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To study the role of sclerostin in periodontal ligament (PDL) as a homeostatic regulator in biophysical-force-induced tooth movement (BFTM).BFTM was performed in rats, followed by microarray, immunofluorescence, in situ hybridization, and real-time polymerase chain reaction for the detection and identification of the molecules. The periodontal space was analysed via micro-computed tomography. Effects on osteoclastogenesis and bone resorption were evaluated in the bone-marrow-derived cells in mice. In vitro human PDL cells were subjected to biophysical forces.In the absence of BFTM, sclerostin was hardly detected in the periodontium except in the PDL and alveolar bone in the furcation region and apex of the molar roots. However, sclerostin was up-regulated in the PDL in vivo by adaptable force, which induced typical transfiguration without changes in periodontal space as well as in vitro PDL cells under compression and tension. In contrast, the sclerostin level was unaffected by heavy force, which caused severe degeneration of the PDL and narrowed periodontal space. Sclerostin inhibited osteoclastogenesis and bone resorption, which corroborates the accelerated tooth movement by the heavy force.Sclerostin in PDL may be a key homeostatic molecule in the periodontium and a biological target for the therapeutic modulation of BFTM.

Introduction Few studies have reported direct pulp capping in inflamed pulp conditions. The purpose of this study was to investigate the in vitro and in vivo responses of dental pulp during direct pulp capping using various pulp capping materials in inflamed conditions. Methods Human dental pulp cells were treated with lipopolysaccharide (LPS) and cultured with Dycal (Dentsply Caulk, Milford, DE), ProRoot MTA (Dentsply Maillefer, Ballaigues, Switzerland), and Endocem MTA (Maruchi, Wonju, South Korea). The expressions of interleukin (IL)-1β, IL-6, dentin matrix protein 1, and dentin sialophosphoprotein were analyzed through real-time polymerase chain reaction. The maxillary molars of Sprague-Dawley rats were exposed for 2 days. The exposed pulps were capped with Dycal, ProRoot MTA, and Endocem MTA and sealed with resin-modified glass ionomer followed by histologic and immunohistochemical analyses. Results The expression of IL-1β and IL-6 was increased with LPS and decreased by Dycal, ProRoot MTA, and Endocem MTA. Dentin matrix protein 1 and dentin sialophosphoprotein levels were decreased with LPS and increased after treatment with pulp capping materials.In the in vivo study, inflammation associated with Dycal was higher than that associated with ProRoot MTA and Endocem MTA at week 1, without any significant difference between the 2. At 4 weeks, inflammation was decreased, and mineralization was increased compared with week 1 in all 3 of the materials. At week 1, IL-6 immunoreactivity was strongly expressed. Dycal exhibited stronger immunoreactivity than ProRoot MTA and Endocem MTA. However, the immunoreactivity was decreased in all groups at week 4. Conclusions Successful direct pulp capping requires more effective pulp capping materials for the treatment of inflamed pulps.

Objective It has been well known that Hedgehog (Hh) signaling plays an important role in bone development, however, its function in cementogenesis has not yet been reported. This study was intended to elucidate the role of Hh signaling in cementoblast differentiation. Design Expression changes of various Hh signaling components and levels of skeletogenic markers (alkaline phosphatase, osteocalcin, osteopontin) and osteogenic transcription factors (RUNX2, Osterix) by Hh signaling modulators during OCCM-30 cementoblast differentiation were determined by quantitative real-time reverse transcriptase polymerase chain reaction. To investigate effects of Hh signaling modulators on the mineralization of cementoblast, alkaline phosphatase and alizarin red S staining were used. Then, the interaction between Hh and BMP signaling during cementoblast differentiation was evaluated using co-treatment of BMP7 and Hh signaling modulators. Results We observed the consistent expression of Hh signaling molecules in the OCCM-30, which were up-regulated during cementoblast differentiation. We also found that the treatment of cells with Purmo, an Hh activator, enhanced cementoblast differentiation by increasing the mRNA expression of skeletogenic markers and osteogenic transcription factors, as well as increasing alkaline phosphate activity and mineralization capability. On the contrary, an Hh antagonist, like Cyclo, effectively inhibited cementoblast differentiation. Furthermore, BMP7 promoted cementoblast differentiation through crosstalk with the Hh signaling. Conclusion These results suggest that Hh signaling is involved in cementoblast differentiation, and Hh signaling molecules may therefore represent new therapeutic targets in periodontal treatment and regeneration.

