Publications by authors named "Hiroaki Miki"

85 Publications

Importance of the renal ion channel TRPM6 in the circadian secretion of renin to raise blood pressure.

Nat Commun 2021 06 17;12(1):3683. Epub 2021 Jun 17.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

Blood pressure has a daily pattern, with higher values in the active period. Its elevation at the onset of the active period substantially increases the risk of fatal cardiovascular events. Renin secretion stimulated by renal sympathetic neurons is considered essential to this process; however, its regulatory mechanism remains largely unknown. Here, we show the importance of transient receptor potential melastatin-related 6 (TRPM6), a Mg-permeable cation channel, in augmenting renin secretion in the active period. TRPM6 expression is significantly reduced in the distal convoluted tubule of hypotensive Cnnm2-deficient mice. We generate kidney-specific Trpm6-deficient mice and observe a decrease in blood pressure and a disappearance of its circadian variation. Consistently, renin secretion is not augmented in the active period. Furthermore, renin secretion after pharmacological activation of β-adrenoreceptor, the target of neuronal stimulation, is abrogated, and the receptor expression is decreased in renin-secreting cells. These results indicate crucial roles of TRPM6 in the circadian regulation of blood pressure.
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http://dx.doi.org/10.1038/s41467-021-24063-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211686PMC
June 2021

Identification and mechanistic analysis of an inhibitor of the CorC Mg transporter.

iScience 2021 Apr 26;24(4):102370. Epub 2021 Mar 26.

State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Shanghai Key Laboratory of Bioactive Small Molecules, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai 200438, China.

The CorC/CNNM family of Na-dependent Mg transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. CorC and CNNM have attracted interest as therapeutic targets, whereas inhibitors targeting the ATP-binding site have not been identified. Here, we performed a virtual screening of CorC by targeting its ATP-binding site, identified a compound named IGN95a with inhibitory effects on ATP binding and Mg export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more toward the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift.
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http://dx.doi.org/10.1016/j.isci.2021.102370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066426PMC
April 2021

Correction: Phosphatase, pseudo-phosphatase, or both? Understanding PRL oncogenicity.

Br J Cancer 2021 May;124(10):1744

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

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http://dx.doi.org/10.1038/s41416-021-01311-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110988PMC
May 2021

Role of adenomatous polyposis coli in proliferation and differentiation of colon epithelial cells in organoid culture.

Sci Rep 2021 Feb 17;11(1):3980. Epub 2021 Feb 17.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.

Adenomatous polyposis coli (APC) is a tumor-suppressing protein whose inactivation triggers the formation of colorectal polyps. Numerous studies using cell lines or genetically engineered mice have revealed its role in suppressing Wnt/β-catenin signaling pathway and regulating cell proliferation and differentiation. Here, we performed genetic analyses of APC using a three-dimensional organoid culture of mouse colon epithelia, which enables the detailed examination of epithelial properties. Analyses of Apc-knockout colon organoids not only confirmed the importance of APC in suppressing Wnt/β-catenin signaling and regulating cell differentiation, but also revealed several novel features: a significant decrease in proliferating speed and an increase in cross-sectional area of cells. Moreover, we found a significant number of lysozyme-positive Paneth-like cells, which were never observed in wild-type colon tissues or organoids, but have been reported to emerge in colon cancers. Therefore, APC autonomously suppresses ectopic differentiation into lysozyme-positive cells, specifically in the colon epithelia. Colon organoids would be an ideal material to investigate the molecular mechanism and biological importance of the ectopic differentiation associated with cancer development.
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http://dx.doi.org/10.1038/s41598-021-83590-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889860PMC
February 2021

Structural basis for the Mg recognition and regulation of the CorC Mg transporter.

Sci Adv 2021 Feb 10;7(7). Epub 2021 Feb 10.

State Key Laboratory of Genetic Engineering, Shanghai Key Laboratory of Bioactive Small Molecules, Collaborative Innovation Center of Genetics and Development, and Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai 200438, China.

