Publications by authors named "Takeharu Sakamoto"

34 Publications

Mint3 depletion-mediated glycolytic and oxidative alterations promote pyroptosis and prevent the spread of Listeria monocytogenes infection in macrophages.

Cell Death Dis 2021 Apr 14;12(4):404. Epub 2021 Apr 14.

Division of Cellular and Molecular Biology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Listeria monocytogenes (LM) infection induces pyroptosis, a form of regulated necrosis, in host macrophages via inflammasome activation. Here, we examined the role of Mint3 in macrophages, which promotes glycolysis via hypoxia-inducible factor-1 activation, during the initiation of pyroptosis following LM infection. Our results showed that Mint3-deficient mice were more resistant to lethal listeriosis than wild-type (WT) mice. Additionally, the mutant mice showed higher levels of IL-1β/IL-18 in the peritoneal fluid during LM infection than WT mice. Moreover, ablation of Mint3 markedly increased the activation of caspase-1, maturation of gasdermin D, and pyroptosis in macrophages infected with LM in vitro, suggesting that Mint3 depletion promotes pyroptosis. Further analyses revealed that Mint3 depletion upregulates inflammasome assembly preceding pyroptosis via glycolysis reduction and reactive oxygen species production. Pharmacological inhibition of glycolysis conferred resistance to listeriosis in a Mint3-dependent manner. Moreover, Mint3-deficient mice treated with the caspase-1 inhibitor VX-765 were as susceptible to LM infection as WT mice. Taken together, these results suggest that Mint3 depletion promotes pyroptosis in host macrophages, thereby preventing the spread of LM infection. Mint3 may serve as a target for treating severe listeriosis by inducing pyroptosis in LM-infected macrophages.
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http://dx.doi.org/10.1038/s41419-021-03691-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046764PMC
April 2021

Mint3 is dispensable for pancreatic and kidney functions in mice.

Biochem Biophys Rep 2020 Dec 8;24:100872. Epub 2020 Dec 8.

Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan.

Munc-18 interacting protein 3 (Mint3) is an activator of hypoxia-inducible factor-1 in cancer cells, macrophages, and cancer-associated fibroblasts under pathological conditions. However, exactly which cells highly express Mint3 in vivo and whether Mint3 depletion affects their physiological functions remain unclear. Here, we surveyed mouse tissues for specific expression of Mint3 by comparing Mint3 expression in wild-type and Mint3-knockout mice. Interestingly, immunohistochemical analyses revealed that Mint3 was highly expressed in islet cells of the pancreas, distal tubular epithelia of the kidney, choroid plexus ependymal cells of the cerebrum, medullary cells of the adrenal gland, and epithelial cells of the seminal gland. We also studied whether Mint3 depletion affects the physiological functions of the islets and kidneys. Mint3-knockout mice did not show any abnormalities in glucose-tolerance and urine-biochemical tests, indicating that Mint3 depletion was compensated for in these organs. Thus, loss of Mint3 might be compensated in the islets and kidneys under physiological conditions in mice.
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http://dx.doi.org/10.1016/j.bbrep.2020.100872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725678PMC
December 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 Nov 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Mint3 depletion restricts tumor malignancy of pancreatic cancer cells by decreasing SKP2 expression via HIF-1.

Oncogene 2020 09 21;39(39):6218-6230. Epub 2020 Aug 21.

Department of System Biology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Takaramachi, Kanazawa, Ishikawa, Japan.

Pancreatic cancer is one of the most fatal cancers without druggable molecular targets. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcriptional factor that promotes malignancy in various cancers including pancreatic cancer. Herein, we found that HIF-1 is accumulated in normoxic or moderate hypoxic areas of pancreatic cancer xenografts in vivo and is active even during normoxia in pancreatic cancer cells in vitro. This prompted us to analyze whether the HIF-1 activator Mint3 contributes to malignant features of pancreatic cancer. Mint3 depletion by shRNAs attenuated HIF-1 activity during normoxia and cell proliferation concomitantly with accumulated p21 and p27 protein in pancreatic cancer cells. Further analyses revealed that Mint3 increased transcription of the oncogenic ubiquitin ligase SKP2 in pancreatic cancer cells via HIF-1. This Mint3-HIF-1-SKP2 axis also promoted partial epithelial-mesenchymal transition, stemness features, and chemoresistance in pancreatic cancer cells. Even in vivo, Mint3 depletion attenuated tumor growth of orthotopically inoculated human pancreatic cancer AsPC-1 cells. Database and tissue microarray analyses showed that Mint3 expression is correlated with SKP2 expression in human pancreatic cancer specimens and high Mint3 expression is correlated with poor prognosis of pancreatic cancer patients. Thus, targeting Mint3 may be useful for attenuating the malignant features of pancreatic cancer.
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http://dx.doi.org/10.1038/s41388-020-01423-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515798PMC
September 2020

EXOSC9 depletion attenuates P-body formation, stress resistance, and tumorigenicity of cancer cells.

