Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures.

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/ western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Inhibition of human mesenchymal stem cell proliferation via Wnt signaling activation.

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 ß-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.

Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-κB signaling pathway in mice.

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-κB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-κB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.

Direct utilization of purple sweet potato by sake yeasts to produce an anthocyanin-rich alcoholic beverage.

OBJECTIVE: To produce an alcoholic beverage containing anthocyanins that can act as antioxidants and have anticarcinogenic activities and antihypertensive effects. RESULTS: High starch-assimilating sake yeast strain of Saccharomyces cerevisiae co-expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis using the double rDNA-integration system was developed. The new strain grew substantially using 5 % (w/v) purple sweet potato flour as the sole carbon source. Its cell yield reached 14.5 mg ml(-1) after 3 days. This value was 2.4-fold higher than that of the parental wild-type strain. It produced 12 % (v/v) ethanol from 20 % (w/v) purple sweet potato flour and consumed 98 % of the starch content in purple sweet potato flour after 5 days of fermentation. CONCLUSION: We have produced a health-promoting alcoholic beverage abundant in anthocyanins from purple sweet potato.

Construction of dextrin and isomaltose-assimilating brewer's yeasts for production of low-carbohydrate beer.

Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer's bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer's yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.

Mechanisms of platelet-activating factor-induced enhancement of VEGF expression.

It has been previously reported that platelet-activating factor (PAF) induces the expression of vascular endothelial growth factor (VEGF) via the downregulation of p53 activity. In this study, we attempted to characterize the mechanism by which p53 activity negatively regulates PAF-induced VEGF expression. PAF increased luciferase activity as well as VEGF mRNA expression in human non-small cell lung cancer cell line H1299 transfected with VEGF luciferase reporter plasmid (VEGF-Luc). Cotransfection of the cells with wt p53, but not mutant p53, effected a blockage of PAF-induced VEGF mRNA expression. The ChIP assay revealed that p53 did not bind to the VEGF promoter. Transfection of Egr-1 or Sp-1 expression vector increased VEGF luciferase activity in VEGF-Luc-transfected cells, and this was inhibited by transfection with wt p53. The results of the Immunoprecipitation and immunoblot analysis showed that p53 binds to Egr-1 and Sp-1. Additionally, our electrophoretic mobility shift assay demonstrated that PAF induced the mobilization of Egr-1 and Sp-1 to the nucleus, and this activity was inhibited by transfection with wt p53. These data indicate that PAF inhibits protein complexes between p53 and Egr-1/Sp-1 via the downregulation of p53 levels, thus increasing the free form levels of Egr-1 and Sp-1, ultimately resulting in the transcriptional activation of VEGF.

Glutamine protects mice from lethal endotoxic shock via a rapid induction of MAPK phosphatase-1.

The nonessential amino acid L-glutamine (Gln) is the most abundant amino acid in plasma. Clinical trials have demonstrated that Gln therapy is safe and improves clinical outcomes in critically ill patients. We have previously shown that Gln protect animals from endotoxic shock through the inhibition of cytosolic phospholipase A(2) activity. In this study, we investigated how Gln regulates MAPK activation, as the molecular mechanism underlying Gln-induced cytosolic phospholipase A(2) inactivation. Gln rapidly (within 10 min) inactivated p38 and JNK, but not ERK, by dephosphorylating them only when these MAPKs were phosphorylated in response to LPS in vivo as well as in vitro. Western blot analysis revealed that Gln administration resulted in rapid ( approximately 5 min) phosphorylation and protein induction of MAP kinase phosphatase-1 (MKP-1). MKP-1 siRNA abrogated the Gln-mediated 1) inactivation of p38 and JNK, 2) induction of MKP-1, and 3) protection against endotoxic shock. The ERK inhibitor U0126 blocked Gln-induced MKP-1 phosphorylation and protein induction, as well as Gln's protective activity against endotoxic shock. These data suggest that Gln exerts a beneficial effect on endotoxic shock by inactivating p38 and JNK via a rapid induction of MKP-1 protein in an ERK-dependent way.

Relaxin is up-regulated in the rat ovary by orthodontic tooth movement.

Relaxin (Rln) is an ovarian hormone that stimulates osteoclastic and osteoblastic activities and connective tissue turnover. To investigate the expression of Rln during orthodontic tooth movement, rats were implanted with orthodontic appliances that connected a spring from the upper incisors to the first molar with a 70 cN force. Rats in each group were killed 6, 48, and 144 h after activating the appliance, and the levels of Rln1 and Rln3 expression in the ovary were determined by real-time RT-PCR, northern blots, western blots, and immunofluorescence analyses. The amount of tooth movement induced by the orthodontic force increased in a time-dependent manner. The levels of Rln1 mRNA increased by 12-, 41-, and 263-fold at 6, 48, and 144 h, respectively, after orthodontic tooth movement. The time-dependent increase in the concentration of Rln 1 protein in the ovary was also confirmed by western blotting. Rln 1 was localized in the granulosa cells of the ovarian follicles, and the immunoreactivity against Rln 1 was increased by the movement. In contrast, the concentration of Rln 3 was below the level of detection. The results of this study suggest that local changes in periodontal tissues induced by orthodontic tooth movement may affect Rln1 expression in the ovary. However, further studies are needed to decipher the mechanisms involved and the possible contribution of the increased level of expression of Rln 1 to the tooth movement.