The CNNM/CorC family proteins are Mg transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms. In humans, CNNM proteins are involved in various biological events, such as body absorption/reabsorption of Mg and genetic disorders. Here, we determined the crystal structure of the Mg-bound CorC TM domain dimer. Each protomer has a single Mg binding site with a fully dehydrated Mg ion. The residues at the Mg binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases. Furthermore, we determined the structures of the CorC cytoplasmic region containing its regulatory ATP-binding domain. A combination of structural and functional analyses not only revealed the potential interface between the TM and cytoplasmic domains but also showed that ATP binding is important for the Mg export activity of CorC.
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http://dx.doi.org/10.1126/sciadv.abe6140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875539PMC
February 2021

Phosphatase, pseudo-phosphatase, or both? Understanding PRL oncogenicity.

Br J Cancer 2021 Mar 3;124(6):1035-1036. Epub 2020 Dec 3.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Phosphatases of regenerating liver (PRL1-3) are among the most oncogenic protein phosphatases but their mechanism of action is poorly understood. Multiple substrates have been proposed as well as a non-catalytic function regulating magnesium transport. Our recent identification of a catalytically inactive PRL mutant that retains oncogenicity in a mouse model promises to resolve the question of whether PRLs act as phosphatases or pseudo-phosphatases in different cancer models.
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http://dx.doi.org/10.1038/s41416-020-01194-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960985PMC
March 2021

Magnesium efflux from Drosophila Kenyon cells is critical for normal and diet-enhanced long-term memory.

Elife 2020 11 26;9. Epub 2020 Nov 26.

Centre for Neural Circuits and Behaviour, The University of Oxford, Tinsley Building, Oxford, United Kingdom.

Dietary magnesium (Mg) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. Here we show that Mg feeding also enhances long-term memory in . Normal and Mg-enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg-efflux transporter encoded by the () gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for from a function in memory. Moreover, UEX localization in mushroom body Kenyon cells (KCs) is altered in memory-defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of KC Mg. We propose that regulated neuronal Mg efflux is critical for normal and Mg-enhanced memory.
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http://dx.doi.org/10.7554/eLife.61339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843133PMC
November 2020

The Oncogenic PRL Protein Causes Acid Addiction of Cells by Stimulating Lysosomal Exocytosis.

Dev Cell 2020 11 11;55(4):387-397.e8. Epub 2020 Sep 11.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

Extracellular pH is usually maintained around 7.4 in multicellular organisms, and cells are optimized to proliferate under this condition. Here, we find cells can adapt to a more acidic pH of 6.5 and become addicted to this acidic microenvironment by expressing phosphatase of regenerating liver (PRL), a driver of cancer malignancy. Genome-scale CRISPR-Cas9 knockout screening and subsequent analyses revealed that PRL promotes H extrusion and acid addiction by stimulating lysosomal exocytosis. Further experiments using cultured cells and Caenorhabditis elegans clarified the molecular link between PRL and lysosomal exocytosis across species, involving activation of lysosomal Ca channel TRPML by ROS. Indeed, disruption of TRPML in cancer cells abolished PRL-stimulated lysosomal exocytosis, acid addiction, and metastasis. Thus, PRL is the molecular switch turning cells addicted to an acidic condition, which should benefit cancer cells to thrive in an acidic tumor microenvironment.
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http://dx.doi.org/10.1016/j.devcel.2020.08.009DOI Listing
November 2020

p18/Lamtor1-mTORC1 Signaling Controls Development of Mucin-producing Goblet Cells in the Intestine.

Cell Struct Funct 2020 ;45(2):93-105

Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University.

Mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in controlling cell growth and metabolism in response to nutrients and growth factors. The activity of mTORC1 is dually regulated by amino acids and growth factor signaling, and amino acid-dependent mTORC1 activity is regulated by mTORC1 interaction with the Ragulator-Rag GTPase complex, which is localized to the surface of lysosomes via a membrane-anchored protein, p18/Lamtor1. However, the physiological function of p18-Ragulator-dependent mTORC1 signaling remains elusive. The present study evaluated the function of p18-mediated mTORC1 signaling in the intestinal epithelia using p18 conditional knockout mice. In p18 knockout colonic crypts, mTORC1 was delocalized from lysosomes, and in vivo mTORC1 activity was markedly decreased. Histologically, p18 knockout crypts exhibited significantly increased proliferating cells and dramatically decreased mucin-producing goblet cells, while overall crypt architecture and enteroendocrine cell differentiation were unaffected. Furthermore, p18 knockout crypts normally expressed transcription factors implicated in crypt differentiation, such as Cdx2 and Klf4, indicating that p18 ablation did not affect the genetic program of cell differentiation. Analysis of colon crypt organoid cultures revealed that both p18 ablation and rapamycin treatment robustly suppressed development of mucin-producing goblet cells. Hence, p18-mediated mTORC1 signaling could promote the anabolic metabolism required for robust mucin production in goblet cells to protect the intestinal epithelia from various external stressors.Key words: mTORC1, p18/lamtor1, intestinal epithelium, goblet cells, mucin.
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http://dx.doi.org/10.1247/csf.20018DOI Listing
January 2020

Stress-responsive MTK1 SAPKKK serves as a redox sensor that mediates delayed and sustained activation of SAPKs by oxidative stress.

Sci Adv 2020 Jun 24;6(26):eaay9778. Epub 2020 Jun 24.

Division of Cell Signaling and Molecular Medicine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Cells respond to oxidative stress by inducing intracellular signaling, including stress-activated p38 and JNK MAPK (SAPK) pathways, but the underlying mechanisms remain unclear. Here, we report that the MAP three kinase 1 (MTK1) SAPK kinase kinase (SAPKKK) functions as an oxidative-stress sensor that perceives the cellular redox state and transduces it into SAPK signaling. Following oxidative stress, MTK1 is rapidly oxidized and gradually reduced at evolutionarily conserved cysteine residues. These coupled oxidation-reduction modifications of MTK1 elicit its catalytic activity. Gene knockout experiments showed that oxidative stress-induced SAPK signaling is mediated by coordinated activation of the two SAPKKKs, MTK1 and apoptosis signal-regulating kinase 1 (ASK1), which have different time and dose-response characteristics. The MTK1-mediated redox sensing system is crucial for delayed and sustained SAPK activity and dictates cell fate decisions including cell death and interleukin-6 production. Our results delineate a molecular mechanism by which cells generate optimal biological responses under fluctuating redox environments.
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http://dx.doi.org/10.1126/sciadv.aay9778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314524PMC
June 2020

PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.

J Biol Chem 2020 08 22;295(33):11682-11692. Epub 2020 Jun 22.

Department of Biochemistry and Centre de Recherche en Biologie Structurale, McGill University, Montreal, Quebec, Canada

Phosphatases of regenerating liver (PRLs) are markers of cancer and promote tumor growth. They have been implicated in a variety of biochemical pathways but the physiologically relevant target of phosphatase activity has eluded 20 years of investigation. Here, we show that PRL3 catalytic activity is not required in a mouse model of metastasis. PRL3 binds and inhibits CNNM4, a membrane protein associated with magnesium transport. Analysis of PRL3 mutants specifically defective in either CNNM-binding or phosphatase activity demonstrate that CNNM binding is necessary and sufficient to promote tumor metastasis. As PRLs do have phosphatase activity, they are in fact -pseudophosphatases. Phosphatase activity leads to formation of phosphocysteine, which blocks CNNM binding and may play a regulatory role. We show levels of PRL cysteine phosphorylation vary in response to culture conditions and in different tissues. Examination of related protein phosphatases shows the stability of phosphocysteine is a unique and evolutionarily conserved property of PRLs. The demonstration that PRL3 functions as a pseudophosphatase has important ramifications for the design of PRL inhibitors for cancer.
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http://dx.doi.org/10.1074/jbc.RA120.014464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450121PMC
August 2020

Excessive Mg Impairs Intestinal Homeostasis by Enhanced Production of Adenosine Triphosphate and Reactive Oxygen Species.

Antioxid Redox Signal 2020 07 8;33(1):20-34. Epub 2020 Apr 8.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.

