Enteric glial cells exert neuroprotection from hyperglycemia-induced damage via Akt/GSK3ß pathway.

OBJECTIVE: Enteric glial cells (EGCs) can activate multiple pathways to inhibit the deleterious effects of acute and chronic insults. Our aim was to test the effect of EGCs on hyperglycemia-induced neuron damage and its underlying intracellular mechanisms. METHODS: A coculture model composed of EGCs and neuroblastoma cells (SH-SY5Y) was established to examine glial-mediated neuroprotection under high glucose conditions. The cell counting assay kit CCK-8 was used to measure cell viability. Flow cytometry was used to measure the induction of reactive oxygen species (ROS), change of mitochondrial membrane potential (MMP), cell cycle distribution, and apoptosis. The expressions of cyclin D1, cyclin E2, Bax, cleaved caspase-3, AKT, p-AKT, GSK-3ß, and p-GSK-3ß were tested using western blot. RESULTS: Exposure to high glucose (≥35 mM) reduced the viability of SH-SY5Y cells in a concentration- and time-dependent manner. Meanwhile, enhanced ROS generation and decrease of MMP were observed in SH-SY5Y cells when treated with high glucose. Furthermore, high glucose also caused SH-SY5Y cells arrest in G2 phase and apoptosis, accompanied by decreasing cyclin D1 and E2, and upregulating Bax and cleaved caspase-3. Coculture EGC lines or EGC-conditioned medium with SH-SY5Y prevented the neurotoxic effects. The p-AKT/AKT and p-GSK-3ß/GSK-3ß ratios were dramatically decreased in SH-SY5Y cells after high glucose incubation, which was restored after coculture with EGCs. CONCLUSIONS: EGCs can protect neurons from hyperglycemia-induced injury by activating the Akt/GSK-3ß pathway.

[Mechanism of Calculus Bovis Sativus in inhibiting hepatocyte lipid deposition based on serum pharmacology].

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.

A Retrospective Analysis on 1330 Adverse Event Reports of Qingkailing in China: Further Perception of Its Risks and Rational Use.

Qingkailing (QKL) is a modern preparation exploited according to the traditional Chinese medicine theory. It becomes the second leading cause of adverse drug events (ADEs) in all traditional Chinese medicine injections. The safety evaluation and rational use of QKL are of special importance. This retrospective study used data from Adverse Drug Reaction Monitoring Center of Hubei Province in China from January 2012 to December 2014. ADE cases induced by QKL were collected and analyzed according to patients' demographics, characteristics of drugs involved, characteristics of ADEs, causality, and outcomes. A total of 1330 qualified ADEs were included. Most ADEs occurred within 30 min after administration and the 0-10 years old age group had the highest number of ADEs. The common ADEs included anaphylactic reaction, dyspnea and nausea. Serious reactions accounted for 5.19%. Combination with cephalosporin (74/146, 50.69%) caused more ADEs than other drugs did. Serious attention should be paid when QKL is used for children, and combination with cephalosporin should be avoided.

Stem cells in cancer therapy: opportunities and challenges.

Metastatic cancer cells generally cannot be eradicated using traditional surgical or chemoradiotherapeutic strategies, and disease recurrence is extremely common following treatment. On the other hand, therapies employing stem cells are showing increasing promise in the treatment of cancer. Stem cells can function as novel delivery platforms by homing to and targeting both primary and metastatic tumor foci. Stem cells engineered to stably express various cytotoxic agents decrease tumor volumes and extend survival in preclinical animal models. They have also been employed as virus and nanoparticle carriers to enhance primary therapeutic efficacies and relieve treatment side effects. Additionally, stem cells can be applied in regenerative medicine, immunotherapy, cancer stem cell-targeted therapy, and anticancer drug screening applications. However, while using stem cells to treat human cancers appears technically feasible, challenges such as treatment durability and tumorigenesis necessitate further study to improve therapeutic performance and applicability. This review focuses on recent progress toward stem cell-based cancer treatments, and summarizes treatment advantages, opportunities, and shortcomings, potentially helping to refine future trials and facilitate the translation from experimental to clinical studies.