Sci Rep 2020 06 9;10(1):9275. Epub 2020 Jun 9.

Department of System Biology, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Takaramachi, Kanazawa, Ishikawa, Japan.

Cancer cells adapt to various stress conditions by optimizing gene expression profiles via transcriptional and translational regulation. However, whether and how EXOSC9, a component of the RNA exosome complex, regulates adaptation to stress conditions and tumorigenicity in cancer cells remain unclear. Here, we examined the effects of EXOSC9 depletion on cancer cell growth under various stress conditions. EXOSC9 depletion attenuated growth and survival under various stress conditions in cancer cells. Interestingly, this also decreased the number of P-bodies, which are messenger ribonucleoprotein particles (mRNPs) required for stress adaptation. Meanwhile, EXOSC2/EXOSC4 depletion also attenuated P-body formation and stress resistance with decreased EXOSC9 protein. EXOSC9-mediated stress resistance and P-body formation were found to depend on the intact RNA-binding motif of this protein. Further, RNA-seq analyses identified 343 EXOSC9-target genes, among which, APOBEC3G contributed to defects in stress resistance and P-body formation in MDA-MB-231 cells. Finally, EXOSC9 also promoted xenografted tumor growth of MDA-MB-231 cells in an intact RNA-binding motif-dependent manner. Database analyses further showed that higher EXOSC9 activity, estimated based on the expression of 343 target genes, was correlated with poorer prognosis in some cancer patients. Thus, drugs targeting activity of the RNA exosome complex or EXOSC9 might be useful for cancer treatment.
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http://dx.doi.org/10.1038/s41598-020-66455-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283315PMC
June 2020

Structures and Molecular Composition of Schmidt-Lanterman Incisures.

Adv Exp Med Biol 2019 ;1190:181-198

Division of Cellular and Molecular Biology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Schmidt-Lanterman incisure (SLI) is a circular-truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system (PNS). The SLI circular-truncated cones elongate like spring at the narrow sites of beaded appearance nerve fibers under the stretched condition. In this chapter, we demonstrate various molecular complexes in SLI, and especially focus on membrane skeleton, protein 4.1G-membrane protein palmitoylated 6 (MPP6)-cell adhesion molecule 4 (CADM4). 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. Motor activity and myelin ultrastructures were abnormal in 4.1G-deficient mice, indicating the 4.1G function as a signal for proper formation of myelin in PNS. Thus, SLI probably has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cell myelin formation.
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http://dx.doi.org/10.1007/978-981-32-9636-7_12DOI Listing
December 2019

Munc18-1-interacting protein 3 mitigates renal fibrosis through protection of tubular epithelial cells from apoptosis.

Nephrol Dial Transplant 2020 04;35(4):576-586

Division of Nephrology and Endocrinology, University of Tokyo, Tokyo, Japan.

Background: Tubulointerstitial fibrosis is a hallmark of chronic kidney disease (CKD), and is initiated by tubular epithelial cell (TEC) injury. Hypoxia promotes tubular cell death, fibrosis and CKD progression. Munc18-1-interacting protein 3 (Mint3) is a molecule that activates hypoxia-inducible factors (HIFs) by binding and suppressing factor inhibiting HIF-1 (FIH). However, the role of Mint3 in tubulointerstitial fibrosis remains unknown.

Methods: We induced fibrosis of the kidney after unilateral ischemia-reperfusion injury (uIRI) in Mint3-knockout and littermate wild-type mice. The duration of ischemia was 23 min and the kidneys were harvested at 24 h and 7 days after ischemia-reperfusion. The function of Mint3 was further investigated by using mouse cortical tubular (MCT) cells, which were treated with Mint3 and/or FIH small interfering RNA and exposed to normoxia or hypoxia.

Results: Knockout of Mint3 did not affect the acute injury induced by uIRI, but exacerbated the tubulointerstitial fibrosis, accompanied by an increase in TEC apoptosis. Consistently, hypoxia-induced apoptosis of MCT cells was aggravated by Mint3 knockdown. Unexpectedly, the additional knockdown of FIH did not suppress the increase in apoptosis by Mint3 knockdown, demonstrating the irrelevance of the FIH/HIF pathway. Therefore, we next focused on nuclear factor (NF)-κB, which has an anti-apoptotic role. Indeed, not only the expression of the inhibitory NF-κB p50 but also the DNA-binding activity of p50/p50 homodimer was increased by knockdown of Mint3 in the TECs, along with the decreased expressions of the NF-κB-targeted anti-apoptotic genes. An increase in NF-κB p50 was also confirmed in Mint3-knockout kidneys.

