OBJECTIVES:The Fat gene family, also known as atypical cadherins, contributes to the formation of planar cell polarity, which determines organ shape, size, and polarity. However, their roles in tooth morphogenesis remain unclear. In this study, the aim was to investigate the spatiotemporal expression patterns of Fat1-Fat4 during murine tooth development. METHODS:Quantitative polymerase chain reaction was performed using total RNA from newborn C57BL/6J murine tissues to determine Fat1-Fat4 expression. In situ hybridization was performed to examine the localization of Fat family transcripts during tooth development from embryonic Day 12.5 to birth. The dental epithelial stem cell line, M3H1, was used as an in vitro differentiation model, and small-interfering-RNA-mediated knockdown of Fat2 was performed to assess its function. Public single-cell RNA-sequence (scRNA-seq) datasets were analyzed to confirm cell-type specificity. RESULTS:All Fat genes were expressed in the teeth, with Fat2 having the highest level among tissues examined. Fat1 was expressed in the epithelial and mesenchymal cells, and Fat2 expression was restricted to the outer enamel epithelium (OEE) and stratum intermedia (SI). Fat3 and Fat4 were expressed in the dental mesenchyme. In M3H1 cells, Fat1 and Fat2 had biphasic expression at undifferentiated and differentiated stages. Fat2 knockdown decreased Sox2 and nephronectin expression, and increased Foxj1 expression. scRNA-seq supported Fat2 specificity in the OEE and SI. CONCLUSION:Fat genes have cell-type-specific and developmentally regulated expression during tooth development. Among them, Fat2 emerges as a novel marker of OEE and SI, and it may contribute to epithelial identity and differentiation during odontogenesis.

Metabolic stress during enamel development may influence ameloblast differentiation and enamel formation, although the underlying mechanisms remain poorly understood. In this study, we investigated transcriptional and cellular responses to palmitic acid (PA) in the human ameloblast-like cell line HAM3 using a custom quantitative reverse transcription polymerase chain reaction panel based on amelogenesis imperfecta-related genes. PA exposure under both sustained low-dose conditions (50 μM for 24 h) and acute high-dose conditions (500 μM for 4 h) induced coordinated transcriptional changes associated with enamel maturation and cellular stress responses. Expression of the maturation-associated gene AMTN was detectable at baseline and consistently upregulated following PA exposure, whereas that of the epithelial transcription factor gene BCL11B was selectively suppressed under acute high-dose conditions. PA also induced expression of stress-responsive genes including GDF15 and HMOX1. In parallel, immunoblotting demonstrated increased levels of cleaved caspase-3 and LC3-II, while immunofluorescence analysis revealed accumulation of p62-positive structures, consistent with stress-associated apoptosis and altered autophagy-related processes. These findings suggest that PA-induced metabolic stress promotes a stress-associated pseudo-maturation-like transcriptional state in ameloblast-like cells while activating coordinated cellular stress responses. This study provides a potential framework linking metabolic stress environments to altered enamel development.

Head and neck squamous cell carcinoma (HNSCC) is a significant healthcare burden globally. Previous research using single-cell transcriptome analysis identified TGFBI as a crucial marker for the partial-epithelial-mesenchymal transition (partial-EMT) program. However, the precise role of TGFBI in HNSCC progression remains unclear. Therefore, our study aimed to clarify the impact of TGFBI on the malignant behavior of HNSCC cells. Through RNA-sequencing data from the TCGA database, we validated that increased TGFBI expression correlates with a higher occurrence of lymph node metastasis and unfavorable prognosis in HNSCC cases. Functional experiments demonstrated that TGFBI overexpression enhances the ability of sphere formation, indicating stem-cell-like properties. Conversely, TGFBI depletion reduces sphere formation and suppresses the expression of cancer stem cell (CSC) markers. RNA-sequencing analysis of TGFBI-overexpressing and control HNSCC cells revealed TAGLN as a downstream effector mediating TGFBI-induced sphere formation. Remarkably, TAGLN depletion abolished TGFBI-induced sphere formation, while its overexpression rescued the suppressed sphere formation caused by TGFBI depletion. Moreover, elevated TAGLN expression showed correlations with the expression of TGFBI and partial-EMT-related genes in HNSCC cases. In conclusion, our findings suggest that TGFBI may promote CSC properties through the upregulation of TAGLN. These novel insights shed light on the involvement of the TGFBI-TAGLN axis in HNSCC progression and hold implications for the development of targeted therapies.

