Involvement of the OTUB1-YAP1 axis in driving malignant behaviors of head and neck squamous cell carcinoma.

This study highlights the critical role of OTUB1 in HNSCC progression via modulating YAP1. Targeting the OTUB1-YAP1 axis holds promise as a potential therapeutic strategy for HNSCC treatment.

In HNSCC patients, USP10, USP14, OTUB1, and STAMBP among the screened DUBs were associated with a poor prognosis. Among them, OTUB1 showed the most aggressive characteristics in both in vitro and in vivo experiments. Additionally, OTUB1 regulated the stability and nuclear localization of YAP1, a substrate involved in cell proliferation and invasion. Notably, OTUB1 expression exhibited a positive correlation with the HNSCC-YAP score in HNSCC cells.


Metallic foreign bodies ingestion by schizophrenic patient: a case report

The ingestion of foreign objects is a widespread health issue, with a higher occurrence in adults with psychosis. The authors present the case of a 39-year-old man who arrived at the hospital with symptoms of abdominal distension and occasional black stools for a week. The patient was known to have schizophrenia but had not received regular hospital follow-up or treatment for the past 5 years. He had a history of exogenous stimulation, which led him to surreptitiously swallow metallic objects. Upon physical examination, he displayed abdominal distension and mild tenderness in the upper abdomen. Radiographs revealed multiple foreign objects in his stomach, leading to the decision for laparotomy, gastric opening, and removal of the foreign objects under general anesthesia. Mental illnesses, including schizophrenia, bipolar disorder, major depression, and multiple substance abuse, are recognized as being significant risk factors for ingesting foreign bodies. In such cases, it is crucial to intervene quickly. For patients presenting with psychiatric symptoms, the involvement of family caregivers is of even greater importance than endoscopic or surgical treatments. Foreign body ingestion is more prevalent in individuals with psychosis, highlighting the importance of ongoing care and follow-up for patients with mental illness.


The Role of Deubiquitinating Enzyme in Head and Neck Squamous Cell Carcinoma

Ubiquitination and deubiquitination are two popular ways for the post-translational modification of proteins. These two modifications affect intracellular localization, stability, and function of target proteins. The process of deubiquitination is involved in histone modification, cell cycle regulation, cell differentiation, apoptosis, endocytosis, autophagy, and DNA repair after damage. Moreover, it is involved in the processes of carcinogenesis and cancer development. In this review, we discuss these issues in understanding deubiquitinating enzyme (DUB) function in head and neck squamous cell carcinoma (HNSCC), and their potential therapeutic strategies for HNSCC patients are also discussed.

International Journal of Molecular Sciences


The Effect of Heterozygous Mutation of Adenylate Kinase 2 Gene on Neutrophil Differentiation.

Mitochondrial ATP production plays an important role in most cellular activities, including growth and differentiation. Previously we reported that Adenylate kinase 2 (AK2) is the main ADP supplier in the mitochondrial intermembrane space in hematopoietic cells, especially in the bone marrow. AK2 is crucial for the production of neutrophils and T cells, and its deficiency causes reticular dysgenesis. However, the relationship between ADP supply by AK2 and neutrophil differentiation remains unclear. In this study, we used CRISPR/Cas9 technology to establish two heterozygous AK2 knock-out HL-60 clones as models for reticular dysgenesis. Their AK2 activities were about half that in the wild-type (WT). Furthermore, neutrophil differentiation was impaired in one of the clones. In silico analysis predicted that the obtained mutations might cause a structural change in AK2. Time course microarray analysis of the WT and mutants revealed that similar gene clusters responded to all-trans retinoic acid treatment, but their expression was lower in the mutants than in WT. Application of fructose partially restored neutrophil differentiation in the heterozygous knock-out HL-60 clone after all-trans retinoic acid treatment. Collectively, our study suggests that the mutation of N-terminal region in AK2 might play a role in AK2-dependent neutrophil differentiation and fructose could be used to treat AK2 deficiency.