Mg is fundamental for life, and its shortage severely impairs vital functions. However, whether excessive Mg has beneficial or adverse effects has remained unknown. To clarify this issue, we analyzed the effect of suppressing the functions of Cyclin M (CNNM) Mg efflux transporters in various experimental systems. Investigation of short-lived worms mutated for CNNM genes revealed reactive oxygen species (ROS) augmentation in intestinal cells, coincidently with high levels of Mg. Knockdown of , encoding Mg-incorporating channel into intestinal cells, reduced ROS levels and restored life span, confirming the causative role of excessive Mg. Also, inactivation of orthologous CNNM in human cultured cells and mice by RNA interference, expression of CNNM-inhibiting protein, phosphatase of regenerating liver 3, or gene knockout resulted in ROS overproduction. Moreover, biochemical analyses revealed that excessive Mg stimulates adenosine triphosphate overproduction and accelerates mitochondrial electron transport, whose suppression shut down ROS generation. These results provide definitive evidence that excessive Mg drives overproduction of ROS by affecting energy metabolism, implying the crucial importance of the tight regulation of intracellular Mg levels.
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http://dx.doi.org/10.1089/ars.2019.7951DOI Listing
July 2020

Phosphatase of regenerating liver sensitizes MET to functional activation by hepatocyte growth factor.

Biochem J 2019 05 21;476(10):1419-1431. Epub 2019 May 21.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Phosphatase of regenerating liver (PRL) is overexpressed in metastatic cancers and actively drives their malignant progression. Many studies on cultured cancer cells have implied PRL overexpression as a stimulant for cellular signaling involved in cell proliferation. However, its role in the tightly adhered and polarized epithelial cells remains largely uncharacterized. In this study, we show that inducible expression of PRL in MDCK normal epithelial cells sensitized MET, the receptor for hepatocyte growth factor (HGF), to functional activation by HGF. We found that PRL expression amplified tyrosine phosphorylation levels of various proteins, among which MET was identified to be the most abundant. This phosphorylation occurred selectively at Y1234/1235 in the activation loop of MET, whereas phosphorylation of Y1349 in the effector-binding site, which is directly involved in downstream signaling, was almost undetectable. Consistently, PRL overexpression by itself did not cause observable alterations at the cellular level. However, when cells were stimulated with HGF, phosphorylation of Y1349 was much more strongly induced in PRL-expressing cells than in control cells. This resulted in robust cell scattering and tubulogenesis, even with low levels of HGF. Collectively, these results demonstrate a unique role of PRL in regulating MET function, which is known to be crucial for remodeling of epithelial tissues and malignant progression of cancers.
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http://dx.doi.org/10.1042/BCJ20190071DOI Listing
May 2019

Cnnm4 deficiency suppresses Ca signaling and promotes cell proliferation in the colon epithelia.

Oncogene 2019 05 22;38(20):3962-3969. Epub 2019 Jan 22.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.

CNNM4 is a Mg transporter highly expressed in the colon epithelia. Its importance in regulating intracellular Mg levels and cancer development has been documented, but how CNNM4 function affects the dynamic homeostasis of the epithelial tissue remains unclear. Here, we show that Cnnm4 deficiency promotes cell proliferation and partly suppresses cell differentiation in the colon epithelia, making them vulnerable to cancer development. Such phenotypic characteristics are highly similar to those of mice lacking Trpv1, which encodes the cation channel involved in capsaicin-stimulated Ca influx. Indeed, Ca-imaging analyses using the organoid culture reveal that Ca influx stimulated by capsaicin is greatly impaired by Cnnm4 deficiency. Moreover, EGF receptor signaling is constitutively activated in the colon epithelia of Cnnm4-deficient mice, as is the case with Trpv1-deficient mice. The administration of gefitinib, a clinically available inhibitor of EGF receptor, cancels the augmented proliferation of cells observed in Cnnm4-deficient mice. Collectively, these results establish the functional interplay between Mg and Ca in the colon epithelia, which is crucial for maintaining the dynamic homeostasis of the epithelial tissue.
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http://dx.doi.org/10.1038/s41388-019-0682-0DOI Listing
May 2019

Molecular function and biological importance of CNNM family Mg2+ transporters.

J Biochem 2019 Mar;165(3):219-225

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.

