2-aminoethoxydiphenylborate inhibited SAS-induced TCIPA. Interestingly, catalase suppressed SAS cells-induced TCIPA, whereas AN extract enhanced this event. CONCLUSIONS: These results suggest that tongue cancer cells may induce TCIPA and enhance tumor metastasis. SAS-induced TCIPA is related to TF secretion, thrombin generation and associated with Phospholipase C-Inositol triphosphate signaling and ROS production. Betel quid chewing may potentially promote tongue cancer metastasis.

Growth and differentiation factor-5 regulates the growth and differentiation of human dental pulp cells.

INTRODUCTION: Growth and differentiation factor-5 (GDF-5) is a multifunctional protein that regulates the development and repair in many tissues. The purpose of this study was to investigate whether GDF-5 may influence the proliferation, differentiation, and collagen turnover of human dental pulp cells. METHODS: Human dental pulp cells were treated with different concentrations of GDF-5 (0-500 ng/mL). Morphology of pulp cells was observed under a microscope. Cell proliferation was evaluated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Immunofluorescent assay was used to observe the percentages of cell mitosis. Collagen content was measured by Sircol collagen assay. Tissue inhibitor of metalloproteinase-1 level in the culture medium was measured with enzyme-linked immunosorbent assay and Western blotting. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining and ALP enzyme activity assay. RESULTS: After exposure of dental pulp cells to various concentrations of GDF-5, cell number was up-regulated significantly in dose-dependent manner. GDF-5 also stimulated mitosis of dental pulp cells as indicated by an increased percentage of binucleated cells from 28% to 35%-45%. GDF-5 did not affect the collagen content and tissue inhibitor of metalloproteinase-1 level of pulp cells. GDF-5 decreased the ALP activity of pulp cells as analyzed by ALP staining and enzyme activity assay, with 14%-44% of inhibition. CONCLUSIONS: GDF-5 revealed mitogenic and proliferative activity to dental pulp cells. GDF-5 showed inhibitory effect on ALP activity but little effect on the collagen turnover. These events are crucial in specific stages of dental pulp repair and regeneration. GDF-5 may be potentially used for tissue engineering of pulp-dentin complex.

Transforming growth factor ß1 down-regulates Runx-2 and alkaline phosphatase activity of human dental pulp cells via ALK5/Smad2/3 signaling.

OBJECTIVE: Transforming growth factor ß1 (TGF-ß1) plays a role in repair and dentinogenesis in dental pulp. The purpose of this study was to study how TGF-ß1 affects 2 differentiation markers, Runt-related transcription factor 2 (Runx-2) and ALP, in dental pulp cells. STUDY DESIGN: Primary-cultured human dental pulp cells were treated with TGF-ß1 with or without pretreatment and coincubation with 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U0126, a mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor), Noggin (a bone morphogenetic protein inhibitor), or 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate (SB431542, an activin receptor-like kinase (ALK) 5/Smad2/3 inhibitor). The differentiation status of pulp cells was evaluated by ALP staining and quantitative ALP activity assay. Changes in ALP and Runx-2 mRNA expression were determined by reverse-transcription polymerase chain reaction. RESULTS: Cells under the treatment of TGF-ß1 (5 and 10 ng/mL) showed a decrease in ALP activity and gene expression of ALP and Runx-2. Pretreatment by U0126 and Noggin was not effective to prevent the TGF-ß1-induced decline of ALP activity. Interestingly, SB431542 prevented the TGF-ß1-induced decline of ALP activity and ALP and Runx-2 gene expression. CONCLUSION: TGF-ß1 down-regulates Runx-2 and ALP in human dental pulp cells via ALK5/Smad2/3 signaling. These events may play important roles at specific stages of pulpal repair and dentinogenesis.

The role of reactive oxygen species and hemeoxygenase-1 expression in the cytotoxicity, cell cycle alteration and apoptosis of dental pulp cells induced by BisGMA.

