Orthodontic force regulates metalloproteinase-3 promoter in osteoblasts and transgenic mouse models.

BACKGROUND/PURPOSE: Previously we demonstrated up-regulation of matrix metalloproteinase-3 (MMP-3) in human osteoblasts under compression and in bony specimens of experimental orthodontic tooth movement (OTM). Here, we studied the temporal characteristics of compression stimulation in human and mouse osteoblast cell lines, and generated a transgenic mouse model for assessing the MMP-3 expression during OTM. MATERIALS AND METHODS: We investigated MMP-3 expressions in human and murine osteoblasts through RT-PCR and luciferase assay, after compressive force loading. Inhibitors were added to identify the possible mechanisms for signal transduction. A human MMP-3 promoter was isolated, cloned and transfected to generate a transgenic mouse with a green fluorescent protein reporter. OTM was then initiated to observe the location and time course of transcriptional regulation of MMP-3 signals. RESULTS: We found changes in the transcription of MMP-3 in response to mechanical force applied to both human and mouse osteoblast cell lines, suggesting that the response is positive across species. Cloned human MMP-3 promoter may cause the response of luciferase to 1% compression. Moreover, p38 inhibitor exerted a down-regulatory effect on MMP-3 promoter expression, although the inhibitory effect didn't reach a significant level. In the transgenic mouse OTM model, we again found increased expression of MMP-3 in response to mechanical force loading around the periodontal ligament. CONCLUSION: Mechanical force can stimulate MMP-3 expression, possibly through the p38 MAPK pathway, with its strongest signal occurring at 24 h. The mechanical responsiveness in MMP-3 promoter regions can be observed in both humans and rodents in vitro and in vivo.

Antiplatelet, antioxidative, and anti-inflammatory effects of hydroquinone.

Platelets play crucial roles in thrombosis and hemostasis through platelet activation and aggregation that are crucial in cardiovascular diseases. Hydroquinone (HQ) and its derivatives are present in many dermatological creams, paints, motor fuels, air, microorganisms, and plant products like wheat bread, fruit, coffee, and red wine. The effect of HQ on humans is not clear. In this study, we found that HQ (>25 µM) inhibited arachidonic acid (AA)-induced platelet aggregation. HQ suppressed AA-induced thromboxane B2 production of platelets. HQ (>10 µM) also attenuated ex vivo platelet-rich plasma aggregation. HQ prevented the interleukin (IL)-1ß-induced 8-isoprostane, and PGE2 production, but not IL-8 production of pulp cells. These results indicate that HQ may have an antiplatelet effect via inhibition of thromboxane production. HQ has antioxidative and anti-inflammatory effects, and possible inhibition of COX. Exposure and consumption of HQ-containing products, food or drugs may have antiplatelet, antioxidative, and anti-inflammatory effects.

Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as Porphyromonas, Fusobacterium, etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.

970 nm low-level laser affects bone metabolism in orthodontic tooth movement.

OBJECTIVE: During orthodontic tooth movement (OTM), the speed of movement depends on the rate of bone turnover. In this study, we used a rat model to investigate the effect of 970 nm low-level laser therapy (LLLT) on OTM under different dose and frequency protocols. METHODS: We first compared the OTM rates between the OTM only control and the OTM + LLLT group (1250 J/cm2) in Experiment 1 and showed that LLLT significantly increased OTM. In Experiment 2, we employed 3 different LLLT protocols: the low-dose group and the high-dose group receiving 5 doses of 750 J/cm2 and 15,000 J/cm2 of LLLT every 3 days, respectively, and the early high-dose group which received 5 daily doses at 15,000 J/cm2 at the beginning of the experiments. The OTM-only control group received no LLLT. Tooth movement rate was measured through sequential silicone impressions. MicroCT was also performed to evaluate bone de-mineralization rate. Bone histmorphometry was used to compare the bone turnover rate between LLLT group and control group. Finally, TRAP, Osteocalcin, and VEGF expression is evaluated by immunohistochemistry (IHC) in tissue sections. RESULTS: When LLLT treatment was given every three days, both the 1250 J/cm2 and 15,000 J/cm2 groups showed significantly increased OTM compared to the control group. No significant difference was observed in the 750 J/cm2 group, or in the early irradiation group, when compared with controls, although 750 J/cm2 showed the same trend of accelerating OTM. The MicroCT result of rat maxilla demonstrated that LLLT increased bone remodeling and showed decreased bone mineral density and bone volume/total volume in the furcation areas of the maxillary first molars at the end of experiment. LLLT without OTM increased bone turnover as evidenced by fluorochrome incorporation. Immunohistochemistry analyses revealed high osteocalcin expression at later stages of OTM in the LLLT group, while VEGF expression was highly induced in the LLLT + OTM group at an early stage. CONCLUSION: Our results suggest that the 970 nm LLLT increases the rate of OTM in a dose-sensitive and frequency-dependent manner. Further animal and human studies are needed to determine the optimal timing and dosage of LLLT for OTM acceleration.

