Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells 1215 1227 EN Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Tetta Takahashi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Toshiki Ochi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Kazumi Sagayama Organization for Research and Innovation Strategy, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector�fs shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2076-3921 14 7 2025 Carnosol, a Rosemary Ingredient Discovered in a Screen for Inhibitors of SARM1-NAD+ Cleavage Activity, Ameliorates Symptoms of Peripheral Neuropathy 808 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuki Ogawa Tama Biochemical Co., Ltd. Yu Yasui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoji Wada Tama Biochemical Co., Ltd. Hiromichi Nakamura Tama Biochemical Co., Ltd. Masahiro Nishibori Department of Translational Research and Drug Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+) hydrolase involved in axonal degeneration and neuronal cell death. SARM1 plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents the degeneration; as a result, SARM1 has been attracting attention as a potent therapeutic target. In recent years, the development of several SARM1 inhibitors derived from synthetic chemical compounds has been reported; however, no dietary ingredients with SARM1 inhibitory activity have been identified. Therefore, we here focused on dietary ingredients and found that carnosol, an antioxidant contained in rosemary, inhibits the NAD+-cleavage activity of SARM1. Purified carnosol inhibited the enzymatic activity of SARM1 and suppressed neurite degeneration and cell death induced by the anti-cancer medicine vincristine (VCR). Carnosol also inhibited VCR-induced hyperalgesia symptoms, suppressed the loss of intra-epidermal nerve fibers in vivo, and reduced the blood fluid level of phosphorylated neurofilament-H caused by an axonal degeneration event. These results indicate that carnosol has a neuroprotective effect via SARM1 inhibition in addition to its previously known antioxidant effect via NF-E2-related factor 2 and thus suppresses neurotoxin-induced peripheral neuropathy. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signaling pathway 1138 1149 EN Jin Zhou Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naoko Mizuta Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuko Yamakawa Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of ƒÁ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11�fs nature in colorectal cancers and others. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-ƒÀ1-NF-ƒÈB-MMP9 axis 1371307 EN Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Kazumi Ninomiya Faculty of Science and Technology, Division of Molecular Science, Gunma University Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-beta 1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-beta 1 hastens the invasive outgrowth of TNBC cells at the molecular level.<br> Methods LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively.<br> Results Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-kappa B (NF-kappa B). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-beta activated kinase 1 (TAK1) were required for the activation of NF-kappa B through I kappa beta kinase kinase (IKK alpha/beta) phosphorylation.<br> Conclusion Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-beta 1-TRAF4-TAK1-IKK alpha/beta-I kappa beta alpha-NF-kappa B-MMP9, is crucial for TNBC cell invasiveness. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0946-2716 101 4 2023 Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells 431 447 EN Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Léna Audebert Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Youyi Chen Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Chikako Yoshizawa Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masami Watanabe Department of Urology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3�\namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0021-924X 174 6 2023 Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration 533 548 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Tha Zin Phoo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ikuko Miyazaki Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Translational Research and Drug Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masato Asanuma Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson�fs disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface 1371342 EN Tetta Takahashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshito Zamami Department of Pharmacy, Okayama University Hospital Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-ƒÀ1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain.<br> <br> Methods: Cell invasion was assessed using a transwell-based assay, protein–protein interactions by an immunoprecipitation–Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography.<br> <br> Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion.<br> <br> Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0898-6568 108 2023 STAT1/3 signaling suppresses axon degeneration and neuronal cell death through regulation of NAD+-biosynthetic and consuming enzymes 110717 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yu Yasui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuma Oiso Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-ƒÁ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-ƒÁ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-ƒÁ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 13 2023 Lysyl oxidase-like 4 exerts an atypical role in breast cancer progression that is dependent on the enzymatic activity that targets the cell-surface annexin A2 1142907 EN Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, TheFourth Affiliated Hospital of Harbin Medical University Yoshihiko Sakaguchi Department of Microbiology, Kitasato University School of Medicine Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ich Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of Dalian University of Technology Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: LOX family members are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer.<br> Methods: We established the MDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells�f activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach.<br> Results: Our in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin ƒÀ-1, resulting in the locking of integrin ƒÀ-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth.<br> Conclusions: LOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin ƒÀ-1 on the cell surface.<br> No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 13 2023 LOXL1 and LOXL4 are novel target genes of the Zn2+-bound form of ZEB1 and play a crucial role in the acceleration of invasive events in triple-negative breast cancer cells 1142886 EN Daisuke Hirabayashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Maruyama Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. <br> Methods: We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (Delta Zn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. <br> Results: MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (Delta Zn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. <br> Conclusions: These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 18 2022 Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells 10300 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hidenori Wake Department of Pharmacology, Kindai University Faculty of Medicine Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University I. Made Winarsa Ruma Faculty of Medicine, Udayana University Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Faculty of Medicine, Udayana University Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 24055808 22 2020 NeuroplastinƒÀ-mediated upregulation of solute carrier family 22 member 18 antisense (SLC22A18AS) plays a crucial role in the epithelial-mesenchymal transition, leading to lung cancer cells' enhanced motility 100768 EN Karolina Bajkowska Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School I. Made Winarsa Ruma University of Surrey Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Our recent study revealed an important role of the neuroplastin (NPTN)ƒÀ downstream signal in lung cancer dissemination in the lung. The molecular mechanism of the signal pathway downstream of NPTNƒÀ is a serial activation of the key molecules we identified: tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) adaptor, nuclear factor (NF)IA/NFIB heterodimer transcription factor, and SAM pointed-domain containing ETS transcription factor (SPDEF). The question of how dissemination is controlled by SPDEF under the activated NPTNƒÀ has not been answered. Here, we show that the NPTNƒÀ-SPDEF-mediated induction of solute carrier family 22 member 18 antisense (SLC22A18AS) is definitely required for the epithelial-mesenchymal transition (EMT) through the NPTNƒÀ pathway in lung cancer cells. In vitro, the induced EMT is linked to the acquisition of active cellular motility but not growth, and this is correlated with highly disseminative tumor progression in vivo. The publicly available data also show the poor survival of SLC22A18AS-overexpressing lung cancer patients. Taken together, these data highlight a crucial role of SLC22A18AS in lung cancer dissemination, which provides novel input of this molecule to the signal cascade of NPTNƒÀ. Our findings contribute to a better understanding of NPTNƒÀ-mediated lung cancer metastasis. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 24055808 18 2019 Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue 100619 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1522-8002 21 7 2019 Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness 627 640 EN Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Yosuke Mitsui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences I Made Winarsa Ruma Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroki Sato Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuta Tomida Department of Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichi Soh Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miyoko Kubo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Endy Widya Putranto Department of Pediatrics, Dr. Sardjito Hospital/Faculty of Medicine, Universitas Gadjah Mada Takashi Murakami Department of Microbiology, Faculty of Medicine, Saitama Medical University Ming Liu Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Toshihiko Hibino Department of Dermatology, Tokyo Medical University Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes. No potential conflict of interest relevant to this article was reported.

