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

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.

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. Case Presentation: 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. Clinical Discussion: 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. Conclusion: 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 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.

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.

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

BACKGROUND/AIMS: Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are involved in hematopoietic differentiation. However, the mechanistic linkage between ER stress/UPR and hematopoietic differentiation remains unclear. METHODS: We used bipotent HL-60 cells as an in vitro hematopoietic differentiation system to investigate the role of ER stress and UPR activity in neutrophil and macrophage differentiation. RESULTS: The in vitro differentiation analysis revealed that ER stress decreased during both neutrophil and macrophage differentiations, and the activities of PERK and ATF6 were decreased and that of IRE1α was increased during neutrophil differentiation in a stage-specific manner. By contrast, the activities of ATF6 and ATF4 decreased during macrophage differentiation. When the cells were treated with oligomycin, the expression of CD11b, a myelocytic differentiation marker, and morphological differentiation were suppressed, and XBP-1 activation was inhibited during neutrophil differentiation, whereas CD11b expression was maintained, and morphological differentiation was not obviously affected during macrophage differentiation. CONCLUSION: In this study, we demonstrated that neutrophil differentiation is regulated by ER stress/UPR that is supported by mitochondrial ATP supply, in which IRE1α-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 in vitro 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.

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.

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.

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.

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.

Localization of adenylate kinase 4 in mouse tissues.

Adenylate kinase (AK) is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. Of its isoforms, AK4 has a similar sequence and subcellular localization to that of AK3 in the mitochondrial matrix. However, unlike AK3, AK4 lacks the guanosine triphosphate: adenosine monophosphate phosphotransferase activity. To elucidate the physiological role of AK4, we explored the protein localization of AK4 in various mouse tissues by immunohistochemical analysis. AK4 protein was detected in the kidney, liver, brain, heart, stomach, intestine, and gonads but not in the lung and spleen. Interestingly, cell-type specific expression was evident in the brain, gastrointestinal tract, and gonads. In the cerebellum, AK4 was detected in granular cells but not in Purkinje cell bodies. In the gastrointestinal tract, AK4 was highly expressed in epithelia. In the ovary, AK4 was detected in oocytes and corpora lutea. In the testis, AK4 was detected in spermatocytes but not in spermatogonia. Our findings demonstrate that AK4 localizes uniquely in a cell-type and tissue-specific manner 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.

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.

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.