Conclusions: Mint3 in epithelial cells protects the cells from apoptosis by up-regulating anti-apoptotic effects of NF-κB, leading to fibrosis suppression. This new pathophysiology of tubulointerstitial fibrosis could be a target of future therapy for CKD.
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http://dx.doi.org/10.1093/ndt/gfz177DOI Listing
April 2020

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules.

Histochem Cell Biol 2019 Nov 13;152(5):333-343. Epub 2019 Aug 13.

Health Science Division, Department of Medical Sciences, Shinshu University Graduate School of Medicine, Science and Technology, 3-1-1 Asahi, Matsumoto City, Nagano, 390-8621, Japan.

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.
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http://dx.doi.org/10.1007/s00418-019-01807-2DOI Listing
November 2019

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high-pressure freezing unit.

Microsc Res Tech 2019 Mar 23;82(3):244-249. Epub 2018 Dec 23.

Health Science Division, Department of Medical Sciences, Shinshu University Graduate School of Medicine, Science and Technology, Matsumoto City, Nagano, Japan.

The high-pressure freezing (HPF) technique is known to cryofix water-containing materials with little ice-crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light-exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight-exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.
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http://dx.doi.org/10.1002/jemt.23166DOI Listing
March 2019

The membrane palmitoylated protein, MPP6, is involved in myelin formation in the mouse peripheral nervous system.

Histochem Cell Biol 2019 May 24;151(5):385-394. Epub 2018 Oct 24.

Health Science Division, Department of Medical Sciences, Graduate School of Medicine, Science and Technology, Shinshu University, 3-1-1 Asahi, Matsumoto City, Nagano, 390-8621, Japan.

A membrane skeletal molecular complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6)-Lin7-cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells, especially in Schmidt-Lanterman incisures (SLIs), in the mouse peripheral nervous system (PNS). MPP6, Lin7, and CADM4 are transported to SLIs by 4.1G. In this study, we created MPP6-deficient mice and evaluated myelin structure and MPP6 protein complexes. In SLIs in MPP6-deficient nerves, Lin7 was rarely detected by immunohistochemistry and western blotting, but the localization and amount of CADM4 and 4.1G were not altered. Motor activity was not significantly impaired in a tail-suspension test, but the sciatic nerves of MPP6-deficient mice had thicker myelin in internodes by electron microscopy compared to that of wild-type mice. These results indicate that the MPP6-Lin7 complex regulates myelin formation.
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http://dx.doi.org/10.1007/s00418-018-1745-yDOI Listing
May 2019

Novel adherent CD11b Gr-1 tumor-infiltrating cells initiate an immunosuppressive tumor microenvironment.

Oncotarget 2018 Feb 29;9(13):11209-11226. Epub 2018 Jan 29.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

The immunosuppressive tumor microenvironment is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs) are CD11b Gr-1 tumor-infiltrating immature myeloid cells that strongly mediate tumor immunosuppression. The CD11b Gr-1 cells are a heterogeneous cell population, and the impacts of each subpopulation on tumor progression are not yet completely understood. In the present study, we identified a novel subpopulation of CD11b Gr-1 cells from murine lung carcinoma tumors according to their strongly adherent abilities. Although strong adherent activity is a unique property of macrophages, their marker expression patterns are similar to those of MDSCs; thus, we named this novel subpopulation MDSC-like adherent cells (MLACs). Unlike known MDSCs, MLACs lack the ability to suppress cytotoxic T lymphocytes and differentiate into tumor-associated macrophages (TAMs), but could still directly facilitate tumor growth and angiogenesis through secreting CCL2, CXCL1/2/5, PAI-1, MMPs, and VEGFA. Furthermore, MLACs recruited MDSCs via the secretion of CCL2/5 and CXCL1/2/5, thereby enhancing the immunosuppressive tumor microenvironment and promoting TAMs-mediated tumor progression. Our findings suggest that MLACs may function as an initiator of the immunosuppressive tumor microenvironment and highlight a new therapeutic target to prevent the onset or delay malignant progression.
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http://dx.doi.org/10.18632/oncotarget.24359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834266PMC
February 2018

Deficiency of a membrane skeletal protein, 4.1G, results in myelin abnormalities in the peripheral nervous system.

Histochem Cell Biol 2017 Dec 28;148(6):597-606. Epub 2017 Jul 28.

Division of Health Sciences, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.