Activation of Lysophosphatidic Acid Receptor 3 Inhibits Megakaryopoiesis in Human Hematopoietic Stem Cells and Zebrafish.

Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that exists in the plasma and platelets. It exerts its functions through activation of various LPA receptors (LPARs), which belong to the family of G protein-coupled receptors. Activation of LPARs has important roles in stem cell differentiation. However, how LPA affects human hematopoietic stem cell (HSC) differentiation remains elusive. In our previous studies, we have suggested that LPA receptor 2 (LPA2) and LPA receptor 3 (LPA3) play opposing roles and may act as a molecular switch during megakaryocytic differentiation in K562 cells. In this study, human CD34+ HSCs and zebrafish are adopted to investigate the roles of LPA3 during megakaryopoiesis/thrombopoiesis in vitro and in vivo. Our results show that LPAR3 mRNA expression level is decreased upon induction by thrombopoietin and stem cell factor in human HSCs. Using pharmacological activators and shRNA knockdown experiments, we demonstrate that activation of LPA3 inhibits megakaryopoiesis in human HSCs. In addition, pharmacological activation of LPA3 suppressed thrombopoiesis in zebrafish. Furthermore, blockage of LPA3 translation by morpholino increased the number of CD41-GFP+ cells in Tg(CD41:eGFP) zebrafish. Moreover, the mRNA expression level of zCD41 increased significantly in LPA3-knockout zebrafish. These results clarify the negative role of LPA3 during megakaryopoiesis and provide important information for potential treatments of related diseases, such as megakaryopenia.

Cytotoxicity and transformation of C3H10T1/2 cells induced by areca nut components.

BACKGROUND/PURPOSE: Betel quid (BQ) chewing is popular in Taiwan and many other countries. There are about 200-600 million BQ chewers in the world. BQ chewing is one major risk factor of oral cancer and oral submucous fibrosis (OSF). While areca nut (AN), a main component of BQ, exhibits genotoxicity, its transformation capacity and its role in the initiation and promotion stages of carcinogenesis are not fully clear. METHODS: Mouse C3H10T1/2 cells were exposed to AN extract (ANE) for 24 hours. Cytotoxicity was evaluated by colony forming efficiency. For the transformation assay, C3H10T1/2 cells were exposed to ANE for 24 hours and then incubated in medium with/without 12-O-tetradecanolylphorbol-13-acetate (TPA; a tumor promoter) for 42 days. Cells were stained with Giemsa and type II and type III transformed foci were counted for analysis of the transformation capacity of ANE. RESULTS: ANE exhibited cytotoxicity to C3H10T/12 cells at concentrations higher than 320 µg/mL as shown by a decrease in colony numbers. ANE (80-640 µg/mL) alone mildly stimulated the transformed foci formation (p > 0.05). In the presence of TPA, ANE (80-640 µg/mL) markedly stimulated the transformed foci formation. The percentage of dishes with foci increased from 0% in controls to 20% in ANE (80 µg/mL and 320 µg/mL)-treated groups and further increased to 65-94% in ANE plus TPA groups. CONCLUSION: These results indicate that ANE is a weak complete carcinogen. ANE is an effective tumor initiator and can induce malignant transformation of C3H10T1/2 cells in the presence of a tumor promoter. ANE may be involved in multistep chemical carcinogenesis by its malignant transformation capacity.