Cyclin M (CNNM) family proteins are evolutionarily conserved Mg2+ transporters. They extrude Mg2+ from cells and maintain intracellular Mg2+ levels within the normal range. Moreover, they play an important role in Mg2+ (re)absorption in the intestine and kidney by mediating the directional transport of Mg2+ across epithelial tissue from the tubular lumen to the body inside. Mg2+ efflux is suppressed by the direct binding with phosphatase of regenerating liver (PRL), and the formation of the complex is dynamically regulated by cysteine phosphorylation of PRL. The dysfunction of CNNM family proteins is responsible for inherited hypomagnesemia, as well as various intractable diseases, such as cancer and hypertension. Through multiple functional analyses of CNNM family proteins, the biomedical significance of the proper regulation of Mg2+ levels has been elucidated.
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http://dx.doi.org/10.1093/jb/mvy095DOI Listing
March 2019

The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg efflux activity.

J Biol Chem 2018 12 19;293(52):19998-20007. Epub 2018 Oct 19.

From the Department of Biochemistry and Centre for Structural Biology, McGill University, Montreal, Quebec H3G 0B1, Canada and. Electronic address:

Proteins of the cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg transporters. CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Although the CBS domain has been extensively characterized, little is known about the CNBH domain. Here, we determined the first crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. Contrary to expectation, these domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. Analytical ultracentrifugation experiments revealed an inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg efflux. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg transport and associated diseases.
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http://dx.doi.org/10.1074/jbc.RA118.005672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311497PMC
December 2018

Visualization of long-term Mg dynamics in apoptotic cells using a novel targetable fluorescent probe.

Chem Sci 2017 Dec 20;8(12):8255-8264. Epub 2017 Oct 20.

Department of Material and Life Science , Graduate School of Engineering , Osaka University , Suita , Osaka 565-0871 , Japan . Email:

Mg plays important roles in many physiological processes. However, the underlying molecular mechanisms, especially in the apoptotic pathway, remain unclear due to the diffusion of Mg probes, which hinders long-term imaging in specific organelles. We developed an immobilized Mg probe, MGH, which is covalently conjugated with the HaloTag protein in various organelles. HaloTag-coupled MGH enabled long-term imaging of intracellular local Mg dynamics for 24 h. To exploit this remarkable property, MGH was applied to the investigation of intracellular Mg dynamics during apoptosis. Time-lapse imaging revealed an increase in the Mg concentration after apoptotic cell shrinkage. Combined imaging analyses of intracellular Mg and ATP concentrations strongly suggested that this Mg concentration increase was caused by the dissociation of Mg from ATP, along with a decrease in the intracellular ATP concentration. Thus, this protein-coupled Mg probe could be a new chemical tool to elucidate intracellular Mg dynamics with high spatiotemporal resolution.
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http://dx.doi.org/10.1039/c7sc03954aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858021PMC
December 2017

Phosphatase of regenerating liver maintains cellular magnesium homeostasis.

Biochem J 2018 03 26;475(6):1129-1139. Epub 2018 Mar 26.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan

Phosphatase of regenerating liver (PRL) is highly expressed in malignant cancers and promotes cancer progression. Recent studies have suggested its functional relationship with Mg, but the importance and molecular details of this relationship remain unknown. Here, we report that PRL expression is regulated by Mg and PRL protects cells from apoptosis under Mg-depleted conditions. When cultured cells were subjected to Mg depletion, endogenous PRL protein levels increased significantly. siRNA-mediated knockdown of endogenous PRL did not significantly affect cell proliferation under normal culture conditions, but it increased cell death after Mg depletion. Imaging analyses with a fluorescent probe for Mg showed that PRL knockdown severely reduced intracellular Mg levels, indicating a role for PRL in maintaining intracellular Mg We also examined the mechanism of augmented expression of PRL proteins and found that mRNA transcription was stimulated by Mg depletion. A series of analyses revealed the activation and the crucial importance of signal transducer and activator of transcription 1 in this process. Collectively, these results implicate PRL in maintaining cellular Mg homeostasis.
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http://dx.doi.org/10.1042/BCJ20170756DOI Listing
March 2018

Rebuttal from Yosuke Funato, Kazuharu Furutani, Yoshihisa Kurachi and Hiroaki Miki.

J Physiol 2018 03 31;596(5):751. Epub 2018 Jan 31.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.