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Introduction Recent reports have shown that the combined use of platelet-rich fibrin (PRF), an autologous fibrin matrix, and mineral trioxide aggregate (MTA) as root filling material is beneficial for the endodontic management of an open apex. However, the potential of the combination of MTA and PRF as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro has not yet been studied. The purpose of this study was to evaluate the effect of the combination of MTA and PRF on odontoblastic maturation in HDPCs. Methods HDPCs extracted from third molars were directly cultured with MTA and PRF extract (PRFe). Odontoblastic differentiation of HDPCs was evaluated by measuring the alkaline phosphatase (ALP) activity, and the expression of odontogenesis-related genes was detected using reverse-transcription polymerase chain reaction or Western blot. Mineralization formation was assessed by alizarin red staining. Results HDPCs treated with MTA and PRFe significantly up-regulated the expression of dentin sialoprotein and dentin matrix protein-1 and enhanced ALP activity and mineralization compared with those with MTA or PRFe treatment alone. In addition, the combination of MTA and PRFe induced the activation of bone morphogenic proteins (BMP)/Smad, whereas LDN193189, the bone morphogenic protein inhibitor, attenuated dentin sialophosphoprotein and dentin matrix protein-1 expression, ALP activity, and mineralization enhanced by MTA and PRFe treatment. Conclusions This study shows that the combination of MTA and PRF has a synergistic effect on the stimulation of odontoblastic differentiation of HDPCs via the modulation of the BMP/Smad signaling pathway.

The present study is aimed at investigating the effects of the exogenous estrogen 17β-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.

Objective This study aimed to determine the expression of chemokine (C-X-C motif) ligand 14 (CXCL14) in pulpal and periodontal cells in vivo and in vitro, and investigate function of CXCL14 and its underlying mechanism in the proliferation and osteogenic differentiation of human periodontal ligament (hPDL) cells. Methods To determine the expression level of CXCL14 in adult rat oral tissues and in hPDL cells after application of biophysical forces, RT-PCR, western blot, and histological analyses were performed. The role of CXCL14 in proliferation and osteogenic differentiation of PDL cells was evaluated by measuring dehydrogenase activity and Alizarin red S staining. Results Strong immunoreactivity against CXCL14 was observed in the PDL tissues and pulpal cells of rat molar, and attenuated apparently by orthodontic biophysical forces. As seen in rat molar, highly expressed CXCL14 was observed in human dental pulp and hPDL cells, and attenuated obviously by biophysical tensile force. CXCL14 expression in hPDL cells was increased in incubation time-dependent manner. Proliferation of hPDL cells was inhibited dramatically by small interfering (si) RNA against CXCL14. Furthermore, dexamethasone-induced osteogenic mineralization was inhibited by recombinant human (rh) CXCL14, and augmented by CXCL14 siRNA. rhCXCL14 increased transforming growth factor-beta1 (TGF- β1) in hPDL cells. Inhibition of the cell proliferation and osteogenic differentiation of hPDL cells by CXCL14 siRNA and rhCXCL14 were restored by rhTGF-β1 and SB431542, respectively. Conclusion These results suggest that CXCL14 may play roles as a growth factor and a negative regulator of osteogenic differentiation by increasing TGF-β1 expression in hPDL cells.