Elastase induces lung epithelial cell autophagy through placental growth factor: a new insight of emphysema pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD.

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.

Toxicity of areca nut ingredients: activation of CHK1/CHK2, induction of cell cycle arrest, and regulation of MMP-9 and TIMPs production in SAS epithelial cells.

BACKGROUND: There are 600 million betel quid chewers around the world. betel quid chewing is a major risk factor of oral cancer. Why betel quid components induce oral cancer is not clear. METHODS: Cytotoxicity of areca nut extract and arecoline (an areca nut alkaloid) to SAS oral epithelial cell line was evaluated by trypan blue dye exclusion and MTT assays. Cell cycle distribution and apoptosis was analyzed by propidium iodide staining flow cytometry. Chk1 and chk2 activation was analyzed by Pathscan phospho-enzyme-linked immunosorbent assay. Metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase (TIMPs) production was measured by enzyme-linked immunosorbent assay. RESULTS: Areca nut extract (800 µg/mL) and arecoline (>0.4 mmol/L) caused cell death, apoptosis, and cell cycle arrest of SAS cells. Areca nut extract and arecoline stimulated Chk1 and Chk2 phosphorylation in SAS cells. Areca nut extract stimulated cellular MMP-9 but suppressed TIMP-1 and TIMP-2 production. CONCLUSIONS: Areca nut components activate Chk1/Chk2, alter cell cycle regulation/apoptosis, MMP-9, and TIMPs production, contributing to the pathogenesis of oral carcinogenesis.

Cloning, expression and characterization of CCL21 and CCL25 chemokines in zebrafish.

Chemokines are a large group of proteins implicated in migration, activation, and differentiation of leukocytes. They are well-surveyed in mammals, but less is known in lower vertebrates about their spatiotemporal expressions and functions. From an evolutionary point of view, comparative analyses may provide some fundamental insights into these molecules. In mammals, CCL21 and CCL25 are crucial for thymocyte homing. Herein, we identified and cloned the zebrafish orthologues of CCL21 and CCL25, and analyzed their expression in embryos and adult fish by in situ hybridization. We found that CCL21 was expressed in the craniofacial region, pharynx, and blood vessels in embryos. In adult fish, CCL21 transcripts were located in the kidney, spinal cord, and blood cells. In contrast, expression of CCL25 was only detected in the thymus primordia in embryos. In adult fish, transcripts of CCL25 were maintained in the thymus, and they were also found in the brain and oocytes. Furthermore, we performed an antisense oligonucleotide experiment to evaluate the biological function of CCL25. Results showed that the recruitment of thymocytes was impeded by morpholino-mediated knockdown of CCL25, suggesting that CCL25 is essential for colonization of T-cells in the thymus in early development. Together, our results demonstrate the basic profiles of two CCL chemokines in zebrafish. The tissue-specific expression patterns may pave the way for further genetic dissection in this model organism.

Antiplatelet effect of phloroglucinol is related to inhibition of cyclooxygenase, reactive oxygen species, ERK/p38 signaling and thromboxane A2 production.

Platelet dysfunction is a major risk factor of cardiovascular diseases such as atherosclerosis, stroke and myocardial infarction. Many antiplatelet agents are used for prevention and treatment of these diseases. In this study, phloroglucinol (2.5-25 µM) suppressed AA-induced platelet aggregation and thromboxane B(2) (TXB(2)) production, but not U46619-induced platelet aggregation. Phloroglucinol (100-250 µM) showed little cytotoxicity to platelets. Phloroglucinol inhibited the COX-1 and COX-2 activities by 45-74% and 49-72% respectively at concentrations of 10-50 µM. At concentrations of 1 and 5 µM, phloroglucinol attenuated the AA-induced ROS production in platelets by 30% and 53%, with an IC(50) of 13.8 µM. Phloroglucinol also inhibited the PMA-stimulated ROS production in PMN. Preincubation of platelets by phloroglucinol (10-25 µM) markedly attenuated the AA-induced ERK and p38 phosphorylation. Intravenous administration of phloroglucinol (2.5 and 5 µmol/mouse) suppressed the ex vivo AA-induced platelet aggregation by 57-71%. Phloroglucinol administration also elevated the mice tail bleeding time. Moreover, phloroglucinol inhibited the IL-1ß-induced PGE(2) production in pulp fibroblasts. These results indicate that antiplatelet and anti-inflammatory effects of phloroglucinol are related to inhibition of COX, ROS and TXA2 production as well as ERK/p38 phosphorylation in platelets. Phloroglucinol further suppress PMA-induced ROS production in PMN. The antiplatelet effect of phloroglucinol was confirmed by ex vivo study. Clinically, the consumption of phloroglucinol-containing food/natural products as nutritional supplement may be helpful to cardiovascular health. Phloroglucinol has potential pharmacological use.