Protective Effects of Ginsenosides on 17[Formula: see text]-Ethynyelstradiol-Induced Intrahepatic Cholestasis via Anti-Oxidative and Anti-Inflammatory Mechanisms in Rats.

The present study was designed to assess the effects and potential mechanisms of ginsenosides on 17[Formula: see text]-ethynyelstradiol (EE)-induced intrahepatic cholestasis (IC). Ginsenoside at doses of 30, 100, 300[Formula: see text]mg/kg body weight was intragastrically (i.g.) given to rats for 5 days to examine the effect on EE-induced IC. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were measured. Hepatic malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Protein expression of proinflammatory cytokines TNF-[Formula: see text], IL-6 and IL-1[Formula: see text] was analyzed by immunohistochemistry and Western blot. Results indicated that ginsenosides remarkably prevented EE-induced increase in the serum levels of AST, ALT, ALP and TBA. Moreover, the elevation of hepatic MDA content induced by EE was significantly reduced, while hepatic SOD activities were significantly increased when treated with ginsenosides. Histopathology of the liver tissue showed that pathological injuries were relieved after treatment with ginsenosides. In addition, treatment with ginsenosides could significantly downregulate the protein expression of TNF-[Formula: see text], IL-6 and IL-1[Formula: see text] compared with EE group. These findings indicate that ginsenosides exert the hepatoprotective effect on EE-induced intrahepatic cholestasis in rats, and this protection might be attributed to the attenuation of oxidative stress and inflammation.

Baicalin may alleviate inflammatory infiltration in dextran sodium sulfate-induced chronic ulcerative colitis via inhibiting IL-33 expression.

AIMS: To investigate the therapeutic effect of baicalin treatment in chronic ulcerative colitis (UC), and explore the potential anti-inflammation mechanism(s) via IL-33 pathway. MAIN METHODS: UC model were established by giving three cycles of 5-day 2% dextran sodium sulfate (DSS) with two intervals of 14-day recovery in mice, totaling 43days. At the 13th day of the UC modeling, mice received baicalin at doses of 50, 100, or 150mg/kg, respectively. Disease activity index (DAI) assessment as well as HE and PAS staining were performed. Serum levels of TNF-α, IL-1ß and IL-6 were determined by ELISA. Myeloperoxidase (MPO) activity and nitric oxide (NO) contents in colon were measured. The expressions of IL-33 and Ly6/G were examined by immunochemistry. And contents of IL-33 protein and NF-κB-related proteins were tested by Western blot. KEY FINDINGS: Morphological and histological analyses revealed that baicalin administration had a significant effect on reducing the severity of DSS-induced UC in mice. Besides, baicalin treatment significantly reduced the levels of MPO and NO. Moreover, increased levels of inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, have been identified in damaged colon tissue, which was noticeably reduced by baicalin treatment. Our data demonstrated that protein levels of IL-33 and NF-κB p65 were elevated in colon tissues of chronic UC mice. Baicalin treatment significantly suppressed levels of IL-33 and NF-κB p65, whereas levels of IκB-α were increased. SIGNIFICANCE: Baicalin treatment effectively alleviated DSS-induced chronic UC, and the protective mechanisms may involve inhibition of IL-33 expression and subsequent NF-κB activation.

Upregulation of PDZK1 by Calculus Bovis Sativus May Play an Important Role in Restoring Biliary Transport Function in Intrahepatic Cholestasis.