Gene-expression profile reveals the genetic and acquired phenotypes of hyperactive mutant SPORTS rad

Spontaneously Running Tokushima Shikoku (SPORTS) rat is a hyperactive rat strain. However, the causative mutation of this phenotype has not yet been identified. To investigate the molecular basis for the unique phenotype of SPORTS rats, we examined gene-expression profiles by microarray analyses. Among adenylate kinase isozymes that maintain the homeostasis of cellular adenine nucleotide composition in the cell, only adenylate kinase 1 is highly up-regulated in both exercised and sedentary SPORTS rats compared with wild-type (WT) rats, 5.5-fold and 3.3-fold, respectively. Further comparative analyses revealed that genes involved in glucose metabolism were up-regulated in skeletal muscle tissue of exercised SPORTS rats compared with sedentary mutants, whereas genes related to extracellular matrix or region were down-regulated compared with WT rats. In brain tissue of sedentary SPORTS rats, genes associated with defense and catecholamine metabolism were highly expressed compared with WT rats. These findings suggest that genetic mutation(s) in SPORTS rat remodels metabolic demands through differentially regulating gene expression regardless of exercise. Therefore, the SPORTS rats are useful animal model not only for further examining the effects of exercise on metabolism but also for deeply studying the molecular basis how mutation affect the psychological motivation with spontaneous voluntary exercise phenotype. J. Med. Invest. 67 : 51-61, February, 2020.

The Journal of Medical Investigation : JMI


Mitochondrial Activity and Unfolded Protein Response are Required for Neutrophil Differentiation.

In this study, we demonstrated that neutrophil differentiation is regulated by ER stress/UPR that is supported by mitochondrial ATP supply, in which IRE1-XBP1 activation is essential. Our findings provide the evidence that mitochondrial energy metabolism may play a critical role in neutrophil differentiation.


Deciphering defective amelogenesis using invitro culture systems.

The conventional two-dimensional (2D) in vitro culture system is frequently used to analyze the gene expression with or without extracellular signals. However, the cells derived from primary culture and cell lines frequently deviate the gene expression profile compared to the corresponding in vivo samples, which sometimes misleads the actual gene regulation in vivo. To overcome this gap, we developed the comparative 2D and 3D in vitro culture systems and applied them to the genetic study of amelogenesis imperfecta (AI) as a model. Recently, we found specificity protein 6 (Sp6) mutation in an autosomal-recessive AI rat that was previously named AMI. We constructed 3D structure of ARE-B30 cells (AMI-derived rat dental epithelial cells) or G5 (control wild type cells) combined with RPC-C2A cells (rat pulp cell line) separated by the collagen membrane, while in 2D structure, ARE-B30 or G5 was cultured with or without the collagen membrane. Comparative analysis of amelogenesis-related gene expression in ARE-B30 and G5 using our 2D and 3D in vitro systems revealed distinct expression profiles, showing the causative outcomes. Bone morphogenetic protein 2 and follistatin were reciprocally expressed in G5, but not in ARE-B30 cells. All-or-none expression of amelotin, kallikrein-related peptidase 4, and nerve growth factor receptor was observed in both cell types. In conclusion, our in vitro culture systems detected the phenotypical differences in the expression of the stage-specific amelogenesis-related genes. Parallel analysis with 2D and 3D culture systems may provide a platform to understand the molecular basis for defective amelogenesis caused by Sp6 mutation.

Journal of Bioscience and Bioengineering


Isolation and characterization of dental epithelial cells derived from amelogenesis imperfecta rat.

OBJECTIVE:Disruption of the third zinc finger domain of specificity protein 6 (SP6) presents an enamel-specific defect in a rat model of amelogenesis imperfecta (AMI rats). To understand the molecular basis of amelogenesis imperfecta caused by the Sp6 mutation, we established and characterized AMI-derived rat dental epithelial (ARE) cells.MATERIALS AND METHODS:ARE cell clones were isolated from the mandibular incisors of AMI rats, and amelogenesis-related gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Localization of wild-type SP6 (SP6WT) and mutant-type SP6 (SP6AMI) was analyzed by immunocytochemistry. SP6 transcriptional activity was monitored by rho-associated protein kinase 1 (Rock1) promoter activity with its specific binding to the promoter region in dental (G5 and ARE) and non-dental (COS-7) epithelial cells.RESULTS:Isolated ARE cells were varied in morphology and gene expression. Both SP6WT and SP6AMI were mainly detected in nuclei. The promoter analysis revealed that SP6WT and SP6AMI enhanced Rock1 promoter activity in G5 cells but that enhancement by SP6AMI was weaker, whereas no enhancement was observed in the ARE and COS-7 cells, even though SP6WT and SP6AMI bound to the promoter in all instances.CONCLUSION:ARE cell clones can provide a useful in vitro model to study the mechanism of SP6-mediated amelogenesis imperfecta.


Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells.

Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.


Sp6 regulation of Rock1 promoter activity in dental epithelial cells

Sp6 is a transcription factor of the SP/KLF family and an indispensable regulator of the morphological dynamics of ameloblast differentiation during tooth development. However, the underlying molecular mechanisms remain unclear. We have previously identified one of the Sp6 downstream genes, Rock1, which is involved in ameloblast polarization. In this study, we investigated the transcriptional regulatory mechanisms of Rock1 by Sp6. First, we identified the transcription start sites (TSS) and cloned the 5'-flanking region of Rock1. Serial deletion analyses identified a critical region for Rock1 promoter activity within the 249-bp upstream region of TSS, and chromatin immunoprecipitation assays revealed Sp6-binding to this region. Subsequent transient transfection experiments showed that Rock1 promoter activity is enhanced by Sp6, but reduced by Sp1. Treatment of dental epithelial cells with the GC-selective DNA binding inhibitor, mithramycin A, affected Rock1 promoter activity in loss of enhancement by Sp6, but not repression by Sp1. Further site-directed mutagenesis indicated that the region from -206 to -150 contains responsive elements for Sp6. Taken together, we conclude that Sp6 positively regulates Rock1 transcription by direct binding to the Rock1 promoter region from -206 to -150, which functionally distinct from Sp1.


Differential expression of adenine nucleotide converting enzymes in mitochondrial intermembrane space: a potential role of adenylate kinase isozyme 2 in neutrophil differentiation.

Adenine nucleotide dynamics in the mitochondrial intermembrane space (IMS) play a key role in oxidative phosphorylation. In a previous study, Drosophila adenylate kinase isozyme 2 (Dak2) knockout was reported to cause developmental lethality at the larval stage in Drosophila melanogaster. In addition, two other studies reported that AK2 is a responsible gene for reticular dysgenesis (RD), a human disease that is characterized by severe combined immunodeficiency and deafness. Therefore, mitochondrial AK2 may play an important role in hematopoietic differentiation and ontogenesis. Three additional adenine nucleotide metabolizing enzymes, including mitochondrial creatine kinases (CKMT1 and CKMT2) and nucleoside diphosphate kinase isoform D (NDPK-D), have been found in IMS. Although these kinases generate ADP for ATP synthesis, their involvement in RD remains unclear and still an open question. In this study, mRNA and protein expressions of these mitochondrial kinases were firstly examined in mouse ES cells, day 8 embryos, and 7-week-old adult mice. It was found that their expressions are spatiotemporally regulated, and Ak2 is exclusively expressed in bone marrow, which is a major hematopoietic tissue in adults. In subsequent experiments, we identified increased expression of both AK2 and CKMT1 during macrophage differentiation and exclusive production of AK2 during neutrophil differentiation using HL-60 cells as an in vitro model of hematopoietic differentiation. Furthermore, AK2 knockdown specifically inhibited neutrophil differentiation without affecting macrophage differentiation. These data suggest that AK2 is indispensable for neutrophil differentiation and indicate a possible causative link between AK2 deficiency and neutropenia in RD.


Ctip2-mediated Sp6 transcriptional regulation in dental epithelium-derived cells.

Tooth development relies on the interaction between the oral ectoderm and underlying mesenchyme, and is regulated by a complex genetic cascade. This transcriptional cascade is regulated by the spatiotemporal activation and deactivation of transcription factors. The specificity proteins 6 (Sp6) and chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (Ctip2) were identified in loss-of-function studies as key transcription factors required for tooth development. Ctip2 binds to the Sp6 promoter in vivo; however, its role in Sp6 expression remains unclear. In this study, we investigated Sp6 transcriptional regulation by Ctip2. Immunohistochemical analysis revealed that Sp6 and Ctip2 colocalize in the rat incisor during tooth development. We examined whether Ctip2 regulates Sp6 promoter activity in dental epithelial cells. Cotransfection experiments using serial Sp6 promoter-luciferase constructs and Ctip2 expression plasmids showed that Ctip2 significantly suppressed the Sp6 second promoter activity, although the Sp6 first promoter activity was unaffected. Ctip2 was able to bind to the proximal region of the Sp6 first promoter, as previously demonstrated, and also to the novel distal region of the first, and second promoter regions. Our findings indicate that Ctip2 regulates Sp6 gene expression through direct binding to the Sp6 second promoter region. J. Med. Invest. 61: 126-136, February, 2014.