Background The association between diabetes mellitus (DM) and bone diseases is acknowledged. However, the mechanistic pathways leading to the alveolar bone (AB) destruction remain unclear. This study aims to elucidate the mechanical forces (MF)-induced AB destruction in DM and its underlying mechanism. Methods In vivo periodontal tissue responses to MF were evaluated in rats with diabetes. In vitro human periodontal ligament (PDL) cells were either treated with advanced glycation end products (AGEs) alone or with AGEs and MF. Results In vivo, the transcription of VEGF-A, colony stimulating factor-1 (CSF-1), and Ager was upregulated in diabetes, whereas changes in DDOST and Glo1 mRNAs were negligible. DM induced VEGF-A protein in the vascular cells of the PDL and subsequent angiogenesis, but DM itself did not induce osteoclastogenesis. MF-induced AB resorption was augmented in DM, and such augmentation was morphologically substantiated by the occasional undermining resorption as well as the frontal resorption of the AB by osteoclasts. The mRNA levels of CSF-1 and vascular endothelial growth factor (VEGF) during MF application were highly elevated in diabetes, compared with those of the normal counterparts. In vitro, AGEs treatment elevated Glut-1 and CSF-1 mRNA levels via the p38 and JNK pathways, whereas OGT and VEGF levels remained unchanged. Compressive MF especially caused upregulation of VEGF, CSF-1, and Glut-1 levels, and such upregulation was further enhanced by AGEs treatment. Conclusions Overloaded MF and AGEs metabolites may synergistically aggravate AB destruction by upregulating CSF-1 and VEGF. Therefore, regulating the compressive overloading of teeth, as well as the levels of diabetic AGEs, may prove to be an effective therapeutic modality for managing DM-induced AB destruction.

Human mesenchymal stem cells (hMSCs), characterized by rapid in vitro expandability and multi-differentiation potential, have been widely used in the clinical field of tissue engineering. Recent studies have shown that various signaling networks are involved in the growth and differentiation of hMSCs. Although Wnts and their downstream signaling components have been implicated in the regulation of hMSCs, the role of Wnt signaling in hMSC self-renewal is still controversial. Here, it was observed that activation of endogenous canonical Wnt signaling with LiCl, which decreased β-catenin phosphorylation, leads to a decrease in hMSC proliferation. The fact that this growth arrest is not linked to apoptosis was verified by annexin V-FITC/propidium iodide assay. It was associated with sealing off of the cells in the G1 phase of the cell cycle accompanied by changes in expression of cell cycle-associated genes such as cyclin A and D. In addition, activation of Wnt signaling during hMSC proliferation seemed to reduce their clonogenic potential. On the contrary, Wnt signaling activation during hMSC proliferation had little effect on the osteogenic differentiation capability of cells. These findings show that canonical Wnt signaling is a critical regulator of hMSC proliferation and clonogenicity.

Introduction Mineral trioxide aggregate (MTA) can induce differentiation of the dental pulp cells into odontoblast-like cells and generate a dentin-like mineral structure. The mechanisms underlying MTA-induced odontoblastic differentiation in human dental pulp cells (HDPCs) are not completely understood. The purpose of this study was to evaluate the effect of nifedipine as calcium channel blocker on MTA-induced odontoblastic differentiation in HDPCs. Methods HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured on MTA with or without nifedipine in the culture medium. Cell growth and expression of odontoblastic differentiation markers were determined by using methyl-thiazol-diphenyl-tetrazolium assay and reverse transcription–polymerase chain reaction analysis, respectively. Phosphorylation of mitogen-activated protein kinase was measured by Western blotting, and calcium deposition was assessed by using alizarin red S staining. Results MTA at a concentration of 1 mg/mL significantly up-regulated the expression of dentin sialophosphoprotein and dentin matrix protein-1 and enhanced mineralized nodule formation. However, nifedipine attenuated the MTA-induced odontoblastic differentiation in HDPCs. In addition, MTA-induced mineralization was blocked by inhibition of extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) by using U0126, SB203580, and SP600125, respectively. Furthermore, phosphorylation of ERK and JNK in response to MTA was inhibited when the medium was supplemented with nifedipine. Conclusions This study showed that calcium ions released from MTA play an important role in odontoblastic differentiation of HDPCs via modulation of ERK and JNK activation.