Raw starch fermentation to ethanol by an industrial distiller's yeast strain of Saccharomyces cerevisiae expressing glucoamylase and α-amylase genes.

Industrial strains of a polyploid, distiller's Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l(-1)) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.

Regulatory role of insulin-like growth factor-binding proteins in odontogenic mineralization in rats.

Much information is currently available for molecules in early odontogenesis, but there is limited knowledge regarding terminal cytodifferentiation of ameloblasts and odontoblasts for the determination of normal crown morphology. The present differential display PCR (DD-PCR) revealed that insulin-like growth factor-binding protein 5 (IGFBP5) was differentially expressed in molar tooth germs between the cap (before crown mineralization) and root formation (after crown mineralization) stages. Real-time PCR confirmed that the expression levels of IGFBP1-4 were not significantly changed but those of IGFBP5-7 were upregulated in a time-dependent manner. Immunoreactivities for IGFBP5-7 were hardly seen in molar germs at the cap/early bell stage and protective-stage ameloblasts at the root formation stage. However, the reactivity was strong in odontoblasts and maturation-stage ameloblasts, which are morphologically and functionally characterized by wide intercellular space and active enamel matrix mineralization. The localization of each IGFBP was temporospatial. IGFBP5 was localized in the nuclei of fully differentiated odontoblasts and ameloblasts, while IGFBP6 was localized in the apical cytoplasm of ameloblasts and odontoblasts with dentinal tubules, and IGFBP7 was mainly found in the whole cytoplasm of odontoblasts and the intercellular space of ameloblasts. IGFBP silencing using specific siRNAs upregulated representative genes for dentinogenesis and amelogenesis, such as DMP1 and amelogenin, respectively, and augmented the differentiation media-induced mineralization, which was confirmed by alizarin red s and alkaline phosphatase staining. These results suggest that IGFBP5-7 may play independent and redundant regulatory roles in late-stage odontogenesis by modulating the functional differentiation of ameloblasts and odontoblasts.

Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, alpha-amylase and debranching enzyme.

To develop a strain of Saccharomyces cerevisiae that produces ethanol directly from starch, two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis alpha-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. The GA1 and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae using the double delta-integration system. The GAM1 gene was constitutively expressed under the corresponding promoter using the double 18S rDNA-integration system. The recombinant industrial strain secreting biologically active alpha-amylase, glucoamylase and debranching enzyme was able to ferment starch to ethanol in a single step. The new strain produced 8% (v/v) ethanol (62.8 g l(-1)) from 20% (w/v) soluble starch after 2 days, fermentation.

Inhibitory effect of C-X-C motif chemokine ligand 14 on the osteogenic differentiation of human periodontal ligament cells through transforming growth factor-beta1.

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- ß1) in hPDL cells. Inhibition of the cell proliferation and osteogenic differentiation of hPDL cells by CXCL14 siRNA and rhCXCL14 were restored by rhTGF-ß1 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-ß1 expression in hPDL cells.

Dual role of phosphatidylserine and its receptors in osteoclastogenesis.

Fusion and apoptosis share a breakdown of the membrane phospholipids asymmetry, modes of which are largely unknown in osteoclastogenesis. Here, we investigated the externalization of phosphatidylserine (PS) and its receptors, and their biological functions in osteoclastogenesis. Strong immunoreactivities in vivo for the PS receptors TIM4, BAI1, and STAB2 were observed in the TRAP-positive multinucleated cells in the alveolar bone that was being remodeled around the developing dental follicles in rats. These receptors were significantly upregulated during M-CSF/RANKL-induced in vitro osteoclastogenesis using mouse bone marrow-derived cells. PS externalization in preosteoclasts was increased by the M-CSF/RANKL treatment. Multinucleation of preosteoclasts was markedly inhibited by antibodies against PS and its receptors. Among the investigated lipid transporter proteins, floppases (Abcb4, Abcc5, and Abcg1) were upregulated, whereas flippases (Atp11c and Atp8a1) downregulated during osteoclastogenesis. Preosteoclast fusion was markedly blocked by the ATPase inhibitor Na3VO4 and siRNAs against Abcc5 and Abcg1, revealing the importance of these lipid transporters in PS externalization. Further, the levels of Cd47 and Cd31, don't-eat-me signal inducers, were increased or sustained in the early phase of osteoclastogenesis, whereas those of AnnexinI and Mfg-e8, eat-me signals inducers, were increased in the late apoptotic phase. In addition, Z-VAD-FMK, a pan caspase inhibitor, had no effect on preosteoclast fusion in the early phase of osteoclastogenesis, whereas Abs against PS, TIM4, and BAI1 decreased osteoclast apoptosis during the late phase. These results suggest that PS externalization is essential for the whole process of osteoclastogenesis and share PS receptors and transporters in the early stage fusion and late stage apoptosis. Therefore, modulation of PS and its receptors could be a useful strategy to develop anti-bone resorptive agents.