Objectives: Periodontitis, commonly associated with Porphyromonas gingivalis ( Pg ), involves intricate alterations of oral intercellular interactions, in which extracellular vesicles (EVs) play a pivotal role. The understanding of the miRNA profiles in the EVs derived from Pg -infected cells ( Pg -EVs) remains incomplete despite acknowledging their importance in intercellular communication during periodontitis. Therefore, our objective was to identify and characterize the miRNAs enriched in Pg -EVs. Methods: Microarray analysis was conducted to examine the miRNA profiles in the EVs derived from Pg -infected THP-1 cells. We compared the identified miRNAs with those upregulated in the EVs after stimulation with LPS. Additionally, we explored how inhibiting TLR signaling during Pg infection affects the transcription of specific miRNAs. We investigated the unique sequence motifs specific to the miRNAs concentrated in Pg -EVs. Results: The levels of eleven miRNAs, including miR-155, were increased in Pg -EVs compared with those elevated after LPS stimulation. The Pg -induced miR-155 upregulation via TLR2 but not TLR4 signaling suggests the influence of TLR signaling on the miRNA composition of EVs. Furthermore, the miRNAs upregulated in Pg -EVs contained AGAGGG and GRGGSGC sequence motifs. Conclusions: Our findings demonstrate that Pg -induced alterations in EV-containing miRNA composition occur in a TLR4-independent manner. Notably, the concentrated miRNAs in Pg -EVs harbor specific motifs with a high G + C content within their sequences. The upregulation of specific miRNAs in EVs under infectious conditions suggests the influence of both innate immune receptor signals and miRNA sequence characteristics.

BACKGROUND:Comprehending the molecular mechanisms underlying head and neck squamous cell carcinoma (HNSCC) is vital for the development of effective treatment strategies. Deubiquitinating enzymes (DUBs), which regulate ubiquitin-dependent pathways, are potential targets for cancer therapy because of their structural advantages. Here we aimed to identify a potential target for HNSCC treatment among DUBs.METHODS:A screening process was conducted using RNA sequencing data and clinical information from HNSCC patients in the TCGA database. A panel of 88 DUBs was analyzed to identify those associated with poor prognosis. Subsequently, HNSCC cells were modified to overexpress specific DUBs, and their effects on cell proliferation and invasion were evaluated. In vivo experiments were performed to validate the findings.RESULTS: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.CONCLUSIONS: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.

Supplementary Data from Novel Calcium-Binding Ablating Mutations Induce Constitutive RET Activity and Drive Tumorigenesis

The precise formation of mineralized dental tissues such as enamel and/or dentin require tight transcriptional control of the secretion of matrix proteins. Here, we have investigated the transcriptional regulation of the second most prominent enamel matrix protein, enamelin, and its regulation through the major odontogenic transcription factor, MSX2.

Glucosylceramide is the primary molecule of glycosphingolipids, and its metabolic regulation is crucial for life. Defects in the catabolizing enzyme, glucocerebrosidase (GCase), cause a lysosomal storage disorder known as Gaucher disease. However, the genetic regulation of GCase has not been fully understood. Here we show the redefined structure of the GCase coding gene ( GBA ), and clarify the regulatory mechanisms of its transcription and translation. First, alternative uses of the two GBA gene promoters were identified in fibroblasts and HL60-derived macrophages. Intriguingly, both GBA transcripts and GCase activities were induced in macrophages but not in neutrophils. Second, we observed cap-independent translation occurs via unique internal ribosome entry site activities in first promoter-driven GBA transcripts. Third, the reciprocal expression was observed in GBA and miR22-3p versus GBAP1 transcripts before and after HL60-induced macrophage differentiation. Nevertheless, these findings clearly demonstrate novel cell-type-specific GBA gene expression regulatory mechanisms, providing new insights into GCase biology.

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.

Abstract Distinguishing oncogenic mutations from variants of unknown significance (VUS) is critical for precision cancer medicine. Here, computational modeling of 71,756 RET variants for positive selection together with functional assays of 110 representative variants identified a three-dimensional cluster of VUSs carried by multiple human cancers that cause amino acid substitutions in the calmodulin-like motif (CaLM) of RET. Molecular dynamics simulations indicated that CaLM mutations decrease interactions between Ca2+ and its surrounding residues and induce conformational distortion of the RET cysteine-rich domain containing the CaLM. RET-CaLM mutations caused ligand-independent constitutive activation of RET kinase by homodimerization mediated by illegitimate disulfide bond formation. RET-CaLM mutants possessed oncogenic and tumorigenic activities that could be suppressed by tyrosine kinase inhibitors targeting RET. This study identifies calcium-binding ablating mutations as a novel type of oncogenic mutation of RET and indicates that in silico–driven annotation of VUSs of druggable oncogenes is a promising strategy to identify targetable driver mutations. Significance: Comprehensive proteogenomic and in silico analyses of a vast number of VUSs identify a novel set of oncogenic and druggable mutations in the well-characterized RET oncogene.