Nitric oxide (NO) is produced by three different isoforms of the enzyme NO synthase (NOS). NOS isoforms are expressed in many cell types, including human dental pulp cells (HDPC). NO acts as an intracellular messenger at physiological levels although it can be cytotoxic at higher concentrations. Epigallocatechin gallate (EGCG), a major green tea polyphenol, has diverse pharmacological activities in cell growth and death. This study is aimed to investigate the apoptotic mechanism by NO and effects of EGCG on NO-induced apoptosis in HDPC. Sodium nitroprusside (SNP), an NO donor, decreased the cell viability of HDPC in a dose- and time-dependent manner. EGCG was administered for 1 hr before the SNP treatment, resulting in increased cell viability and reactive oxygen species (ROS) production inhibition. Expression of Bax, a pro-apoptotic Bcl-2 family, was upregulated, whereas expression of Bcl-2, an anti-apoptotic Bcl-2 family, was downregulated in SNP-treated HDPC. SNP augmented the release of cytochrome c from mitochondria into cytosol and enhanced caspase-9, and -3 activities, a marker of the apoptotic executing stage. EGCG ameliorated caspase-9 and -3 activities and cytochrome c release increased by SNP. These results suggest that EGCG has a protective effect against NO-induced apoptosis in HDPC by scavenging ROS and modulating the Bcl-2 family.

Thiolated chitosan (Thio-CS) is a well-established pharmaceutical excipient for drug delivery. However, its use as a scaffold for bone formation has not been investigated. The aim of this study was to evaluate the potential of Thio-CS in bone morphogenetic protein-2 (BMP-2) delivery and bone formation.In vitrostudy showed that BMP-2 interacted with the Thio-CS and did not affect the swelling behavior. The release kinetics of BMP-2 from the Thio-CS was slightly delayed (70%) within 7 days compared with that from collagen gel (Col-gel, 85%), which is widely used in BMP-2 delivery. The BMP-2 released from Thio-CS increased osteoblastic cell differentiation but did not show any cytotoxicity until 21 days. Analysis of thein vivoectopic bone formation at 4 weeks of posttransplantation showed that use of Thio-CS for BMP-2 delivery induced more bone formation to a greater extent (1.8 fold) than that of Col-gel. However, bone mineral density in both bones was equivalent, regardless of Thio-CS or Col-gel carrier. Taken together, Thio-CS system might be useful for delivering osteogenic protein BMP-2 and present a promising bone regeneration strategy.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, was tested for in vitro cytotoxicity against human laryngeal epidermoid carcinoma of the larynx Hep2 cells. EGCG-induced apoptotic cell death accompanied by a change in the cell cycle. However, EGCG did not result in caspase activation, nor did a caspase inhibitor block cell death. Furthermore, EGCG caused no change in the intracellular levels of reactive oxygen species (ROS). The levels of p53 were increased in the EGCG-treated cells, with a corresponding decrease in Bcl-2 and Bid protein levels as well as an increase in the Bax level. In addition, EGCG induced the cytoplasmic release of cytochrome c from the mitochondria accompanied by a decreased mitochondrial membrane potential, and subsequently upregulated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus during the apoptotic process. Taken together, these findings indicate that the p53-mediated mitochondrial pathway and the nuclear translocation of AIF and EndoG play a crucial role in EGCG-induced apoptosis of human laryngeal epidermoid carcinoma Hep2 cells, which proceeds through a caspase-independent pathway.