American Society for Biochemistry and Molecular Acta Medica Okayama 0021-9258 289 34 2014 DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes. 23389 23402 EN Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yumi Aoyama Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshihiko Hibino Shiseido Research Center, Advanced Science Research Endy Widya Putranto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Yoshihiko Sakaguchi Interdisciplinary Research Organization, University of Miyazaki Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Biotechnology, Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University Junichiro Futami Department of Biotechnology, Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University Ken Kataoka Department of Life Science, Faculty of Science, Okayama University of Science Keiji Iwatsuki Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nam-ho Huh Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes. No potential conflict of interest relevant to this article was reported.

‰ªŽRˆãŠw‰ï Acta Medica Okayama 0030-1558 126 2 2014 •½�¬25”N“x‰ªŽRˆãŠw‰ï�Ü ‘��‡Œ¤‹†�§—ã�Ü�iŒ‹�é�Ü�j 83 86 EN Hitoshi Murata No potential conflict of interest relevant to this article was reported.

Spandidos Publications Ltd. Acta Medica Okayama 1107-3756 32 4 2013 Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization 938 944 EN Endy Widya Putranto Hitoshi Murata Ken-Ichi Yamamoto Ken Kataoka Hidenori Yamada Jun-Ichiro Futami Masakiyo Sakaguchi Nam-Ho Huh The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 0020-7136 126 7 2010 Down-regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene-therapeutic overexpression of REIC/Dkk-3 1562 1569 EN Ryuta Tanimoto Masakiyo Sakaguchi Fernando Abarzua Ken Kataoka Kaoru Kurose Hitoshi Murata Yasutomo Nasu Hiromi Kumon Nam-Ho Huh We have recently shown that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) exhibits a potent tumor-specific cell-killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad-REIC-induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad-REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad-REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk-3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad-REIC-induced apoptosis, we found that BiP/GRP78, an ER-residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad-REIC were inversely correlated. Down-regulation of BiP with siRNA sensitized the resistant cells to Ad-REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad-REIC-induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad-REIC. No potential conflict of interest relevant to this article was reported.

The American Society for Biochemistry and Molecular Biology, Inc. Acta Medica Okayama 0021-9258 284 21 2009 Overexpression of REIC/Dkk-3 in Normal Fibroblasts Suppresses Tumor Growth via Induction of Interleukin-7 14236 14244 EN Masakiyo Sakaguchi Ken Kataoka Fernando Abarzua Ryuta Tanimoto Masami Watanabe Hitoshi Murata Swe Swe Than Kaoru Kurose Yuji Kashiwakura Kazuhiko Ochiai Yasutomo Nasu Hiromi Kumon Nam-ho Huh We previously showed that the tumor suppressor gene REIC/Dkk-3, when overexpressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on human cancers through a mechanism triggered by endoplasmic reticulum stress. Adenovirus vectors show no target cell specificity and thus may elicit unfavorable side effects through infection of normal cells even upon intra-tumoral injection. In this study, we examined possible effects of Ad-REIC on normal cells. We found that infection of normal human fibroblasts (NHF) did not cause apoptosis but induced production of interleukin (IL)-7. The induction was triggered by endoplasmic reticulum stress and mediated through IRE1 alpha, ASK1, p38, and IRF-1. When Ad-REIC-infected NHF were transplanted in a mixture with untreated human prostate cancer cells, the growth of the cancer cells was significantly suppressed. Injection of an IL-7 antibody partially abrogated the suppressive effect of Ad-REIC-infected NHF. These results indicate that Ad-REIC has another arm against human cancer, an indirect host-mediated effect because of overproduction of IL-7 by mis-targeted NHF, in addition to its direct effect on cancer cells. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2008 Development of a protein transduction technology using polyethylenimine-cationization, and application to regulation of cellular function EN Hitoshi Murata No potential conflict of interest relevant to this article was reported.