Role of glucose-regulated Protein 78 in embryonic development and neurological disorders.

Glucose-regulated protein 78 (GRP78) is an important chaperone protein that is predominantly expressed in the endoplasmic reticulum. The multifunctional roles of GRP78 in protein folding, endoplasmic reticulum calcium binding, cytoprotection, and anti-apoptosis, as well as its function as a receptor on the cell surface, disclose its major involvement in physiological and numerous pathological conditions. Recent advances in mouse models targeting GRP78 allele have revealed the essential roles of GRP78 in development and neurological disorders, as well as accurate neural migration and neuroprotection. This review of correlation between GRP78 and embryogenesis and neurological disorders provides further directions for investigation, as well as potential therapeutics for clinical use.

Recapitulation of human betaB1-crystallin promoter activity in transgenic zebrafish.

Development of the eye is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process may have been highly conserved during evolution. Herein we analyzed the promoter of the human betaB1-crytallin gene in zebrafish by transgenic experiments. To delineate the evolutionarily conserved regulatory elements, we performed serial deletion assays in the promoter region. The results demonstrated that the -90/+61-bp upstream proximal promoter region is sufficient to confer lens-tissue specificity to the human betaB1-crystallin gene in transgenic zebrafish. Through phylogenetic sequence comparisons and an electrophoretic mobility shift assay (EMSA), a highly conserved cis-element of a six-base pair sequence TG(A/C)TGA, the consensus sequence for the Maf protein binding site, within the proximal promoter region was revealed. Further, a site-mutational assay showed that this element is crucial for promoter activity. These data suggest that the fundamental transcriptional regulatory mechanism of the betaB1-crystallin gene has been well conserved between humans and zebrafish, and plausibly among all vertebrates, during evolution.

Developmental toxicity of arecoline, the major alkaloid in betel nuts, in zebrafish embryos.

BACKGROUND: The major alkaloid in the betel nut, arecoline, has been reported to be potent in inducing developmentally toxic effects by generally lowering the embryo weight and retarding development of the embryo. This study examined the adverse effects of arecoline and tried to unravel the mechanism through the tools of molecular biology. METHODS: Arecoline was administered to zebrafish embryos by incubation at concentrations ranging from 0.01-0.04% (wt/vol) and lethality and morphological changes were recorded. The expression of genes was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. In addition, the protective effects of several antioxidants were tested. RESULTS: The survival rate of treated embryos during a three-day incubation significantly declined as the arecoline concentration increased. Treated embryos showed general growth retardation and lower rate of heartbeat. When examined at the 24-hr stage, the relative amounts of transcripts of p53, p21, and cyclin D1, and the spatial expression patterns of these genes in treated groups, were comparable to those of the untreated early stages of embryos. Finally, the addition of glutathione (GSH) or its precursor, N-acetyl-L-cysteine (NAC), ameliorated the developmental retardation of embryos by arecoline. CONCLUSIONS: Arecoline-treated embryos exhibited general developmental retardation in a dose-dependent manner. Our results from RT-PCR, in situ hybridization, and antioxidant-protection experiments indicate that the mechanism underlying growth retardation by arecoline in embryos is predominantly due to a general cytotoxic effect induced by depletion of intracellular thiols.