Adenylate kinase 2 deficiency limits survival and regulates various genes during larval stages of Drosophila melanogaster.

Adenylate kinase isozyme 2 (AK2) is located in mitochondrial intermembrane space and regulates energy metabolism by reversibly converting ATP and AMP to 2 ADPs. We previously demonstrated that disruption of the Drosophila melanogaster AK2 gene (Dak2) resulted in growth arrest during the larval stage and subsequent death. Two other groups found that human AK2 mutations cause reticular dysgenesis, a form of severe combined immunodeficiency (SCID) that is associated with severe hematopoietic defects and sensorineural deafness. However, the mechanisms underlying differential outcomes of AK2 deficiency in Drosophila and human systems remain unknown. In this study, effects of tissue-specific inactivation of the Dak2 gene on Drosophila development were analyzed using RNAi-mediated gene knockdown. In addition, to investigate the roles of AK2 in the regulation of gene expression during development, microarray analysis was performed using RNA from first and second instar larvae of Dak2-deficient mutant and wild-type D. melanogaster. Knockdown of Dak2 in all germ layers caused cessation of growth and subsequent death of flies. Microarray analysis revealed that Dak2 deficiency downregulates various genes, particularly those involved in the proteasomal function and in mitochondrial translation machinery. These data indicate that adenine nucleotide interconversion by Dak2 is crucial for developmental processes of Drosophila melanogaster. J. Med. Invest. 61: 137-150, February, 2014.

The Journal of Medical Investigation : JMI


Novel genetic linkage of rat Sp6 mutation to Amelogenesis imperfecta

ABSTRACT: BACKGROUND: Amelogenesis imperfecta (AI) is an inherited disorder characterized by incomplete formation of tooth enamel. Although several genes responsible for AI have been reported, not all causative genes for human AI have been identified to date. AMI rat has been reported as an autosomal recessive mutant with hypoplastic AI isolated from a colony of stroke-prone spontaneously hypertensive rat strain, but the causative gene has not yet been clarified. Through a genetic screen, we identified the causative gene of autosomal recessive AI in AMI and analyzed its role in amelogenesis. METHODS: cDNA sequencing of possible AI-candidate genes so far identified using total RNA of day 6 AMI rat molars identified a novel responsible mutation in specificity protein 6 (Sp6). Genetic linkage analysis was performed between Sp6 and AI phenotype in AMI. To understand a role of SP6 in AI, we generated the transgenic rats harboring Sp6 transgene in AMI (Ami/Ami + Tg). Histological analyses were performed using the thin sections of control rats, AMI, and Ami/Ami + Tg incisors in maxillae, respectively. RESULTS: We found the novel genetic linkage between a 2-bp insertional mutation of Sp6 gene and the AI phenotype in AMI rats. The position of mutation was located in the coding region of Sp6, which caused frameshift mutation and disruption of the third zinc finger domain of SP6 with 11 cryptic amino acid residues and a stop codon. Transfection studies showed that the mutant protein can be translated and localized in the nucleus in the same manner as the wild-type SP6 protein. When we introduced the CMV promoter-driven wild-type Sp6 transgene into AMI rats, the SP6 protein was ectopically expressed in the maturation stage of ameloblasts associated with the extended maturation stage and the shortened reduced stage without any other phenotypical changes. CONCLUSION: We propose the addition of Sp6 mutation as a new molecular diagnostic criterion for the autosomal recessive AI pateints. Our findings expand the spectrum of genetic causes of autosomal recessive AI and sheds light on the molecular diagnosis for the classification of AI. Furthermore, tight regulation of the temporospatial expression of SP6 may have critical roles in completing amelogenesis. .