Dental implants made of titanium are commonly used. Although titanium implants succeed by osseointegration with bone, the detailed molecular mechanism of osseointegration is unclear. To clarify the involvement of microRNA (miRNA) in the acquisition of osseointegration on titanium, here we compared the miRNA expression profiles of mouse osteoblast-like cells (MC3T3-E1) cultured on titanium-, gold-, and stainless steel–coating glass dishes by microarray analysis. Three kinds of metals, namely titanium, gold, and stainless steel, were coated on the surface of the glass dishes by sputtering with similar roughness and shape of their surface. After MC3T3-E1 cells were cultured on the dishes without coating or coating with titanium, gold, or stainless steel for 6 h, total RNA was extracted, and miRNA expression was analyzed by microarray. To confirm the expression of the selected miRNA during osteogenic differentiation of MC3T3-E1 cells, real-time PCR analysis was performed. Furthermore, the effects of selected miRNA were examined by ectopic overexpression in MC3T3-E1 cells. The microarray analysis revealed that the expressions of miR-155-5p and miR-7023-3p were significantly increased in MC3T3-E1 cells cultured on titanium-coating glass dishes, compared to non-coating, gold-, and stainless steel–coating glass dishes. Interestingly, miR-155-5p was upregulated during osteogenic differentiation of MC3T3-E1 cells. Furthermore, overexpression of miR-155-5p enhanced the expression of Runx2 and Col1a1. In this study, miR-155-5p may be involved in the acquisition of osseointegration on titanium implant via upregulating osteogenic differentiation–related genes.

Background: It is well-established that cervical lymph node metastasis is the most important prognostic factor in head and neck squamous cell carcinoma (HNSCC). Cancer cells invade the underlying stroma during metastasis by breaching the basement membrane. Highlight: The ability to metastasize is a key hallmark of cancer progression and this characteristic can be attained by undergoing epithelial-to-mesenchymal transition (EMT). EMT is a biological process in which epithelial cells lose their epithelial features and gain mesenchymal features. Recent evidence reports the intermediate state in the induction of EMT and partial-EMT. Notably, the partial-EMT phenotype is more aggressive than the complete EMT phenotype. However, the role of partial-EMT is not fully understood. Conclusion: In this review, we highlight the features of partial-EMT in HNSCC by summarizing previous studies. Moreover, we discuss the therapeutic potential for targeting partial-EMT. (c) 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Considering that every tissue/organ has the most suitable microenvironment for its functional cells, controlling induced pluripotent stem cell (iPSC) differentiation by culture on frozen sections having a suitable microenvironment is possible. Induced PSCs were cultured on frozen sections of the liver, the brain, the spinal cord, and cover glasses (control) for 9 days. The iPSCs cultured on the sections of the liver resembled hepatocytes, whereas those on sections of the brain and the spinal cord resembled neuronal cells. The percentage of hepatocytic marker-positive cells in the iPSCs cultured on the sections of the liver was statistically higher than that of those in the iPSCs cultured on the sections of the brain and the spinal cord or on cover glasses. In contrast, the iPSCs cultured on the sections of the brain and the spinal cord revealed a high percentage of neural marker-positive cells. Thus, iPSCs can be differentiated into a specific cell lineage in response to specific factors within frozen sections of tissues/organs. Differentiation efficacy of the frozen sections markedly differed between the iPSC clones. Therefore, our induction method could be simple and effective for evaluating the iPSC quality.

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

Background Bronchioalveolar stem cells (BASCs) located at the bronchioalveolar-duct junction (BADJ) are stem cells residing in alveoli and terminal bronchioles that can self-renew and differentiate into alveolar type (AT)-1 cells, AT-2 cells, club cells, and ciliated cells. Following terminal-bronchiole injury, BASCs increase in number and promote repair. However, whether BASCs can be differentiated from mouse-induced pluripotent stem cells (iPSCs) remains unreported, and the therapeutic potential of such cells is unclear. We therefore sought to differentiate BASCs from iPSCs and examine their potential for use in the treatment of epithelial injury in terminal bronchioles. Methods BASCs were induced using a modified protocol for differentiating mouse iPSCs into AT-2 cells. Differentiated iPSCs were intratracheally transplanted into naphthalene-treated mice. The engraftment of BASCs into the BADJ and their subsequent ability to promote repair of injury to the airway epithelium were evaluated. Results Flow cytometric analysis revealed that BASCs represented ~ 7% of the cells obtained. Additionally, ultrastructural analysis of these iPSC-derived BASCs via transmission electron microscopy showed that the cells containing secretory granules harboured microvilli, as well as small and immature lamellar body-like structures. When the differentiated iPSCs were intratracheally transplanted in naphthalene-induced airway epithelium injury, transplanted BASCs were found to be engrafted in the BADJ epithelium and alveolar spaces for 14 days after transplantation and to maintain the BASC phenotype. Notably, repair of the terminal-bronchiole epithelium was markedly promoted after transplantation of the differentiated iPSCs. Conclusions Mouse iPSCs could be differentiated in vitro into cells that display a similar phenotype to BASCs. Given that the differentiated iPSCs promoted epithelial repair in the mouse model of naphthalene-induced airway epithelium injury, this method may serve as a basis for the development of treatments for terminal-bronchiole/alveolar-region disorders.

BackgroundAmeloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis.MethodsTwo different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites.ResultsUsing the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts, (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts, and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid, up-regulated Sp6 and downregulated Fst expression. Additionally, using MatInspector, we identified several potential putative Msx2 binding sites, 3.5 kb upstream of the murine Sp6 promoter region. By chIP, we confirmed the binding of Msx2 to Sp6 promoter at these sites, thus suggesting that Sp6 is a direct target of Msx2.ConclusionCollectively, these results show that Sp6 and Msx2 work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.

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.