Intrahepatic cholestasis is a main cause of hepatic accumulation of bile acids leading to liver injury, fibrosis, and liver failure. Our previous studies proved that Calculus Bovis Sativus (CBS) can restore biliary transport function through upregulating the multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) in 17α-ethynylestradiol- (EE-) induced intrahepatic cholestasis rats. The regulation mechanism of CBS on these transporters, however, remains unclear. This study was designed to evaluate the possible relationship between the effect of CBS on transport activities and the regulation of CBS on the expression of PDZK1, a mainly scaffold protein which can regulate MRP2 and BCRP. Intrahepatic cholestasis model was induced in rats with injection of EE for five consecutive days and then the biliary excretion rates and cumulative biliary excretions were measured. The mRNA and protein expression levels of PDZK1 were detected by reverse transcription-quantitative real-time polymerase chain reaction, western blot, and immunohistochemical analysis. When treated with CBS, cumulative biliary excretions and mRNA and protein expressions of PDZK1 were significantly increased in intrahepatic cholestasis rats. This study demonstrated that CBS exerted a beneficial effect on EE-induced intrahepatic cholestasis rats by restoring biliary transport function, which may result from the upregulation of PDZK1 expression.

Intestinal injury in the rat model of 17α-ethynylestradiol-induced intrahepatic cholestasis.

OBJECTIVE: Although the intimate relationship between liver and gut has been previously reported under physiological and pathological conditions, intestinal involvement in the process of intrahepatic cholestasis of pregnancy remains unclear. The aim of this study was to investigate intestinal changes in 17α-ethynylestradiol (EE)-induced cholestatic rat model. METHODS: Liver injury was assessed by HE stain and serum biochemical parameters were measured. Intestinal transit was determined using ink marks. Neuronal protein expressions in the intestine were analyzed by Western blot. RESULTS: EE treatment induced liver damage, including severe bile duct hyperplasia, portal edema, portal infiltration, a loss of hepatic structure in periportal areas and increased serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total bilirubin. Large areas of inflammatory cell infiltration and increased myeloperoxidase activity were observed in the intestine of EE-induced cholestatic rats. The EE-treated group showed increased intestinal transit and malondialdehyde levels, while the glutathione content and superoxide dismutase activity were notably decreased, together with decreased protein gene product 9.5 and neuronal nitric oxide synthase expression in the ileum and colon. Furthermore, choline acetyltransferase expression was significantly decreased in the ileum, whereas no change was observed in the colon of EE-treated rats. CONCLUSION: EE-induced liver damage is associated with oxidative stress, inflammation and neural loss in the intestine, which may lead to altered intestinal motility.

Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways.

INTRODUCTION: Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. METHODS: hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. RESULTS: Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. CONCLUSIONS: Biodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways.

Effect of Biodentine™ on the proliferation, migration and adhesion of human dental pulp stem cells.

OBJECTIVES: To investigate the proliferative, migratory and adhesion effect of Biodentine™, a new tricalcium silicate cement formulation, on the human dental pulp stem cells (hDPSCs). METHODS: The cell cultures of hDPSCs obtained from impacted third molars were treated with Biodentine™ extract at four different concentrations: Biodentine™ 0.02mg/ml (BD 0.02), Biodentine™ 0.2mg/ml (BD 0.2), Biodentine™ 2mg/ml (BD 2) and Biodentine™ 20mg/ml (BD 20). Human dental pulp stem cells proliferation was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) viability analysis at different times. Migration was investigated by microphotographs of wound healing and transwell migration assays. Adhesion assay was performed as well in presence of BD 0.2, BD 2 and blank control, while qRT-PCR (quantitative real-time reverse-transcriptase polymerase chain) was used for further analysis of the mRNA expression of chemokine and adhesion molecules in hDPSCs. RESULTS: Biodentine™ significantly increased proliferation of stem cells at BD 0.2 and BD 2 concentrations while decreased significantly at higher concentration of BD 20. BD 0.2 concentration had a statistically significant increased migration and adhesion abilities. In addition, qRT-PCR results showed that BD 0.2 could have effect on the mRNA expression of chemokines and adhesion molecules in human dental pulp stem cells. CONCLUSIONS: The data imply that Biodentine™ is a bioactive and biocompatible material capable of enhancing hDPSCs proliferation, migration and adhesion abilities. CLINICAL SIGNIFICANCE: Biodentine™ when placed in direct contact with the pulp during pulp exposure can positively influence healing by enhancing the proliferation, migration and adhesion of human dental pulp stem cells.

[Design and implementation of a long wavelength near infrared spectrometer based on MEMS scanning mirror].