We previously demonstrated that a membrane skeletal molecular complex, 4.1G-membrane palmitoylated protein 6 (MPP6)-cell adhesion molecule 4, is incorporated in Schwann cells in the peripheral nervous system (PNS). In this study, we evaluated motor activity and myelin ultrastructures in 4.1G-deficient (-/-) mice. When suspended by the tail, aged 4.1G mice displayed spastic leg extension, especially after overwork. Motor-conduction velocity in 4.1G mice was slower than that in wild-type mice. Using electron microscopy, 4.1G mice exhibited myelin abnormalities: myelin was thicker in internodes, and attachment of myelin tips was distorted in some paranodes. In addition, we found a novel function of 4.1G for sorting a scaffold protein, Lin7, due to disappearance of the immunolocalization and reduction of the production of Lin7c and Lin7a in 4.1G sciatic nerves, as well as the interaction of MPP6 and Lin7 with immunoprecipitation. Thus, we herein propose 4.1G functions as a signal for proper formation of myelin in PNS.
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http://dx.doi.org/10.1007/s00418-017-1600-6DOI Listing
December 2017

Mint3 in bone marrow-derived cells promotes lung metastasis in breast cancer model mice.

Biochem Biophys Res Commun 2017 08 19;490(3):688-692. Epub 2017 Jun 19.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, 108-8639 Tokyo, Japan; Division of Molecular Pathology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, 108-8639 Tokyo, Japan. Electronic address:

Breast cancer is one of the most common cancers in women in the world. Although breast cancer is well treatable at the early stage, patients with distant metastases show a poor prognosis. Data from recent studies using transplantation models indicate that Mint3/APBA3 might promote breast cancer malignancy. However, whether Mint3 indeed contributes to tumor development, progression, or metastasis in vivo remains unclear. To address this, here we examined whether Mint3 depletion affects tumor malignancy in MMTV-PyMT breast cancer model mice. In MMTV-PyMT mice, Mint3 depletion did not affect tumor onset and tumor growth, but attenuated lung metastases. Experimental lung metastasis of breast cancer Met-1 cells derived from MMTV-PyMT mice also decreased in Mint3-depleted mice, indicating that host Mint3 expression affected lung metastasis of MMTV-PyMT-derived breast cancer cells. Further bone marrow transplant experiments revealed that Mint3 in bone marrow-derived cells promoted lung metastasis in MMTV-PyMT mice. Thus, targeting Mint3 in bone marrow-derived cells might be a good strategy for preventing metastasis and improving the prognosis of breast cancer patients.
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http://dx.doi.org/10.1016/j.bbrc.2017.06.102DOI Listing
August 2017

Control of metastatic niche formation by targeting APBA3/Mint3 in inflammatory monocytes.

Proc Natl Acad Sci U S A 2017 05 15;114(22):E4416-E4424. Epub 2017 May 15.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan;

Cancer metastasis is intricately orchestrated by both cancer and normal cells, such as endothelial cells and macrophages. Monocytes/macrophages, which are often co-opted by cancer cells and promote tumor malignancy, acquire more than half of their energy from glycolysis even during normoxic conditions. This glycolytic activity is maintained during normoxia by the functions of hypoxia inducible factor 1 (HIF-1) and its activator APBA3. The mechanism by which APBA3 inhibition partially suppresses macrophage function and affects cancer metastasis is of interest in view of avoidance of the adverse effects of complete suppression of macrophage function during therapy. Here, we report that APBA3-deficient mice show reduced metastasis, with no apparent effect on primary tumor growth. APBA3 deficiency in inflammatory monocytes, which strongly express the chemokine receptor CCR2 and are recruited toward chemokine CCL2 from metastatic sites, hampers glycolysis-dependent chemotaxis of cells toward metastatic sites and inhibits VEGFA expression, similar to the effects observed with HIF-1 deficiency. Host APBA3 induces VEGFA-mediated E-selectin expression in the endothelial cells of target organs, thereby promoting extravasation of cancer cells and micrometastasis formation. Administration of E-selectin-neutralizing antibody also abolished host APBA3-mediated metastatic formation. Thus, targeting APBA3 is useful for controlling metastatic niche formation by inflammatory monocytes.
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http://dx.doi.org/10.1073/pnas.1703171114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465932PMC
May 2017

Integrated functions of membrane-type 1 matrix metalloproteinase in regulating cancer malignancy: Beyond a proteinase.

Cancer Sci 2017 Jun 22;108(6):1095-1100. Epub 2017 May 22.

Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed in different types of invasive and proliferative cells, including cancer cells and stromal cells. MT1-MMP cleaves extracellular matrix proteins, membrane proteins and other pericellular proteins, thereby changing the cellular microenvironment and regulating signal activation. Critical roles of protease activity in cancer cell proliferation, invasion and metastasis have been demonstrated by many groups. MT1-MMP also has a non-protease activity in that it inhibits the oxygen-dependent suppression of hypoxia-inducible factors (HIFs) via Munc18-1-interacting protein 3 (Mint3) and thereby enhances the expression of HIF target genes. Elevated HIF activity in MT1-MMP-expressing cancer cells is a fundamental mechanism underlying the Warburg effect, a well-known phenomenon where malignant cancer cells exhibit a higher rate of glucose metabolism. Because specific intervention of HIF activation by MT1-MMP suppresses tumor formation by cancer cells in mice, both the proteolytic and non-proteolytic activities of MT1-MMP are important for tumor malignancy and function in an integrated manner. In this review, we summarize recent findings relating to how MT1-MMP activates HIF and its effects on cancer cells and stromal cells.
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http://dx.doi.org/10.1111/cas.13231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480062PMC
June 2017

Mint3/Apba3 depletion ameliorates severe murine influenza pneumonia and macrophage cytokine production in response to the influenza virus.

Sci Rep 2016 11 24;6:37815. Epub 2016 Nov 24.

Division of Molecular Pathology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Influenza virus (IFV) infection is a common cause of severe pneumonia. Studies have suggested that excessive activation of the host immune system including macrophages is responsible for the severe pathologies mediated by IFV infection. Here, we focused on the X11 protein family member Mint3/Apba3, known to promote ATP production via glycolysis by activating hypoxia inducible factor-1 (HIF-1) in macrophages, and examined its roles in lung pathogenesis and anti-viral defence upon IFV infection. Mint3-deficient mice exhibited improved influenza pneumonia with reduced inflammatory cytokines/chemokine levels and neutrophil infiltration in the IFV-infected lungs without alteration in viral burden, type-I interferon production, or acquired immunity. In macrophages, Mint3 depletion attenuated NF-κB signalling and the resultant cytokine/chemokine production in response to IFV infection by increasing IκBα and activating the cellular energy sensor AMPK, respectively. Thus, Mint3 might represent one of the likely therapeutic targets for the treatment of severe influenza pneumonia without affecting host anti-viral defence through suppressing macrophage cytokine/chemokine production.
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http://dx.doi.org/10.1038/srep37815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121658PMC
November 2016

Mint3 potentiates TLR3/4- and RIG-I-induced IFN-β expression and antiviral immune responses.

Proc Natl Acad Sci U S A 2016 10 3;113(42):11925-11930. Epub 2016 Oct 3.

Department of Immunology and Key Laboratory of Infection and Immunity of Shandong Province, Shandong University School of Medicine, Jinan, Shandong 250012, China;

Type I IFNs (IFN-α/β) play crucial roles in the elimination of invading viruses. Multiple immune cells including macrophages recognize viral infection through a variety of pattern recognition receptors, such as Toll-like receptors (TLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors, and initiate type I IFN secretion and subsequent antiviral immune responses. However, the mechanisms by which host immune cells can produce adequate amounts of type I IFNs and then eliminate viruses effectively remain to be further elucidated. In the present study, we show that munc18-1-interacting protein 3 (Mint3) expression can be markedly induced during viral infection in macrophages. Mint3 enhances TLR3/4- and RIG-I-induced IRF3 activation and IFN-β production by promoting K63-linked polyubiquitination of TNF receptor-associated factor 3 (TRAF3). Consistently, Mint3 deficiency greatly attenuated antiviral immune responses and increased viral replication. Therefore, we have identified Mint3 as a physiological positive regulator of TLR3/4 and RIG-I-induced IFN-β production and have outlined a feedback mechanism for the control of antiviral immune responses.
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http://dx.doi.org/10.1073/pnas.1601556113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081575PMC
October 2016

The ERK signaling target RNF126 regulates anoikis resistance in cancer cells by changing the mitochondrial metabolic flux.

Cell Discov 2016 26;2:16019. Epub 2016 Jul 26.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Division of Molecular Pathology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Loss of anchorage to the extracellular matrix leads to apoptosis (anoikis) in normal cells, but cancerous cells are usually resistant to such stress. Here we report the pivotal role of an E3 ubiquitin ligase, ring-finger protein 126 (RNF126), in the resistance of cancer cells to the stress associated with non-adherent conditions. Non-adherent cancer cells exhibited increased flux through the tricarboxylic acid cycle via increased conversion of pyruvate to acetyl-CoA. RNF126 was found to act as a ubiquitin ligase for pyruvate dehydrogenase kinases (PDKs), resulting in their proteasomal degradation. This decrease in PDK levels allowed pyruvate dehydrogenases to catalyze the conversion of pyruvate to acetyl-CoA. Moreover, depletion of RNF126 or increased expression of PDK1 in cancer cells suppressed colony formation in soft agar as well as tumorigenicity in mice. RNF126 expression in cancer cells was found to be under the control of the extracellular signal-regulated kinase signaling pathway, which is essential for anoikis resistance. Thus, RNF126 is an attractive molecule for treating cancer by selectively targeting anchorage-independent growth.
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http://dx.doi.org/10.1038/celldisc.2016.19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960523PMC
July 2016

NECAB3 Promotes Activation of Hypoxia-inducible factor-1 during Normoxia and Enhances Tumourigenicity of Cancer Cells.

