ABSTRACT
Melatonin is the major hormone released from the pineal gland and regulates a variety of physiological and pathophysiological processes. According to the recent studies the melatonin plays an important role in regulation of bone growth. The purpose of this study was to determine whether melatonin promotes the cell differentiation and nodules formation in MDPC-23 pre-odontoblast cell line. MDPC-23 cells were cultured for up to 15 days in growth media containing differentiation medium with melatonin or without melatonin. Cultures were stained with Alizarin-S. The expression of the mRNAs for DSPP, OC, ALP and NFI-C were analyzed by RT-PCR. The results were as follows. Cultures containing melatonin at day 15 showed extensive mineralization as compared with control cultures. Melatonin increased the expression of DSPP and OC mRNAs in MDPC-23 cells in a concentration-dependent manner. However, melatonin did not changed ALP expression. Melatonin markedly decreased mRNA expression of NFI-C in early stage cultures as compared with control cultures. These results demonstrated that melatonin is capable of promoting MDPC-23 cells differentiation and mineralization and suggested that melatonin may play an important role in dentin formation.
Subject(s)
Bone Development , Cell Differentiation , Cell Line , Dentin , Melatonin , Pineal Gland , RNA, MessengerABSTRACT
This study was aimed to elucidate the biological function of OD314 (Apin protein), which is related toameloblast differentiation and amelogenesis. Apin protein, calcifying epithelial odontogenic (pindborg) tumors (CEOTs)-associated amyloid, were isolated from CEOTs, and has similar nucleotide sequences to OD314. We examined expression of the OD314 mRNA using in-situ hybridization during tooth development in mice. Expression of OD314 and several enamel matrix proteins were examined in the cultured ameloblast cell line up to 28 days by reverse transcription-polymerase chain reaction (RT-PCR) amplification. After inactivation and over-expression of the OD314 gene in ameloblast cell lines using U6 vector-driven RNA interference and CMV-OD314 construct, RT-PCR were performed to evaluate the effect of the OD314 during amelogenesis. The results were as follows: 1. In in-situ hybridization, OD314 mRNAs were more strongly expressed in ameloblast than odontoblast. 2. When ameloblast cells were cultured in the differentiation and mineralization medium for 28 days, the tuftelin mRNA expression was maintained from the beginning to day 14, and then gradually decreased to day 28. The expressions of amelogenin and enamelin were gradually decreased according to the ameloblast differentiation. 3. Inactivation of OD314 by U6-OD314 siRNA construct down-regulated the expression of OD314, MMP-20, and tuftelin, whereas over-expression of OD314 by CMV-OD314 construct up-regulated the expression of OD314 and MMP-20 without change in tuftelin. These results suggest that OD314 is considered as an ameloblast-enriched gene and may play the important roles in ameloblast differentiation and mineralization.
Subject(s)
Animals , Mice , Ameloblasts , Amelogenesis , Amelogenin , Amyloid , Base Sequence , Cell Line , Dental Enamel , Odontoblasts , RNA Interference , RNA, Messenger , RNA, Small Interfering , ToothABSTRACT
Ameloblasts are responsible for the formation and maintenance of enamel which is an epithelially derived protective covering for teeth. Ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions. However, little is known about the differentiation and maturation mechanisms. OD314 was firstly identified from odontoblasts by subtraction between odontoblast/pulp cells and osteoblast/dental papilla cells, even though OD314 protein was also expressed in ameloblast during tooth formation. In this study, to better understand the biological function of OD314 during amelogenesis, we examined expression of the OD314 mRNA and protein in various stages of ameloblast differentiation using in-situ hybridization and immunohistochemistry. The results were as follows : 1. The ameloblast showed 4 main morphological and functional stages referred to as the presecretory, secretory, smooth-ended, and ruffle-ended. 2. OD314 mRNA was expressed in secretory ameloblast and increased according to the maturation of the cells. 3. OD314 protein was not expressed in presecretory ameloblast but expressed in secretory ameloblast and maturative ameloblast. OD314 protein was distributed in entire cytoplasm of secretory ameloblast. However, OD314 was localized at the proxiamal and distal portion of the cytoplasm of smooth-ended and ruffle-ended ameloblast. These results suggest that OD314 may play important roles in the ameloblast differentiation and maturation.
Subject(s)
Ameloblasts , Amelogenesis , Cytoplasm , Dental Enamel , Immunohistochemistry , Odontoblasts , RNA, Messenger , ToothABSTRACT
A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidy-lcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.