Long Wavelength Near InfraRed (LW-NIR) spectrometer has wide applications. Miniaturization and low-cost are two major goals of the development of LW-NIR spectrometer in the industrial or research community. Under the background that having a trend of spectrometer miniaturization and integration, method and main problems involved in miniaturization of LW-NIR spectrometer through MEMS scanning mirror, such as the design strategy of the light-splitting optical system, selection considerations of the MEMS scanning mirror, design method of the preamplifier circuit, etc, have been presented in detail. A prototype of miniaturized LW-NIR spectrometer, with the spectrum range of detection of 900-2,055 nm, is designed and implemented using MEMS scanning mirror, InGaAs single detector unit with high sensitivity. Littrow optical layout is used for its light-splitting optical system, and the spectral resolution is between 9.4-16 nm at 1,000-1,965 nm detection wavelength range. The prototype is successfully applied in LW-NIR spectrum measurement on pure water and ethanol aqueous solution, and a forecast analysis on ethanol aqueous solution concentration is also demonstrated. Through adopting MEMS scanning mirror into the spectrometer system, the complexity of the mechanical scanning fixtures and its controlling mechanism is greatly reduced therefore the size of the spectrometer is reduced. Furthermore, due to MEMS scanning mirror technology, LW-NIR spectrometer with single InGaAs detector is achieved, thus the cost reduction of the NIR spectrometer system is also realized because the expensive InGaAs arrays are avoided.

[Study of cbfalpha1 on the expression of extracellular matrix proteins in dental papilla cells induced by BMP-2 in vitro].

PURPOSE: To evaluate the function of cbfalpha1 on BMP-2 signaling to extracellular matrix proteins in dental papilla cells in vitro. METHODS: RT-PCR and Western blot were performed to detect the expression of ALP, OC, ON, OPN, BSP, DMP-1 and DSPP in cultured dental papilla cells induced by 200ng/mL BMP-2 and/or down-regulated by cbfalpha1 antisense technology, the results were analysed with SPSS 11.0 software package. RESULTS: We found that the amount of ALP and OC and the expression of OPN, BSP and ON were upregulated significantly after the cells were treated with BMP-2. After transfected with antisense cbfalpha1, the cells downregulated the expression of ALP, OC, OPN and BSP significantly(P<0.01). CONCLUSIONS: As a kind of transcription factor, cbfalpha1 could be an important tache in the BMP-2 signal networks controlling cells differentiation and mineralization.

[Transcriptional regulation of dentin sialophosphoprotein by c-Jun/c-Fos].

OBJECTIVE: To investigate the role of c-Jun and c-Fos as transcriptional factors in regulation of dentin sialophosphoprotein (DSPP) gene by a promoter-luciferase reporter gene construct in odontoblast cell line MDPC-23. METHODS: Endogenous c-Jun or c-Fos protein was determined by immunocytochemistry. The role of c-Jun or c-Fos in transcription of DSPP was investigated in co-transfection experiments using promoter-luciferase reporter gene construct containing the sequence between -791 bp and +54 bp of mouse DSPP gene. RESULTS: c-Jun and c-Fos was expressed by MDPC-23 cells, and located in the nucleus of MDPC-23 cells. Overexpression of c-Jun or c-Fos significantly inhibited luciferase activity of DSPP promoter. CONCLUSION: These findings suggest c-Jun and c-Fos down-regulated the transcription of DSPP gene as a transcriptional factor in odontoblast.

Smad protein mediated transforming growth factor beta1 induction of apoptosis in the MDPC-23 odontoblast-like cell line.