Sci Rep 2016 Mar 7;6:22784. Epub 2016 Mar 7.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Unlike most cells, cancer cells activate hypoxia inducible factor-1 (HIF-1) to use glycolysis even at normal oxygen levels, or normoxia. Therefore, HIF-1 is an attractive target in cancer therapy. However, the regulation of HIF-1 during normoxia is not well characterised, although Mint3 was recently found to activate HIF-1 in cancer cells and macrophages by suppressing the HIF-1 inhibitor, factor inhibiting HIF-1 (FIH-1). In this study, we analysed Mint3-binding proteins to investigate the mechanism by which Mint3 regulates HIF-1. Yeast two-hybrid screening using Mint3 as bait identified N-terminal EF-hand calcium binding protein 3 (NECAB3) as a novel factor regulating HIF-1 activity via Mint3. NECAB3 bound to the phosphotyrosine-binding domain of Mint3, formed a ternary complex with Mint3 and FIH-1, and co-localised with Mint3 at the Golgi apparatus. Depletion of NECAB3 decreased the expression of HIF-1 target genes and reduced glycolysis in normoxic cancer cells. NECAB3 mutants that binds Mint3 but lacks an intact monooxygenase domain also inhibited HIF-1 activation. Inhibition of NECAB3 in cancer cells by either expressing shRNAs or generating a dominant negative mutant reduced tumourigenicity. Taken together, the data indicate that NECAB3 is a promising new target for cancer therapy.
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http://dx.doi.org/10.1038/srep22784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780031PMC
March 2016

Investigation of a MMP-2 activity-dependent anchoring probe for nuclear imaging of cancer.

PLoS One 2014 10;9(7):e102180. Epub 2014 Jul 10.

Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.

Purpose: Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments.

Methods: We designed and synthesized MDAP(1000), MDAP(3000), and MDAP(5000), which consist of 4 independent moieties: RI unit (111)In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates.

Results: MDAP(1000), MDAP(3000), and MDAP(5000) were cleaved by MMP-2 in a concentration-dependent manner. MDAP(3000) pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP(3000) exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP(3000) exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP(3000) existed in tumor xenografts in vivo.

Conclusions: The results indicate the possible usefulness of our MDAP strategy for tumor imaging.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102180PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092090PMC
November 2015

Hypoxia-inducible factor 1 regulation through cross talk between mTOR and MT1-MMP.

Mol Cell Biol 2014 Jan 28;34(1):30-42. Epub 2013 Oct 28.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.
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http://dx.doi.org/10.1128/MCB.01169-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911284PMC
January 2014

MT1-MMP plays a critical role in hematopoiesis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells.

Blood 2012 Jun 27;119(23):5405-16. Epub 2012 Apr 27.

Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP(-/-) mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.
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http://dx.doi.org/10.1182/blood-2011-11-390849DOI Listing
June 2012

Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library.

PLoS One 2012 16;7(4):e35590. Epub 2012 Apr 16.

Division of Cancer Cell Research, Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo, Japan.

Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035590PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327663PMC
August 2012

Genetic dissection of proteolytic and non-proteolytic contributions of MT1-MMP to macrophage invasion.

Biochem Biophys Res Commun 2011 Sep 27;413(2):277-81. Epub 2011 Aug 27.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

MT1-MMP/MMP-14 is a major invasion-promoting membrane protease expressed in macrophages. In addition to its proteolytic activity that degrades the extracellular matrix, MT1-MMP also boosts ATP production in cells in a manner independent of its proteolytic activity. It remains unclear to what extent the proteolytic and energy-boosting activities of MT1-MMP contribute to macrophage invasion. Recently, we demonstrated that the cytoplasmic tail of MT1-MMP makes use of APBA3/Mint3 to activate HIF-1 and thereby boosts glycolysis for ATP production. Here, we used Apba3(-/-) macrophages to dissect the contribution of the proteolytic and the energy-boosting activities of MT1-MMP. The proteolytic activity of MT1-MMP was not affected by the lack of APBA3 in macrophages. Apba3(-/-) and Mmp14(-/-) macrophages exhibited a 55% reduction of ATP levels compared to wild-type (WT) cells and the rate of motility of the mutant cells was accordingly reduced. In contrast, matrigel invasion by Mmp14(-/-) and Apba3(-/-) macrophages was reduced to 24% and 55.4%, respectively, of the level observed in WT cells. These results represent the first attempt to dissect the contribution of the two invasion-promoting activities of MT1-MMP to macrophage invasion.
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http://dx.doi.org/10.1016/j.bbrc.2011.08.085DOI Listing
September 2011

Deletion of the Mint3/Apba3 gene in mice abrogates macrophage functions and increases resistance to lipopolysaccharide-induced septic shock.