Introduction Angiogenesis is closely associated with bone formation, especially endochondral ossification. Angiopoietin 1 (Ang1) is a specific growth factor functioning to generate a stable and matured vasculature through the Tie2 receptor/PI3K/AKT pathway. Recently cartilage oligomeric matrix protein (COMP)-Ang1, an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor and AKT, was developed. This study was designed to examine the effects of angiogenic COMP-Ang1 on BMP2-induced osteoblast differentiation and bone formation. Methods Expression of endogenous Ang-1 and its binding receptor Tie 2 mRNA was examined in osteoblast-like cells and primary mouse calvarial cells by RT-PCR analysis, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid and β-glycerophosphate. Effects of COMP-Ang-1 on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and osteocalcin (OC) production, and Alizarin red stain. For a molecular mechanism, Western blot and OG2 and 6xOSE promoter assays were done. For in vivo evaluation, adenoviral (Ad) vectors containing COMP-Ang-1 or BMP-2 gene were administered into thigh muscle of mice, and after 2 weeks bone formation was analyzed by micro-computed tomography and histology. Angiogenic event of COMP-Ang1 was confirmed by immunofluorescence analysis with anti-CD31 antibody. Results Expression of Tie2 receptor was significantly increased in the course of osteoblast differentiation. Treatment or overexpression of COMP-Ang1 enhanced BMP2-induced ALP activity, OC production, and mineral deposition in a dose-dependent manner. In addition, COMP-Ang1 synergistically increased OG2 and 6xOSE promoter activities of BMP2, and sustained p38, Smad and AKT phosphorylation of BMP2. Notably, in vivo intramuscular injection of COMP-Ang1 dose-dependently enhanced BMP2-induced ectopic bone formation with increases in CD31 reactivity. Conclusions These results suggest that COMP-Ang1 synergistically enhanced osteoblast differentiation and bone formation through potentiating BMP2 signaling pathways and angiogenesis. Combination of BMP2 and COMP-Ang1 should be clinically useful for therapeutic application to fracture and destructive bone diseases.

Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.

Cyclosporin A (CsA) has been used as an immunosuppressive drug to prevent organ transplant rejection and to treat autoimmune diseases. CsA has a proliferative effect on human gingival fibroblasts (HGF) in vitro. However, the molecular mechanisms underlying CsA-induced proliferation in HGF remain to be elucidated. This study was aimed to investigate the CsA responsive proteins in HGF using systematic proteomic approach. Cell viability was determined by MTT assay and reactive oxygen species (ROS) was measured by fluorescent spectrometer. Proteins profiled by two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadrupole time-of-flight mass spectrometry (EIQ-TOF MS). To confirm the expression changes of proteins by proteomics analysis, Western blot was performed using specific antibody. CsA increased the cell viability of HGF in a dose- and time-dependent manner. Significantly, seventeen proteins were overexpressed in the CsA-treated HGF, whereas three proteins were found to be expressed less than the untreated cells. The identified proteins were mainly related with cell proliferation, metabolism, and oxidation. The overexpression of peroxiredoxin 1 (Prx 1) confirmed by Western blotting and reduction of cytosolic reactive oxygen species (ROS) levels in the CsA-treated HGF demonstrated that Prx 1 may play a crucial role in the HGF proliferation induced by CsA. Upregulation of Galectin 3 in CsA-treated HGF indicated that it is related to CsA-induced proliferation. These proteomic analysis data will provide an efficient approach in understanding the mechanisms of HGF proliferation by CsA.