OBJECTIVE: The function of apoptosis and its regulation in odontoblasts remain unclear. In this study, we characterize the possible role of transforming growth factor (TGF)-beta 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-beta1-induced apoptosis in odontoblasts. METHODS: Annexin V/propidium iodide staining, cell Death Detection ELISA and DNA ladder were used to examine the effect of TGF-beta1 on apoptosis in a mouse odontoblast-like cell line, MDPC-23. Stable cell clones expressing Smad2 or Smad3 dominant negative mutants, or wild-type Smad7 were constructed to investigate the role of Smad proteins in the mediation of apoptosis by TGF-beta1 in MDPC-23 cells. The TGF-beta1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-beta-responsive reporter gene, p3TP-Lux. RESULTS: TGF-beta1 can induce apoptotic cell death in MDPC-23 cells in a dose-dependent manner. Transfection of dominant negative mutant forms of Smad2 or Smad3 blocked TGF-beta1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-beta1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-beta1-induced transcriptional activity. CONCLUSION: These results suggest that Smad proteins are involved in TGF-beta1-induced apoptosis of odontoblast cells.

[Role of Smad signaling in transcription of Smad7 gene mediated by TGF-beta1 in odontoblast cell line MDPC-23].

PURPOSE: To investigate the role of Smad signaling in transcription of Smad7 gene mediated by TGF-beta1 in odontoblast cell line MDPC-23, and to explore the molecular mechanism of Smad7 gene expression mediated by TGF-beta1 at the transcriptional level. METHODS: Smad function and its role in transcription of Smad7 were investigated in cotransfection experiments using Smad7 promoter-luciferase reporter construct containing the sequence between -408 bp and +112 bp of mouse Smad7 gene. The data were analysed by one-way ANOVA. RESULTS: When the Smad7 promoter-luciferase reporter gene construct was expressed in MDPC-23 cells, its transcriptional activity was significantly induced by TGF-beta1 treatment, whereas not by BMP-2 treatment. Overexpression of Smad1, 2, 4, or 5 had no effect on transcriptional activity of Smad7 promoter. Overexpression of Smad3 markedly promoted transcriptional activity of Smad7 promoter, whereas co-transfection of Smad3 and Smad4 doubled the effect of Smad3. Overexpression of Smad3 dominant negative mutant or Smad3 antisense cDNA (AS-Smad3) significantly inhibited transcriptional activity of Smad7 promoter induced by TGF-beta1. CONCLUSION: TGF-beta1 regulated transcription of Smad7 gene through association of Smad3 and Smad4 in MDPC-23 cells.

[Bone morphogenetic protein-2-induced alpha 2 (I) collagen expression in odontoblastic MDPC-23 cells mediated by Smad proteins].

OBJECTIVE: To characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23. METHODS: Endogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct. RESULTS: MDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant. CONCLUSIONS: Smad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

TGF-beta activated Smad signalling leads to a Smad3-mediated down-regulation of DSPP in an odontoblast cell line.

OBJECTIVE: Transforming growth factor-beta (TGF-beta) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-beta have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-beta. In this study, we characterise the role of Smad proteins as mediators of TGF-beta in a mouse odontoblast cell line MDPC-23. METHODS: Transcription of Smads was detected by RT-PCR. The change of intracellular location of Smad proteins treated by TGF-beta1 was evaluated immunocytochemically. Smad function and its role in transcription of dentin sialophosphoprotein (DSPP) were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: MDPC-23 cells expressed Smad2, Smad3 and Smad4 mRNA. Endogenous Smad2, Smad3 and Smad4 rapidly translocated from the cytoplasm into the nucleus in response to TGF-beta1. The activity of the TGF-beta-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-beta1 treatment. Over-expression of wild-type Smad3 promoted TGF-beta1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-beta1 also inhibited the activity of DSPP promoter luciferase reporter construct containing the sequence between -791 bp and +54 bp of the mouse DSPP gene. Over-expression of wild-type Smad3 potentiate the inhibitory effect of TGF-beta1 on transcriptional regulation of DSPP, while dominant negative Smad3 decreased the effect. In contrast to Smad3, wild-type Smad2 or its dominant negative mutant had little effect on TGF-beta1 regulation of the promoter activity of DSPP. CONCLUSIONS: Smad2, Smad3 and Smad4 are present and activated by TGF-beta1 in MDPC-23 cells. The Smad pathway is functional in these cells and Smad3 appears to be involved in down-regulation of DSPP by TGF-beta1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.