J Biol Chem 2011 Sep 21;286(37):32542-51. Epub 2011 Jul 21.

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Two major metabolic systems are usually used to generate ATP: oxidative phosphorylation (OXPHOS) in the mitochondria and glycolysis. Most types of cells employ OXPHOS for ATP production during normoxia but then shift energy production from OXPHOS to glycolysis when exposed to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is the master transcription factor regulating this metabolic shift. On the other hand, macrophages are unique in making use of glycolysis for ATP generation constitutively even during normoxia. We recently proposed that in macrophages, Mint3/APBA3 inhibits factor inhibiting HIF-1 (FIH-1) during normoxia, which in turn releases the suppression of HIF-1 activity by FIH-1. To demonstrate the physiological function of APBA3 in macrophages, we established Apba3(-/-) mice. The mutant mice presented no apparent gross phenotype but exhibited significant resistance against LPS-induced septic shock. The level of ATP in macrophages obtained from the mutant mice was reduced to 60% of the level observed in wild type cells, which in turn led to reduced ATP-dependent activities such as glycolysis, cytokine production, and motility. We also generated mutant mice with the Apba3 gene deleted specifically from cells of the myeloid lineage and confirmed that LPS-induced septic shock is mitigated significantly. Thus, we show cell type-specific regulation of energy production by APBA3 in macrophages using genetically manipulated mice. The specific function of APBA3 in macrophages might allow us to develop therapeutics to regulate aberrant macrophage function during infection and diseases.
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http://dx.doi.org/10.1074/jbc.M111.271726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173149PMC
September 2011

Targeting the Warburg effect that arises in tumor cells expressing membrane type-1 matrix metalloproteinase.

J Biol Chem 2011 Apr 3;286(16):14691-704. Epub 2011 Mar 3.

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for cellular adaptation to hypoxia, although its physiological roles and activation mechanisms during normoxia have not been studied sufficiently. The Warburg effect, which is a hallmark of malignant tumors that is characterized by increased activity of aerobic glycolysis, accompanies activation of HIF-1 during normoxia. Besides tumor cells that have multiple genetic and epigenetic alterations, normal macrophages also use glycolysis for ATP production by depending upon elevated HIF-1 activity even during normoxia. We recently found that activity of factor inhibiting HIF-1 (FIH-1) is specifically suppressed in macrophages by a nonproteolytic activity of membrane type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Thus, MT1-MMP expressed in macrophages plays a significant role in regulating HIF-1 activity during normoxia. In the light of this finding, we examined here whether MT1-MMP contributes to the Warburg effect of tumor cells. All the tumor cell lines that express MT1-MMP exhibit increased glycolytic activity, and forced expression of MT1-MMP in MT1-MMP-negative tumor cells is sufficient to induce the Warburg effect. The cytoplasmic tail of MT1-MMP mediates the stimulation of aerobic glycolysis by increasing the expression of HIF-1 target genes. Specific intervention of the MT1-MMP-mediated activation of HIF-1 in tumor cells retarded tumor growth in mice. Systemic administration of a membrane-penetrating form of the cytoplasmic tail peptide in mice to inhibit HIF-1 activation competitively also exhibited a therapeutic effect on tumors.
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http://dx.doi.org/10.1074/jbc.M110.188714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077666PMC
April 2011

ZF21 is a new regulator of focal adhesion disassembly and a potential member of the spreading initiation center.

Cell Adh Migr 2011 Jan-Feb;5(1):23-8. Epub 2011 Jan 1.

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.

Adherent cells migrate on extracellular matrices (ECM) by repeated spreading and contraction of the cell body. Focal adhesions (FAs) play a major role in the adherence of cells to the ECM and in the generation of the cellular forces that maintain morphology and allow cells to move. FAs also mediate bidirectional transmembrane signals in conjunction with growth factor receptors and signaling molecules. Although the mechanisms that regulate cell migration are not yet fully understood, the regulation of the formation and turnover of FAs is a key factor determining the rate and direction of cell migration. We recently identified a component of FAs termed ZF21, which is a member of a family of proteins characterized by the presence of a conserved phosphoinositide-binding motif. ZF21 promotes dephosphorylation of FAK at Tyr ( 397) upon microtubule extension to FAs and thereby regulates the disassembly of FAs in a microtubules-dependent manner. To obtain further insight into the regulation of cell adhesion by ZF21, we analyzed proteins associating with ZF21 by proteomic analysis. We identified 45 proteins including FA-related proteins and multiple RNA binding proteins that have been shown recently to be components of the spreading initiation center (SIC). SICs are cell adherent structures that can be observed only in the early stages of cell spreading and have been implicated in regulating the rate of cell spreading. In this article, we report new ZF21-binding proteins identified by proteomic analysis and discuss the potential functions of ZF21 in regulating disassembly of FAs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038092PMC
http://dx.doi.org/10.4161/cam.5.1.13492DOI Listing
April 2011

A membrane protease regulates energy production in macrophages by activating hypoxia-inducible factor-1 via a non-proteolytic mechanism.