Dissection of morphological and metabolic differentiation of amelobrasts via ectopic SP6 expression

Tooth enamel is the hardest organ in the body. In rodent incisor, the enamel is exclusively produced by ameloblasts with yellowish-brown pigmentation, indicating normal enamel formation. However, the molecular mechanisms of ameloblast differentiation and amelogenesis are not fully understood. Specificity protein (Sp) 6 has been reported as one of the critical factors for tooth development. To explore SP6 function, we generated Sp6 transgenic (Tg) rats. Unexpectedly, the enamel surfaces of the incisors in Tg rats were discolored, even though enamel formation and serum iron concentrations were normal. Histological analysis of incisors from 6-week-old Tg rats demonstrated that the ameloblast layer at the pigmentation stage was elongated up to the gingival margin with ectopic SP6 expression in longitudinal incisor sections. In contrast, the incisors from 10-week-old Tg rats revealed that the pigmented ameloblasts were morphologically changed to those of the reduced stage, concomitant with the sporadic disappearance of ectopic SP6 expression. Here we report that morphological differentiation and metabolism of the iron-containing pigment in ameloblasts are independently regulated during amelogenesis by means of ectopic SP6 expression.


Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization.

Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.


Dynamic changes of Sp6 Transgene Expression in Dental Epithelial Cells during Long-term Culture

The Indonesian Journal of Dental Research


Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6) is a member of the SP/Krüppel-like transcription factor family and plays key roles in tooth development. To study its biological roles, it is important to understand the spatiotemporal regulation of Sp6 gene expression. For this purpose, we first identified two separate 5' ends of the Sp6 cDNA by 5' RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly, we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last, we also found that bone morphogenetic protein and wingless signals could enhance Sp6 promoter activity in dental epithelial cells, suggesting the regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions.


Localization of adenylate kinase 4 in mouse tissues.


Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster.

Adenylate kinases are phylogenetically widespread, highly conserved, and involved in energy metabolism and energy transfer. Of these, adenylate kinase (AK) isozyme 2 is uniquely localized in the mitochondrial intermembrane space and its physiological role remains largely unknown. In this study, we selected Drosophila melanogaster to analyze its role in vivo. AK isozyme cDNAs were cloned and their gene expressions were characterized in D. melanogaster. The deduced amino acid sequences contain highly conserved motifs for P-loop, NMP binding, and LID domains of AKs. In addition, the effects of AK2 gene knockout on phenotype of AK2 mutants were examined using P-element technology. Although homozygous AK2 mutated embryos developed without any visible defects, their growth ceased and they died before reaching the third instar larval stage. Maternally provided AK2 mRNA was detected in fertilized eggs, and weak AK2 activity was observed in first and second instar larvae of the homozygous AK2 mutants, suggesting that maternally provided AK2 is sufficient for embryonic development. Disappearance of AK2 activity during larval stages resulted in growth arrest and eventual death. These results demonstrate that AK2 plays a critical role in adenine nucleotide metabolism in the mitochondrial intermembrane space and is essential for growth in D. melanogaster.

Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology


BMP2-induced gene profiling in dental epithelial cell line.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (BMP2) is known as one of the inducers for tooth development. To analyze the molecular mechanisms of BMP2 on ameloblast differentiation (amelogenesis), we performed microarray analyses using rat dental epithelial cell line, HAT-7. After confirming that BMP2 could activate the canonical BMP-Smads signaling in HAT-7 cells, we analyzed the effects of BMP2 on 14,815 gene expressions and profiled them. Seventy-three genes were up-regulated and 28 genes were down-regulated by BMP2 treatment for 24 hours in HAT-7 cells. Functional classification revealed that 18% of up-regulated genes were ECM/adhesion molecules present in the enamel organ. Furthermore, we examined the expression of several differentiation markers in dental epithelial four cell-lineages including inner enamel epithelium (ameloblasts), stratum intermedium, stratum reticulum, and outer enamel epithelium. The results indicated that BMP2 might induce at least two different cell-lineage markers including a BMP antagonist expressed in HAT-7 cells, suggesting that BMP2 could accelerate amelogenesis via BMP signaling.

The Journal of Medical Investigation : JMI


Sp6 downregulation of follistatin gene expression in ameloblasts.