Sodium fluoride (NaF) has been shown to be cytotoxic and produces inflammatory responses in humans. However, the cellular mechanisms underlying the NaF-induced cytotoxicity in periodontal tissues are unclear. This study examined whether or not NaF induces apoptosis in human gingival fibroblasts (HGF), and its underlying mechanisms by monitoring various apoptosis-associated processes. NaF reduced the cell viability of HGF in a dose- and time-dependent manner. NaF increased TUNEL-positive cell and induced apoptosis with concomitant chromatin condensation and DNA fragmentation in HGF. In addition, NaF increased the level of cytochrome c released from the mitochondria into the cytosol, enhanced the caspase-9, -8 and -3 activities, the cleavage of poly (ADP-ribose) polymerase (PARP), and up-regulated the voltage-dependent anion channel (VDAC) 1. However, NaF did not affect the production of reactive oxygen species (ROS) which is a strong apoptotic inducer. Furthermore, NaF up-regulated the Fas-ligand (Fas-L), a ligand of death receptor. Bcl-2, a member of the anti-apoptotic Bcl-2 family, was down-regulated, whereas the expression of Bax, a member of the pro-apoptotic Bcl-2 family, was unaffected in the NaF-treated HGF. These results suggest that NaF induces apoptosis in HGF through both the mitochondria-mediated pathways regulated by the Bcl-2 family and death receptor-mediated pathway.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Objective To determine the diameter of the crown, total crown area, individual cusp area, and occlusal table area in Korean maxillary permanent molars, as well as dental characteristics relevant to the hypocone reduction trait. Materials and methods Subjects included 121 dental school students in Korea (81 men and 40 women). A digital image analysis system was used for measurements and we relied on visual scoring to assign categories of hypocone expression. Results The mean crown dimension was larger in the first molar (M1) than in the second molar (M2). Regarding differences according to gender, the crown diameter, total crown area, and individual cusp area were significantly larger in the men than in the women. The mean occlusal table area ratios were 61% for M1 and 57% for M2, and these ratios increased in proportion to the total crown area. With respect to the hypocone and other features of the maxillary molars, differences between the men and women were more prominent for M1 than for M2. The M2 hypocone tended to be smaller than the M1 hypocone, and hypocone reduction was inversely related to the cusp area of the protocone. The protocone area may be considered a reliable parameter for comparing race and/or gender differences in maxillary molars. Conclusion This study concerning characteristics of the maxillary molar occlusal surface in Koreans provides useful information for comparative studies among human races.

Objectives: Cyclosporin A (CsA) is known to elicit fibrous gingival overgrowth with changes of blood vessel profiles. In this study, we examined the expression of several angiogenic and angiostatic genes during the development of CsA-induced gingival overgrowth. Methods: For the development of gingival overgrowth, Sprague-Dawley rats received subcutaneous injections of CsA in daily doses of 5, 10, 15 mg/kg body weight for 6 weeks, and another group received 10 mg/kg of CsA for 3, 6, and 12 weeks. Human gingival tissues were obtained from three CsA-treated patients following the gingivectomy procedure and from three healthy patients following the crown-lengthening procedure as a control. Gingival fibroblasts were isolated from the healthy gingival tissues of the rat or the human, and cultured with 250–1000 ng/ml of CsA. Results: Reverse transcription–polymerase chain reaction (RT–PCR) analyses showed that expressions of some angiogenic genes such as angiopoietin 1, basic fibroblast growth factor, and vascular endothelial growth factor, and angiostatic genes such as angiopoietin 2, brain-specific angiogenesis inhibitor 1 and 2, and thrombospondin 1 were not changed significantly in both gingival tissues and cultured fibroblast cells under the CsA treatments. However, expression of thrombospondin 2 (TSP2) decreased dose- and time-dependently in rat and human gingival tissues. Western blot analyses showed that the expression of TSP2 protein was dose-dependently reduced by the CsA treatments in human cultured gingival fibroblasts. Conclusions: These results indicate that the decrease in angiostatic TSP2 expression may be attributed to the CsA-induced gingival vascularization rather than to the increased expression of angiogenic genes. It suggests that TSP2 is involved in the development of CsA-induced gingival overgrowth with the gingival vascularization.

Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1β- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.

Summary: Compared with HIV-negative individuals, HIV-positive individuals have a higher prevalence of anogenital human papillomavirus (HPV) infection, as well as a higher incidence of HPV-associated anal cancer. Little is currently known of chromosomal changes occurring in anal intraepithelial neoplasia (AIN), the probable precursor to anal cancer. Genetic changes in AIN were characterized by comparative genomic hybridization (CGH) in a study of samples obtained from 19 HIV-positive and 11 HIV-negative men. The proportion with genetic changes significantly increased with the severity of the histopathologic grade with none diagnosed as (0%) AIN 1; 5 of 17 (29%) as AIN 2; and 5 of 9 (56%) AIN 3 showing genetic changes (p = .02). This correlation was also found in study subjects who had multiple biopsies with different grades of pathology concurrently or serially over time. The most common regional DNA copy number change was gain mapped to chromosome arm 3q (12% of AIN 2 and 33% of AIN 3). This alteration was previously reported to be commonest alteration in cervical cancer, which suggests a common molecular pathway for these two HPV-associated anogenital neoplasias.

Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.

This article reports the experiences of women aged 55 to 75 with mobility impairments who attributed aspects of their limitations to workplace injuries and provides insight into worker’s compensation policies. The study sample includes Mexican American (MA) and non-Hispanic White (NHW) women aged 55 to 75 who participated in a 4-year ethnographic study of disablement. Ninety-two of the 122 participants in the study attributed aspects of their functional limitations to employment, and their experiences were analyzed using data from 354 meetings. Using Lipscomb and colleagues’ conceptual model of work and health disparities, the women’s experiences were grouped into three categories according to type of injury, assistance gained, and the consequences of a workplace injury; the results have broad implications for policies that influence aging outcomes. Workplace injuries causing permanent functional limitations compound the effects of age and gender on employment outcomes. Policies addressing health disparities should consider work related influences.

Background and Objective Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-oxidizing enzyme with immune-inhibitory effects. The aim of this study was to investigate the expression of IDO by lipopolysaccharide (LPS), a component of gram-negative bacteria, in human periodontal ligament (PDL) cells. Material and Methods Human PDL cells and gingival fibroblasts (GFs) were prepared from explants of human PDLs and from gingival tissues of clinically healthy donors, respectively. Real-time RT-PCR, western blotting and the IDO enzyme assay were performed to determine the expression of IDO following LPS treatment of cells. LPS was injected into mice tail veins to evaluate the effects of LPS in vivo in the maxillary first molar. Immunofluorescence staining and histological analysis were followed to localize IDO in mouse PDL. Results The level of expression of IDO mRNA in primary human PDL cells after LPS treatment was increased in a dose-dependent manner, reaching a peak 8 h after LPS treatment. The expression and activities of IDO protein were significantly increased in comparison with those of the control. In addition, the increased production of kynurenine in culture medium was observed 72 h after LPS treatment. In the immunofluorescence findings, stronger immunoreactivities were shown in PDL than in gingival tissues in the maxillae. In accordance with the immunofluorescence findings, LPS treatment induced a strong up-regulation of IDO mRNA in human PDL cells, whereas human GFs showed only a weak response to LPS. Conclusion These results clearly show that IDO was induced by LPS in primary human PDL cells, suggesting that PDL might be involved in the regulation of oral inflammatory disease.

Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.

Background/Aims: Cartilage oligomeric matrix protein (COMP)-angiopoietin 1 (Ang1) is a soluble and stable form of Ang1 which plays important roles in vessel formation and the survival of endothelial cells, neurons and cardiomyocytes. However, the effects of COMP-Ang1 on the survival of mesenchymal cells are unknown. Mesenchymal cells have been transplanted with some scaffolds for bone tissue regeneration, but they occasionally underwent cell death due to a lack of nutrient supply. This study examined the effects of COMP-Ang1 on the survival of mesenchymal cells under nutrient-deprived conditions. Methods: Primary and C3H10T1/2 mesenchymal cells were cultured under serum deprivation with or without COMP-Ang1. The effects of COMP-Ang1 on mesenchymal cell survival and its molecular mechanism were determined using a viability test, RT-PCR, Western blotting and fluorescence-activated cell sorting analysis. Results and Conclusion: COMP-Ang1 inhibited the nutrient-deprived apoptotic cell death of mesenchymal cells through the Akt, p38 and extracellular-signal-regulated kinase (ERK) pathways. In addition, COMP-Ang1 reversed the nutrient-deprived suppression of cyclin D1 mRNA expression. These results suggest that COMP-Ang1 has a protective role in the survival of nutrient-deprived mesenchymal cells. The use of COMP-Ang1 with some scaffolds might be useful for bone tissue engineering.