J Biol Chem 2010 Sep 27;285(39):29951-64. Epub 2010 Jul 27.

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Most cells produce ATP in the mitochondria by oxidative phosphorylation. However, macrophages, which are major players in the innate immune system, use aerobic glycolysis to produce ATP. HIF-1 (hypoxia-inducible factor-1) regulates expression of glycolysis-related genes and maintains macrophage glycolytic activity. However, it is unclear how HIF-1 activity is maintained in macrophages during normoxia. In this study, we found that macrophages lacking membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14), a potent invasion-promoting protease, exhibited considerably lower ATP levels than wild-type cells. HIF-1 was activated by an unanticipated function of MT1-MMP, which led to the stimulation of ATP production via glycolysis. The cytoplasmic tail of MT1-MMP bound to FIH-1 (factor inhibiting HIF-1), which led to the inhibition of the latter by its recently identified inhibitor, Mint3/APBA3. We have thus identified a new function of MT1-MMP to mediate production of ATP so as to support energy-dependent macrophage functions by a previously unknown non-proteolytic mechanism.
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http://dx.doi.org/10.1074/jbc.M110.132704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943292PMC
September 2010

ZF21 protein regulates cell adhesion and motility.

J Biol Chem 2010 Jul 3;285(27):21013-22. Epub 2010 May 3.

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Cell migration on an extracellular matrix (ECM) requires continuous formation and turnover of focal adhesions (FAs) along the direction of cell movement. However, our knowledge of the components of FAs and the mechanism of their regulation remains limited. Here, we identify ZF21, a member of a protein family characterized by the presence of a phosphatidylinositol 3-phosphate-binding FYVE domain, to be a new regulator of FAs and cell movement. Knockdown of ZF21 expression in cells increased the number of FAs and suppressed cell migration. Knockdown of ZF21 expression also led to a significant delay in FA disassembly following induction of synchronous disassembly of FAs by nocodazole treatment. ZF21 bound to focal adhesion kinase, localized to FAs, and was necessary for dephosphorylation of FAK at Tyr(397), which is important for disassembly of FAs. Thus, ZF21 represents a new component of FAs, mediates disassembly of FAs, and thereby regulates cell motility.
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http://dx.doi.org/10.1074/jbc.M110.106443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898296PMC
July 2010

Mint3 enhances the activity of hypoxia-inducible factor-1 (HIF-1) in macrophages by suppressing the activity of factor inhibiting HIF-1.

J Biol Chem 2009 Oct 2;284(44):30350-9. Epub 2009 Sep 2.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Tokyo 108-8639, Japan.

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor regulating cellular responses to hypoxia and is composed of alpha and beta subunits. During normoxia, factor inhibiting HIF-1 (FIH-1) inhibits the activity of HIF-1 by preventing HIF-1alpha binding to p300/CBP via modification of the Asn(803) residue. However, it is not known whether FIH-1 activity can be regulated in an oxygen-independent manner. In this study, we survey possible binding proteins to FIH-1 and identify Mint3/APBA3, which has been reported to bind Alzheimer beta-amyloid precursor protein. Purified Mint3 binds FIH-1 and inhibits the ability of FIH-1 to modify HIF-1alpha in vitro. In a reporter assay, the activity of HIF-1alpha is suppressed because of endogenous FIH-1 in HEK293 cells, and expression of Mint3 antagonizes this suppression. Macrophages are known to depend on glycolysis for ATP production because of elevated HIF-1 activity. FIH-1 activity is suppressed in macrophages by Mint3 so as to maintain HIF-1 activity. FIH-1 forms a complex with Mint3, and these two factors co-localize within the perinuclear region. Knockdown of Mint3 expression in macrophages leads to redistribution of FIH-1 to the cytoplasm and decreases glycolysis and ATP production. Thus, Mint3 regulates the FIH-1-HIF-1 pathway, which controls ATP production in macrophages and therefore represents a potential new therapeutic target to regulate macrophage-mediated inflammation.
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http://dx.doi.org/10.1074/jbc.M109.019216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781590PMC
October 2009