Sp6 is a member of the Sp family of transcription factors that regulate a wide range of cellular functions, such as cell growth and differentiation. Sp6, also called epiprofin, is specifically expressed in tooth germ, limb bud, and hair follicle, but there is little information on its function.To investigate the possible role of Sp6 in tooth development, first we established an Sp6-overproducing clone, CHA9, and analyzed the features of the cell, including cell proliferation and gene expression. The parental cells of CHA9 are the ameloblast-lineage G5 cells that we previously established from rat dental epithelia of lower incisor. Sp6 overproduction accelerated cell proliferation and induced the expression of ameloblastin mRNA, a marker of ameloblast differentiation. Second, we performed genome-wide screening of Sp6 target genes by microarray analysis. Out of a total 20,450 genes, 448 genes were up-regulated and 500 genes were down-regulated by Sp6. We found the expression of follistatin, a BMP antagonist, to be 22.4-fold lower in CHA9 than in control cells. Transfection of the Sp6-antisense construct into CHA9 cells restored follistatin expression back to equivalent levels seen in control cells, indicating that Sp6 regulates follistatin gene expression in ameloblasts.Our findings demonstrate that the follistatin gene is one of the Sp6 target genes in ameloblasts and suggest that Sp6 promotes amelogenesis through inhibition of follistatin gene expression.


Establishment and characterization of rat dental epithelial derived ameloblast-lineage clones.

Teeth are the hardest tissues covered with enamel produced by ameloblasts. The ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions during tooth morphogenesis. However, the molecular mechanism of ameloblast differentiation remains unclear. To address this question, we developed an in vitro assay system to evaluate the molecular mechanism of amelogenesis. First, we established dental epithelium-derived clones from 6-day-old rat incisors and established that cells of the clone SRE-G5 were the largest producers of amelogenin mRNA. Next, we analyzed the effects of several chemicals on the amelogenin expression in SRE-G5 cells. Only mitogen-activated protein kinase (MAPK) activators enhanced amelogenin mRNA expression. This finding corresponded to the immunohistochemical data showing the presence of phosphorylated forms of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) during ameloblast differentiation. To examine the roles of MAPK signals, we compared the effects of anisomycin and sodium salicylate on the expression of tooth-related differentiation markers. Both anisomycin and sodium salicylate induced amelogenin, Abcg2, and Bmp4 mRNA and down-regulated p75NGFR mRNA. On the other hand, ALP, ectodin, Bmp2 and Fgf8 mRNA were up-regulated only by anisomycin. These results indicate that MAPK signaling functions, at least in part, as the inducer of ameloblast differentiation.

Journal of Bioscience and Bioengineering


Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.


Soluble matrix from osteoblastic cells induces mineralization by dental pulp cells

Dental pulp cells have a capacity to differentiate into mineralization-inducing cells. To clarify the molecular mechanism, we established an in vitro mineralization-inducing system by rat clonal dental pulp cell line, RPC-C2A, and tried to purify a mineralization-inducing factor in conditioned medium (CM) from pre-osteoblastic MC3T3-E1 cells. The active factor was impermeable to an ultrafiltration membrane, and sedimented by ultracentrifugation. The sedimented factor was found as a needle-like structure about 1.3 microm in average length as observed by transmission electron microscopy. The factor contained type I collagen, suggesting not a matrix vesicle, but a soluble matrix. The mineralization-inducing activity was also detected in CM from primary culture of rat calvaria (RC) cells. These results suggested that the soluble matrices from osteoblastic cells serve, at least in part, as differentiation-inducing agents.


Effects of TGF-beta1 on Extracellular Matrix Formation in Rat Clonal Dental Pulp Cells


Three-dimensional Reconstruction of Transcription Termination Factor rho: Orientation of the N-terminal Domain and Visualization of an RNA-binding Site

The Escherichia coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid. Unlike hexameric DNA helicases, where a single strand of DNA passes through the central channel, it has been proposed that the RNA wraps around the outside of the ring. We have generated a three-dimensional reconstruction of rho, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring. An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring. Although rho shares a common structural core with the F1-ATPase and other hexameric helicases, there has been a divergence in function due to rho's N-terminal domain, which has no homology to other helicases.


The Quaternary Geometry of Transcription Termination Factor rho: Assignment by Chemical Cross-linking


Structural and Functional Dissections of Transcription Termination Factor Rho by Random Mutagenesis


Microarray Data Analysis Mainly on a Functional Analysis using DAVID

マイクロアレイデータ解析法 ─DAVIDによる機能解析を中心に─