Keiji Tanimoto | Hiroshima University (original) (raw)

Papers by Keiji Tanimoto

Cancers

Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More ... more Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More effective and safer drugs are needed that are based on molecular mechanisms. A disintegrin and metalloproteases (ADAMs) are expressed with high frequency in various human carcinomas and play an important role in cancer progression. In this study, we analyzed expression of ADAMs in HBL with a cDNA microarray dataset and found that the expression level of ADAM32 is particularly high. To investigate the role of ADAM32 in cancer, forced expression or knockdown experiments were conducted with HepG2 and HBL primary cells. Colony formation, cell migration and invasion, and cell viability were increased in HepG2 expressing ADAM32, whereas knockdown of ADAM32 induced a decrease in these cellular functions. Quantitative RT-PCR demonstrated an association between ADAM32 expression and the expression of genes related to cancer stem cells and epithelial–mesenchymal transition (EMT), suggesting a role...

International Journal of Molecular Sciences, 2022

Sodium fluoride (NaF) is widely used in clinical dentistry. However, the administration of high o... more Sodium fluoride (NaF) is widely used in clinical dentistry. However, the administration of high or low concentrations of NaF has various functions in different tissues. Understanding the mechanisms of the different effects of NaF will help to optimize its use in clinical applications. Studies of NaF and epithelial cells, osteoblasts, osteoclasts, and periodontal cells have suggested the significant roles of fluoride treatment. In this review, we summarize recent studies on the biphasic functions of NaF that are related to both soft and hard periodontal tissues, multiple diseases, and clinical dentistry.

npj Microgravity, 2018

The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic... more The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite ® may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was significantly attenuated under simulated microgravity (10-3 G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was specifically observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were significantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated with 5-AzaC, and further incubated with differentiation medium under 1 G or 10 −3 G conditions. The results showed differences in expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10 −3 G conditions, completely disappeared in this experimental condition. Modified HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation.

Journal of Biological Chemistry, 2002

Under normoxic conditions the hypoxia-inducible factor-1␣ (HIF-1␣) protein is targeted for degrad... more Under normoxic conditions the hypoxia-inducible factor-1␣ (HIF-1␣) protein is targeted for degradation by the von Hippel-Lindau (pVHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Binding of pVHL to HIF-1␣ is dependent on hydroxylation of specific proline residues by O 2-dependent prolyl 4-hydroxylases. Upon exposure to hypoxia the hydroxylase activity is inhibited, resulting in stabilization of HIF-1␣ protein levels and activation of transcription of target genes. One of the two critical proline residues, Pro 563 in mouse HIF-1␣, is located within a bifunctional domain, the Nterminal transactivation domain (N-TAD), which mediates both pVHL-dependent degradation at normoxia and transcriptional activation at hypoxia. Here we have identified two N-TAD residues, Tyr 564 and Ile 565 , which, in addition to Pro 563 , were critical for pVHL-mediated degradation at normoxia. We have also identified D568A/ D569A/D570A, F571A, and L573A as mutations of the N-TAD that abrogated binding to pVHL both in vitro and in vivo, and constitutively stabilized N-TAD against degradation. Moreover, the mutations Y564G, L556A/L558A, and F571A/L573A drastically reduced the transactivation function of either the isolated N-TAD or full-length HIF-1␣ in hypoxic cells. Interestingly, the P563A mutant exhibited a constitutively active and potent transactivation function that was enhanced by functional interaction with the transcriptional coactivator protein CREBbinding protein. In conclusion, we have identified by mutation analysis several residues that are critical for either one or both of the interdigitated and conditionally regulated degradation and transactivation functions of the N-TAD of HIF-1␣.

Biochemical and Biophysical Research Communications, 2013

GLI-similar 1 (GLIS1) is important for the reprogramming of fibroblasts into induced pluripotent ... more GLI-similar 1 (GLIS1) is important for the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs). However, the molecular mechanisms of regulation of GLIS1 expression remain unclear. We have therefore examined GLIS1 expression in various cancer cell lines and demonstrated that GLIS1 expression was dramatically increased under hypoxic conditions. Importantly, GLIS1 expression was significantly attenuated in VHL-overexpressing renal cell carcinoma cells compared to the VHL-deficient parent control. Moreover, promoter analysis demonstrated that GLIS1 transcription was regulated by hypoxia through a hypoxia-inducible factors (HIFs)-dependent mechanism. Co-transfection experiments revealed that HIF-2a had greater potency on the GLIS1 promoter activation than HIF-1a. Subsequent studies using wild-type and mutant HIF-2a demonstrated that DNA binding activity was not necessary but TADs were critical for GLIS1 induction. Finally, co-transfection experiments indicated that HIF-2a cooperated with AP-1 family members in upregulating GLIS1 transcription. These results suggest that the hypoxic signaling pathway may play a pivotal role in regulating the reprogramming factor GLIS1, via non-canonical mechanisms involving partner transcription factor rather than by direct HIF transactivation.

The EMBO Journal, 2000

In normoxic cells the hypoxia-inducible factor-1a (HIF-1a) is rapidly degraded by the ubiquitin-p... more In normoxic cells the hypoxia-inducible factor-1a (HIF-1a) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1a to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1a under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1a. The region of VHL mediating interaction with HIF-1a overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1a and subsequent degradation. Interestingly, the VHL binding site within HIF-1a overlapped with one of the minimal transactivation domains. Protection of HIF-1a against degradation by VHL was a multistep mechanism, including hypoxiainduced nuclear translocation of HIF-1a and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1a during this process. Finally, stabilization of HIF-1a protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.

Cells

The biological effects of low-dose-rate (LDR) radiation exposure in nuclear power plant accidents... more The biological effects of low-dose-rate (LDR) radiation exposure in nuclear power plant accidents and medical uses of ionizing radiation (IR), although being a social concern, remain unclear. In this study, we evaluated the effects of LDR-IR on global gene expression in human cells and aimed to clarify the mechanisms. RNA-seq analyses demonstrated that relatively low dose rates of IR modify gene expression levels in TIG-3 cells under normoxic conditions, but those effects were attenuated under hypoxia-mimicking conditions. Gene set enrichment analysis demonstrated that LDR-IR significantly decreased gene expression related to cell division, cell cycle, mitosis, and the Aurora kinase B and FOXM1 pathways. Quantitative RT-PCR confirmed the down-regulation of AURKB and FOXM1 genes in TIG-3 cells with LDR-IR or hypoxia-mimicking treatments without any dose-rate effect. Knock-down experiments suggested that HIF-1α and HIF-2α, as well as DEC1, participated in down-regulation of AURKB and ...

Cancer research, 2005

Dihydropyrimidine dehydrogenase is the most extensively investigated predictive marker for indivi... more Dihydropyrimidine dehydrogenase is the most extensively investigated predictive marker for individual response to 5-fluorouracil. Clinical responses to the anticancer agent, along with various reports, have clearly shown that dihydropyrimidine dehydrogenase activity is closely correlated to its mRNA levels, but the regulatory mechanisms of its expression have remained unclear. We attempted to clarify the mechanisms and found that activator protein (AP-1) is probably one of the key factors in the transcriptional regulation of DPYD in cancer cells, and that phorbol 12-myristate 13-acetate (PMA) plus ionomycin treatment enhances transcription of DPYD via AP-1 activation. In this study, we characterized our previously subcloned 5' region of human DPYD, an approximately 3.0-kb fragment (accession no. AB162145). Luciferase reporter assay showed that the clone showed strong promoter activities in 293T and HSC42 cells, and comparative analysis using 5' deletion mutants suggested the...

International Journal of Molecular Sciences

Periodontal inflammation is a common inflammatory disease associated with chronic inflammation th... more Periodontal inflammation is a common inflammatory disease associated with chronic inflammation that can ultimately lead to alveolar attachment loss and bone destruction. Understanding autophagy and pyroptosis has suggested their significant roles in inflammation. In recent years, studies of differentiated embryo-chondrocyte expressed genes 1 and 2 (Dec1 and Dec2) have shown that they play important functions in autophagy and in pyroptosis, which contribute to the onset of periodontal inflammation. In this review, we summarize recent studies on the roles of clock genes, including Dec1 and Dec2, that are related to periodontal inflammation and other diseases.

PLOS ONE

Although the biological systems in the human body are affected by the earth's gravity, informatio... more Although the biological systems in the human body are affected by the earth's gravity, information about the underlying molecular mechanisms is limited. For example, apoptotic signaling is enhanced in cancer cells subjected to microgravity. We reasoned that signaling regulated by p53 may be involved because of its role in apoptosis. Therefore, we aimed to clarify the molecular mechanisms of modified cis-diamminedichloroplatinum (CDDP)-sensitivity under simulated microgravity by focusing on p53-related cell death mechanisms. Immunoblotting analyses indicated that, under microgravity, CDDP-induced ATM/p53 signaling increased and caspase-3 was cleaved earlier. However, microgravity decreased the levels of expression of p53 targets BAX and CDKN1A. Interestingly, microgravity increased the PTEN, DRAM1, and PRKAA1 mRNA levels. However, microgravity decreased the levels of mTOR and increased the LC3-II/I ratio, suggesting the activation of autophagy. The CDDP-induced cleavage of caspase-3 was increased during the early phase in Group MG (+), and cleaved caspase-3 was detected even in Group MG (+) with constitutive expression of a mutant type of p53 (hereafter, "+" indicates CDDP treatment). These results interestingly indicate that microgravity altered CDDP sensitivity through activation of caspase-3 by p53-independent mechanism.

Circulation Journal Official Journal of the Japanese Circulation Society, Mar 1, 2008

Immunology Letters, 2011

Differentiated embryo-chondrocyte 2 (DEC2), a basic-helix-loop-helix transcriptional factor, is i... more Differentiated embryo-chondrocyte 2 (DEC2), a basic-helix-loop-helix transcriptional factor, is involved in various biological reactions by regulating the expression of its target genes. In the present study, we demonstrated DEC2 expression in response to the treatment with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, in cultured human mesangial cells. RNA interference against DEC2 enhanced the poly IC-induced expression of IFN-β and its downstream genes, retinoic acid-inducible gene-I (RIG-I) and CCL5. Knockdown of TLR3 abolished the poly IC-induced DEC2 expression. DEC2 expression may constitute a negative feedback system for the IFN-β/RIG-I/CCL5 pathway in the glomerulus, which may play a role in controlling protracted inflammatory reactions in the kidney.

Hypoxia-inducible factor-2α (HIF-2α, or EPAS1) is important for cancer progression, and is a puta... more Hypoxia-inducible factor-2α (HIF-2α, or EPAS1) is important for cancer progression, and is a putative biomarker for poor prognosis for non-small cell lung cancer (NSCLC). However, molecular mechanisms underlying the EPAS1 overexpression are not still fully understood. We explored a role of a single nucleotide polymorphism (SNP), rs13419896 located within intron 1 of the EPAS1 gene in regulation of its expression. Bioinformatic analyses suggested that a region including the rs13419896 SNP plays a role in regulation of the EPAS1 gene expression and the SNP alters the binding activity of transcription factors. In vitro analyses demonstrated that a fragment containing the SNP locus function as a regulatory region and that a fragment with A allele showed higher transactivation activity than one withG, especially in the presence of overexpressed c-Fos or c-Jun. Moreover, NSCLC patients with the A allele showed poorer prognosis than those withG at the SNP even after adjustment with various...

Scientific Reports

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for devel... more Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 ...

Carcinogenesis

We previously demonstrated that expression of a Krüppel-like zinc finger transcription factor, GL... more We previously demonstrated that expression of a Krüppel-like zinc finger transcription factor, GLIS1, dramatically increases under hypoxic conditions via a transcriptional mechanism induced by HIF-2α co-operating with AP-1 members. In this study, we focused on the functional roles of GLIS1 in breast cancer. To uncover its biological function, the effects of altered levels of GLIS1 in breast cancer cell lines on cellular growth, wound-healing, and invasion capacities were assessed. Knockdown of GLIS1 using siRNA in BT-474 cells resulted in significant growth stimulation under normoxia, while attenuation was found in the cell invasion assay under hypoxic conditions. In MDA-MB-231 cells expressing exogenous 3xFLAG-tagged GLIS1, GLIS1 attenuated cell proliferation and enhanced cell mobility and invasion capacities under normoxia. In addition, breast cancer cells expressing GLIS1 acquired resistance to irradiation. Whole transcriptome analysis clearly demonstrated that downstream signals...

PloS one, 2018

Tumor hypoxia contributes to a biologically aggressive phenotype and therapeutic resistance. Rece... more Tumor hypoxia contributes to a biologically aggressive phenotype and therapeutic resistance. Recent studies have revealed that hypoxia reduces expression of several DNA damage recognition and repair (DRR) genes via both hypoxia-inducible factor (HIF)-independent and -dependent pathways, and this induced genomic instability in cancer cells. We show here that one of the HIF-target genes-differentiated embryo chondrocyte (DEC)-plays a role in DNA damage response via transcriptional repression. Comprehensive gene expression and database analyses have revealed systemic repression of DNA-DRR genes in cancer and non-cancer cells under hypoxic conditions. Hypoxic repression in typical cases was confirmed by quantitative RT-PCR and promoter reporter experiments, and knockdown experiments indicated the critical role of DEC2 in such repression. Assessment of histone H2AX phosphorylation revealed that recognition and repair of DNA double-strand breaks (DSBs) induced by bleomycin or γ-ray irradi...

Molecular Cancer Therapeutics

Biochemical and Biophysical Research Communications, Feb 1, 2010

Vascular endothelial growth factor (Vegf) was previously shown to be expressed specifically in th... more Vascular endothelial growth factor (Vegf) was previously shown to be expressed specifically in the condylar cartilage of temporomandibular joint-osteoarthritis (TMJ-OA) model rats. Here we demonstrate for the first time that hypoxia-inducible factor-1alpha (Hif-1alpha) is activated in mature chondrocytes of temporomandibular joint-osteoarthritis (TMJ-OA) model rat by mechanical overload, and that activated Hif-1 in chondrocytes can induce osteoclastogenesis via repression of osteoprotegerin (Opg) expression. In rat TMJs, degeneration of the condylar cartilage became prominent in proportion to the duration of overloading. Hif-1alpha expression was observed specifically in mature and hypertrophic chondrocytes, and Hif-1alpha-positivity, level of Vegf expression, and tartrate-resistant acid phosphatase (TRAP)-positive cell numbers all increased in the same manner. When ATDC5 cells induced differentiation by insulin were cultured under hypoxia, Hif-1alpha induction was observed in mature stage, but not in immature stage. Inductions of Hif-1-target genes showed a similar expression pattern. In addition, expression of Opg decreased in hypoxia, and Hif-1alpha played a role, in part, in its regulation.

Cancer Letters, Feb 8, 2009

EMP3, epithelial membrane protein 3, was recently reported to be a tumor suppressor gene in neuro... more EMP3, epithelial membrane protein 3, was recently reported to be a tumor suppressor gene in neuroblastomas and gliomas. We found that EMP3 was commonly repressed in esophageal squamous cell carcinoma (ESCC) cell lines by oligonucleotide microarrays. Its overexpression in ESCC cell lines caused growth inhibition with morphological changes and TERT repression. In addition to promoter hypermethylation, TSA caused repression of EMP3, indicating the involvement of histone deacetylase-regulated repressors. The post-recurrent survival after radical surgery was poorer in ESCC patients with lower EMP3 expression. We propose that EMP3 may be a tumor suppressor gene at the late step of ESCC carcinogenesis.