Synergistic alveolar bone resorption by diabetic advanced glycation end products and mechanical forces.

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.

Nitric oxide plays a key role in the platelet-activating factor-induced enhancement of resistance against systemic candidiasis.

Platelet-activating factor (PAF) has been demonstrated to augment resistance against Candida albicans infection. In this study, the role of nitric oxide (NO) in PAF-induced resistance in the kidneys was investigated. Pretreatment of the C. albicans-infected mice with PAF resulted in strong expression of messenger RNA (mRNA) and the protein synthesis of inducible nitric oxide synthase (iNOS). These PAF effects were inhibited to a significant degree by pretreatment with the nuclear factor-kappaB inhibitor, pyrrolidinedithiocarbamate. Pretreatment with PAF protected the mice from death caused by C. albicans infection and reduced the growth of fungus in the kidneys. The protective activity of PAF was abrogated by pretreatment with the iNOS inhibitor, aminoguanidine, and in the iNOS(-/-) mice. The PAF markedly increased the infiltration of neutrophils, but not macrophages, and also enhanced the mRNA expression levels of the CXC chemokine, keratinocyte-derived chemokine, in C. albicans-infected kidneys. These effects of PAF were attenuated in the aminoguanidine-treated mice and the iNOS(-/-) mice. These data show that NO plays an important role in PAF-induced protection against C. albicans.

Tumor necrosis factor-alpha develops late anaphylactic reaction through cytosolic phospholipase A(2) activation.

BACKGROUND: We have recently reported that tumor necrosis factor (TNF)-alpha plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-alpha remains unclear. OBJECTIVE: It was the aim of this study to examine whether TNF-alpha induces late-phase anaphylaxis via the activation of cytosolic phospholipase A(2) (cPLA(2)). METHODS: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-alpha knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA(2) was measured by immunoprecipitation. cPLA(2) activity was assessed by using 1-stearoyl-2-[1-(14)C] arachidonyl-sn-glycero-3-phosphocholine as the substrate. RESULTS: Phosphorylation and enzymatic activity of cPLA(2), and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-alpha-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA(2) inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA(2) phosphorylation and activity, but also the late-phase anaphylaxis. CONCLUSION: TNF-alpha-induces cPLA(2) activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis.

Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and alpha-amylase.

Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis alpha-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the delta-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and alpha-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.

Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation.

In this study, we investigated the influence of platelet-activating factor (PAF) on the induction of apoptosis-regulating factors in B16F10 melanoma cells. PAF increased the expression of mRNA and the protein synthesis of antiapoptotic factors, such as Bcl-2 and Bcl-xL, but did not increase the expression of the proapoptotic factor, Bax. A selective nuclear factor-kappaB (NF-kappaB) inhibitor, parthenolide, inhibited the effects of PAF. Furthermore, PAF inhibited etoposide-induced increases in caspase-3, caspase-8, and caspase-9 activities, as well as cell death. p50/p65 heterodimer increased the mRNA expression of Bcl-2 and Bcl-xL and decreased etoposide-induced caspase activities and cell death. In an in vivo model in which Matrigel was injected s.c., PAF augmented the growth of B16F10 cells and attenuated etoposide-induced inhibition of B16F10 cells growth. These data indicate that PAF induces up-regulation of antiapoptotic factors in a NF-kappaB-dependent manner in a melanoma cell line, therefore suggesting that PAF may diminish the cytotoxic effect of chemotherapeutic agents.

Twist1 Suppresses Cementoblast Differentiation.

The transcription factor Twist1 is known to be closely associated with the formation of bone by mesenchymal stem cells and osteoblasts; however, the role of Twist1 in cementogenesis has not yet been determined. This study was undertaken to elucidate the roles of Twist1 in cementoblast differentiation by means of the gain- or loss-of-function method. We used alkaline phosphatase (ALP) and alizarin red S staining and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) to determine whether the forced transient expression or knock-down of Twist1 in a mouse cementoblast cell line, OCCM-30, could affect cementogenic differentiation. Silencing Twist1 with small interference RNA (siRNA) enhanced the formation of mineralized tissue. The expression of several cementogenesis markers, such as bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein1 (DMP1), and dentin sialophosphoprotein (DSPP) mRNA, were upregulated. Transient Twist1 overexpression in OCCM-30 consistently suppressed mineralization capacity and downregulated the differentiation markers. These results suggest that the Twist1 transcription factor may play a role in regulating cementoblast differentiation.

The role of Hedgehog signaling in cementoblast differentiation.

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.