Subject(s)
Animals , Mice , Antibodies, Monoclonal/immunology , fas Receptor/immunology , Base Sequence , Carrier Proteins/metabolism , Clone Cells , Cross-Linking Reagents/pharmacology , Diglycerides/metabolism , Enzyme Activation/drug effects , Lipids/metabolism , Molecular Sequence Data , Phospholipase D/metabolism , Phosphorylation/drug effects , Protein Kinase C/metabolism , Signal Transduction/drug effects , Sphingomyelin Phosphodiesterase/metabolism , Tumor Cells, CulturedABSTRACT
Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.
Subject(s)
Mice , Animals , Antibodies, Monoclonal/immunology , fas Receptor/immunology , Bridged-Ring Compounds/pharmacology , Cell Line , Cross-Linking Reagents , Dose-Response Relationship, Immunologic , Enzyme Activation , Genistein/pharmacology , Hydrolysis , Lymphoma/pathology , Type C Phospholipases/antagonists & inhibitors , Phospholipase D/metabolism , Phosphorylation , Phosphorylcholine/metabolism , Solubility , Thiones/pharmacology , Tumor Cells, Cultured , Tyrosine/metabolism , Water/chemistryABSTRACT
Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.
Subject(s)
Mice , Animals , Antibodies, Monoclonal/immunology , fas Receptor/immunology , Bridged-Ring Compounds/pharmacology , Cell Line , Cross-Linking Reagents , Dose-Response Relationship, Immunologic , Enzyme Activation , Genistein/pharmacology , Hydrolysis , Lymphoma/pathology , Type C Phospholipases/antagonists & inhibitors , Phospholipase D/metabolism , Phosphorylation , Phosphorylcholine/metabolism , Solubility , Thiones/pharmacology , Tumor Cells, Cultured , Tyrosine/metabolism , Water/chemistryABSTRACT
Objective : To investigate the influence of smoking, alcohol ingestion, and physical activity on copper and zinc in RBC and serum and serum ceruloplasmin, this study was performed in a cross-sectional study in 113 healthy men aged 20 to 40 years who had no symptomatic liver, heart, gastrointestinal, and other chronic diseases. METHODS: At the men's entry into the study, blood samples were drawn from each subject and immediately centrifuged for analysis of copper, zinc, iron, ceruloplasmin, total cholesterol, and hematocrit. Each man completed a questionnaire that provided information on smoking, amount of alcohol intake, and physical activity. Partial regres sion analysis was performed on confounding variables such as age, body mass index, hematocrit, serum cholesterol, and serum iron. RESULTS: In general linear models, adjustment for confounding variables did not show statistical differences, and there was only an increasing tendency in serum copper in heavy smoker (P=0.0678). There was no difference between high physical activity with mild smokers and lower physical activfty with, heavy smokers. CONCLUSIONS: This study suggested that copper, zinc and eeruloplasmin were not good biomarker for early effect by smoking, alcohol intake and physical activity in young adult. However, selection bias should be considered in evaluation of this result, and a large prospective study will be needed in advance on usefulness of copper, zinc and ceruloplasmin as a marker for risk factors and early change of atherosclerosis.
Subject(s)
Adult , Humans , Male , Young Adult , Atherosclerosis , Body Mass Index , Ceruloplasmin , Cholesterol , Chronic Disease , Copper , Cross-Sectional Studies , Eating , Heart , Hematocrit , Iron , Linear Models , Liver , Motor Activity , Surveys and Questionnaires , Risk Factors , Selection Bias , Smoke , Smoking , ZincABSTRACT
The changes of phospholipase D (PLD) activity were investigated during the courses of apoptotic process induced by tumor necrosis factor (TNF)-alpha or anti-Fas/Apo1 antibody in human premyelocyte HL-60 and murine B cell lymphoma A20 cells. The treatment of recombinant TNF-alpha to HL-60 cells resulted in the increased PLD activity as determined by the phosphatidylethanol formation in the presence of 1% ethanol. The enhancement of PLD activity was also observed in the anti-Fas/Apo1 monoclonal antibody-treated A20 cells. However, the activity of PLD was maximized when HL-60 and A20 cells were treated with either TNF-alpha or anti-Fas/Apo1 monoclonal antibody for 6 h. Both TNF-alpha and anti-Fas/Apo1 monoclonal antibody increased PLD activity in a dose-dependent manner up to 200 U/ml and 200 ng/ml, respectively. When the intracellular activity of protein kinase C (PKC) was interrupted by treatment of calphostin-C, both the PLD activation and the apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody appeared to be inhibited. Since PKC is reported to activate PLD, the results indicate that the intracellular signaling cascade via PLD may play a role in the induction of apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody.