• Fisheries and Aquatic Sciences
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• v.20 no.6
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• pp.10.1-10.7
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• 2017

Small GTPases are well known as one of the signal transduction factors of immune systems. The ADP-ribosylation factors (ARFs) can be classified into three groups based on the peptide sequence, protein molecular weight, gene structure, and phylogenetic analysis. ARF1 recruits coat proteins to the Golgi membranes when it is bound to GTP. The class I duplicated ARF gene was cloned and characterized from the olive flounder (Paralichthys olivaceus) for this study. PoARF1b contains the GTP-binding motif and the switch 1 and 2 regions. PoARF1b and PoARF1b mutants were transfected into a Hirame natural embryo cell to determine the distribution of its GDP/GTP-bound state; consequently, it was confirmed that PoARF1b associates with the Golgi body when it is in a GTP-binding form. The results of the qPCR-described PoARF1b were expressed for all of the P. olivaceus tissues. The authors plan to study the gene expression patterns of PoARF1b in terms of immunity challenges.

• Fisheries and Aquatic Sciences
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• v.15 no.1
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• pp.29-36
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• 2012

Osteosarcoma is the most common primary malignancy of bone, and patients often develop pulmonary metastasis. The mechanisms underlying osteosarcoma metastasis remain to be elucidated. Recently, anti-inflammatory agents were shown to be useful in the treatment of tumor progression. We previously isolated a natural anti-inflammatory peptide from the seahorse Hippocampus kuda bleeler. Here, we examined the antitumor metastatic activity of this peptide and investigated its mechanism. The peptide significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasive migration of human osteosarcoma MG-63 cells. Its inhibitory effect on invasive migration was associated with reduced expression of matrix metalloproteinases (MMP1 and MMP2). In addition, TPA stimulation increased intracellular reactive oxygen species (ROS) generation and small GTPase Rac1 expression, whereas the peptide decreased ROS generation and Rac1 activation. Taken together, these results suggest that the peptide inhibits invasive migration of MG-63 osteosarcoma cells by inhibiting MMP1 and MMP2 expression through downregulation of Rac1-ROS signaling.

• Biomolecules & Therapeutics
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• v.30 no.1
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• pp.98-116
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• 2022

The small GTPase RhoA has been studied extensively for its role in actin dynamics. In this study, multiple bioinformatics tools were applied cooperatively to the microarray dataset GSE64714 to explore previously unidentified functions of RhoA. Comparative gene expression analysis revealed 545 differentially expressed genes in RhoA-null cells versus controls. Gene set enrichment analysis (GSEA) was conducted with three gene set collections: (1) the hallmark, (2) the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and (3) the Gene Ontology Biological Process. GSEA results showed that RhoA is related strongly to diverse pathways: cell cycle/growth, DNA repair, metabolism, keratinization, response to fungus, and vesicular transport. These functions were verified by heatmap analysis, KEGG pathway diagramming, and direct acyclic graphing. The use of multiple gene set collections restricted the leakage of information extracted. However, gene sets from individual collections are heterogenous in gene element composition, number, and the contextual meaning embraced in names. Indeed, there was a limit to deriving functions with high accuracy and reliability simply from gene set names. The comparison of multiple gene set collections showed that although the gene sets had similar names, the gene elements were extremely heterogeneous. Thus, the type of collection chosen and the analytical context influence the interpretation of GSEA results. Nonetheless, the analyses of multiple collections made it possible to derive robust and consistent function identifications. This study confirmed several well-described roles of RhoA and revealed less explored functions, suggesting future research directions.

• Journal of Integrative Natural Science
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• v.16 no.2
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• pp.47-51
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• 2023

Ras small GTPases are involved in regulating various cellular signaling pathways including cell migration, proliferation, and differentiation. Ras GTPase subfamily is comprised of 15 proteins; 11 Ras, 3 Rap, and one Rheb related protein. Some Ras proteins, such as RasC and RasG, have been identified for their major functions, but there are proteins whose functions have not been studied yet, such as RasU and RasX. Here, we investigated the roles of RasU in cell motility and development. RasU shows the highest homology with RasX. To investigate the functions of RasU, rasU null cells were used to observe the phenotype. Cells lacking RasU were larger and more spread than wild-type cells. These results indicate that RasU plays a negative role in cell spreading. In addition, we investigated the roles of RasU in cell motility and development of Dictyostelium cells and found that rasU null cells exhibited decreased random migration speed and delayed developmental process. These results suggest that RasU plays an important role in cell motility and development.

• BMB Reports
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• v.45 no.3
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• pp.153-158
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• 2012

Geft is a guanine nucleotide exchange factor, which can specifically activate Rho family of small GTPase by catalyzing the exchange of bound GDP for GTP. Geft is highly expressed in the excitable tissue as heart and skeletal muscle and plays important roles in many cellular processes, such as cell proliferation, migration, and cell fate decision. However, the in vivo role of Geft remains unknown. Here, we generated a Geft conditional knockout mouse by flanking exons 5-17 of Geft with loxP sites. Cre-mediated deletion of the Geft gene in heart using Mef2c-Cre transgenic mice resulted in a dramatic decrease of Geft expression. Geft knockout mice develop normally and exhibit no discernable phenotype, suggesting Geft is dispensable for the development of the second heart field in mouse. The Geft conditional knockout mouse will be a valuable genetic tool for uncovering the in vivo roles of Geft during development and in adult homeostasis.

• BMB Reports
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• v.53 no.11
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• pp.576-581
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• 2020

Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5-Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells.

• Biomolecules & Therapeutics
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• v.27 no.6
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• pp.553-561
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• 2019

Rab25, a member of the Rab11 small GTPase family, is central to achieving cellular polarity in epithelial tissues. Rab25 is highly expressed in epithelial cells of various tissues including breast, vagina, cervix, the gastrointestinal tract, and skin. Rab25 plays key roles in tumorigenesis, mainly by regulating epithelial differentiation and proliferation. However, its role in skin physiology is relatively unknown. In this study, we demonstrated that Rab25 knock-out (KO) mice show a skin barrier dysfunction with high trans-epidermal water loss and low cutaneous hydration. To examine this observation, we investigated the histology and epidermal differentiation markers of the skin in Rab25 KO mice. Rab25 KO increased cell proliferation at the basal layer of epidermis, whereas the supra-basal layer remained unaffected. Ceramide, which is a critical lipid component for skin barrier function, was not altered by Rab25 KO in its distribution or amount, as determined by immunohistochemistry. Notably, levels of epidermal differentiation markers, including loricrin, involucrin, and keratins (5, 14, 1, and 10) increased prominently in Rab25 KO mice. In line with this, depletion of Rab25 with single hairpin RNA increased the expression of differentiation markers in a human keratinocyte cell line, HaCaT. Transcriptomic analysis of the skin revealed increased expression of genes associated with skin development, epidermal development, and keratinocyte differentiation in Rab25 KO mice. Collectively, these results suggested that Rab25 is involved in the regulation of epidermal differentiation and proliferation.

• BMB Reports
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• v.57 no.6
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• pp.293-298
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• 2024

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPβ is a major regulator of RHOA expression. Interestingly, the majority of C/EBPβ in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPβ^p27) lacking the N-terminus of C/EBPβ. Overexpression of a gene encoding a C/EBPβ^p27-mimicking protein via an N-terminal deletion in C/EBPβ led to competitive binding with wild-type C/EBPβ at the C/EBPβ binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPβ^p27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPβ^p27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPβ^p27 in the nucleus through CTSL. We propose that CTSL and C/EBPβ^p27 may represent a novel therapeutic target for breast cancer treatment.

• Korean Journal of Ichthyology
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• v.23 no.1
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• pp.1-9
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• 2011

Differentially Expressed Gene (DEG) was obtained from Differential Display Reverse Transcription (DDRT)-PCR using Annealing Control Primer (ACP) to search and clone genes related to developmental stages of Sebastes inermis. By using 120 ACPs, the nucleotide sequences obtained from 16 DEGs showing higher expression in 6-month-old skeletal muscle than 18-month-old ones and from 22 DEGs displaying stronger expression in 18-month-old than 6-month-old were analyzed and BLAST was conducted. The results identified that DEGs shared 69~95% homology with genes of parvalbumin (PVALB), nucleoside diphosphate kinase (NDK) B, tropomyosin (TPM), troponin I (TnI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), muscle-type creatine kinase (CKM2), small EDRK-rich factor 2 (SERF2), adenosine monophosphate deaminase (AMPD), Trimeric intracellular cation channel type A (TRICA), Rho GTPase-activating protein 15 (ARHGAP15), S-formylglutathione hydrolase (Esterase D; ESD), heat shock protein 70 (hsp70), type 1 collagen alpha 2 (COL1A2), glutathione S-transferase, Mid1-interacting protein 1 (Mid1lip1), myosin light chain 1 (MYL1), sarcoplasmic/endoplasmic reticulum calcium ATPase 1B (SERCA1B), and ferritin heavy subunit (FTH1). Expression pattern by developmental stage of DEG14 and PVALB exhibiting strong expression in 6-month-old skeletal muscle was investigated using real time PCR. Expression was reduced as Sebastes inermis grew. Expression of PVALB gene was extremely low after 6 months of age. Expression of CKM2 showed higher expression in 18-month-old skeletal muscle than in 6-month-old muscles, and increased continuously until 4 years old, after which CKM2 expression became gradually reduced. By analysis of tissue-specific expression patterns of DEG, DEG14 was expressed mainly in skeletal muscle, liver, kidney and spleen tissues, whereas PVALB expression was expressed in skeletal muscle and kidney, but not in liver and spleen tissues. CKM2 was expressed in skeletal muscle, kidney, and spleen tissues, but not in liver tissues. PVALB gene was composed of 110 amino acids, which constituted 659 bp nucleotides. The results reported here demonstrate that the expression patterns of parvalbumin and CKM2 could be used as molecular markers for selecting fishes exhibiting fast growth.

• Journal of Life Science
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• v.26 no.12
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• pp.1383-1391
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• 2016

Anti-estrogen drugs such as tamoxifen have been used for treating patients with ER-positive, early breast cancer. However, resistance to anti-estrogen treatment is inevitable in most patients. Breast cancer anti-estrogen resistance-3 (BCAR3) has been identified as the protein responsible for the induction of tamoxifen resistance in estrogen-dependent human breast cancer. We have previously reported that BCAR3 regulates the cell cycle progression and the signaling pathway of EGF and insulin leading to DNA synthesis. In this study, we investigated the functional role of BCAR3 in regulating c-Jun transcription in non-tumorigenic human breast epithelial MCF-12A cells. A transient transfection of BCAR3 increased both the mRNA and protein of c-Jun expression, and stable expression of BCAR3 increased c-Jun protein expression. The overexpression of BCAR3 directly activated the promoter of c-jun, AP-1, and SRE but not that of $NF-{\kappa}B$. Furthermore, single-cell microinjection of BCAR3 expression plasmid in the cell cycle-arrested MCF-12A cells induced c-Jun protein expression, and co-injection of dominant negative mutants of Ras, Rac, and Rho suppressed the transcriptional activity of c-Jun in the presence of BCAR3. Furthermore, stable expression of BCAR3 increased the proliferation of MCF-12A cells. The microinjection of inhibitory materials such as anti-BCAR3 antibody and siRNA BCAR3 inhibited EGF-induced c-Jun expression but did not affect IGF-1 induced upregulation of c-Jun. Taken together, we propose that BCAR3 plays a crucial role in c-Jun protein expression and cell proliferation and that small GTPases (e.g., Ras, Rac, and Rho) are required for the BCAR3-mediated activation of c-Jun expression.