Biocompatibility of dentin bonding agents (DBAs) and resin composite is important to preserve the pulp vitality after operative restoration. Bisphenol-glycidyl-methacrylate (BisGMA) is one common monomer adding into DBAs and resin. In this study, we found that exposure of human dental pulp cells to BisGMA (>0.1 mM) led to cytotoxicity, G2/M cell cycle arrest and apoptosis as analyzed by propidium iodide (PI) and PI/annexin V dual fluorescent flow cytometry. These events were associated with a decline of cdc2, cdc25C and cyclinB1 expression at both mRNA and protein levels. BisGMA also induced the expression of hemeoxygenase-1 (HO-1), an oxidative stress responsive gene, in pulp cells. Catalase could prevent the BisGMA-induced alteration of cell cycle-related genes (cdc2, cdc25C, cyclinB1) and HO-1 expression in dental pulp cells. Interestingly, Zn-protoporphyrin (2.5-5 microM), a HO inhibitor, enhanced the BisGMA-induced reactive oxygen species (ROS) production and cytotoxicity. These results suggest that exposure to higher concentrations of BisGMA may stimulate ROS production, cell cycle arrest, apoptosis and cell death. Inducing the expression of HO-1 in dental pulp cells by BisGMA is mediated by ROS production and important to protect dental pulp against the toxicity by monomers present in composite resin and DBAs.

Prostaglandin F(2alpha)-induced interleukin-8 production in human dental pulp cells is associated with MEK/ERK signaling.

Prostaglandin F(2alpha) (PGF(2alpha)) and interleukin-1beta (IL-1beta) levels are elevated in inflamed dental pulp. The roles of IL-1beta and PGF(2alpha) in the pathogenesis of pulpal inflammation await investigation. We found that IL-1beta stimulated PGF(2alpha) production of human dental pulp cells. IL-1beta and PGF(2alpha) (0.5-10 mumol/L) also induced IL-8 production and mRNA expression in pulp cells. Aspirin inhibited IL-1beta-induced PGF(2alpha), but not IL-8 production. PGF(2alpha)-induced IL-8 production and mRNA expression were inhibited by U0126 (an inhibitor of mitogen-activated protein kinase kinase [MEK1/2]) inhibitor), whereas SQ22536 (an adenylate cyclase inhibitor) enhanced this event. These results indicate that IL-1beta-induced IL-8 production in pulp cells is not mainly via direct activation of cyclooxygenase and PGF(2alpha) generation. PGF(2alpha)-induced IL-8 production is possibly via activation of MEK/extracellular signal-regulated kinase signaling, but not by activation of adenylate cyclase. IL-1beta and PGF(2alpha) might involve the pathogenesis of pulpal inflammation via induction of IL-8 production.

The effect of BisGMA on cyclooxygenase-2 expression, PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and -independent pathways.

After operative restoration, some monomers released from dentin bonding agents or composite resin may induce tissue inflammation and affect the vitality of dental pulp. Whether BisGMA, a major monomer of composite resin, may induce prostaglandin release and cytotoxicity to pulp cells and their mechanisms awaits investigation. We found that BisGMA induced cytotoxicity to human dental pulp cells at concentrations higher than 0.075 mm as analyzed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. BisGMA (0.1 mm) also stimulated ERK phosphorylation, PGE(2) production, COX-2 mRNA and protein expression as well as ROS production (as indicated by an increase in cellular DCF fluorescence) in dental pulp cells. Catalase (500 and 1000 U/ml) and U0126 (10 and 20 microm, a MEK inhibitor) effectively prevented the BisGMA-induced ERK activation, PGE(2) production and COX-2 expression. Moreover, catalase can protect the pulp cells from BisGMA cytotoxicity, whereas aspirin and U0126 lacked of this protective activity. These results suggest that BisGMA released from composite resin may potentially affect the vitality of dental pulp and induce pulpal inflammation via stimulation of ROS production, MEK/ERK1/2 activation and subsequent COX-2 gene expression and PGE(2) production. Cytotoxicity of BisGMA to dental pulp cells is related to ROS production, but not directly mediated by MEK activation and PGE(2) production.