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http://dx.doi.org/10.1113/JP275706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830423PMC
March 2018

CrossTalk proposal: CNNM proteins are Na /Mg exchangers playing a central role in transepithelial Mg (re)absorption.

J Physiol 2018 03 31;596(5):743-746. Epub 2018 Jan 31.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.

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http://dx.doi.org/10.1113/JP275248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830418PMC
March 2018

Renal function of cyclin M2 Mg2+ transporter maintains blood pressure.

J Hypertens 2017 03;35(3):585-592

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Objectives: Epidemiological studies have shown that magnesium intake/excretion is inversely correlated with blood pressure (BP), and artificial supplementation of magnesium was able to prevent hypertension. However, there has been no molecular genetic study showing the importance of magnesium homeostasis in BP regulation.

Methods: We analyzed magnesium content and BP of mice lacking genes encoding cyclin M (CNNM) Mg transporter family proteins.

Results: Systemic heterozygotes and the kidney-specific homozygotes for Cnnm2-deficient alleles are both viable and show hypomagnesemia, indicating the important function of CNNM2 in maintaining magnesium homeostasis in the kidney. Endogenous CNNM2 localizes at the basolateral membrane of kidney distal convoluted tubule cells, which play important roles not only in magnesium reabsorption but also in BP control. The BP of these viable strains is significantly reduced; the SBP values by telemetric measurements are 121.7 ± 2.8 mmHg in wild-type, and 110.2 ± 2.7 and 109.7 ± 3.6 mmHg in systemic heterozygotes and kidney-specific homozygotes, respectively.

Conclusion: Analyses of mice lacking CNNM Mg transporters clearly demonstrated abnormalities in BP values, confirming the importance of magnesium homeostasis in maintaining BP.
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http://dx.doi.org/10.1097/HJH.0000000000001211DOI Listing
March 2017

Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis.

EMBO Rep 2016 12 17;17(12):1890-1900. Epub 2016 Nov 17.

Department of Biochemistry and Groupe de recherche axé sur la structure des protéines, McGill University, Montreal, Quebec, Canada

PRLs (phosphatases of regenerating liver) are frequently overexpressed in human cancers and are prognostic markers of poor survival. Despite their potential as therapeutic targets, their mechanism of action is not understood in part due to their weak enzymatic activity. Previous studies revealed that PRLs interact with CNNM ion transporters and prevent CNNM4-dependent Mg transport, which is important for energy metabolism and tumor progression. Here, we report that PRL-CNNM complex formation is regulated by the formation of phosphocysteine. We show that cysteine in the PRL catalytic site is endogenously phosphorylated as part of the catalytic cycle and that phosphocysteine levels change in response to Mg levels. Phosphorylation blocks PRL binding to CNNM Mg transporters, and mutations that block the PRL-CNNM interaction prevent regulation of Mg efflux in cultured cells. The crystal structure of the complex of PRL2 and the CBS-pair domain of the Mg transporter CNNM3 reveals the molecular basis for the interaction. The identification of phosphocysteine as a regulatory modification opens new perspectives for signaling by protein phosphatases.
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http://dx.doi.org/10.15252/embr.201643393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283600PMC
December 2016

Mg2+ Extrusion from Intestinal Epithelia by CNNM Proteins Is Essential for Gonadogenesis via AMPK-TORC1 Signaling in Caenorhabditis elegans.

PLoS Genet 2016 08 26;12(8):e1006276. Epub 2016 Aug 26.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK.
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http://dx.doi.org/10.1371/journal.pgen.1006276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001713PMC
August 2016

Complementary role of CNNM2 in sperm motility and Ca(2+) influx during capacitation.

Biochem Biophys Res Commun 2016 06 2;474(3):441-446. Epub 2016 May 2.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Ca(2+) plays a central role in the regulation of sperm motility. We recently reported an unexpected role of CNNM4, a Mg(2+) transporter, in this process by demonstrating perturbed Ca(2+) influx and gradual loss of motility of Cnnm4-deficient sperm. However, Cnnm4-deficient male mice were not entirely infertile, and a significant Ca(2+) response was still observed in their sperm. In the present study, we generated Cnnm4-deficient mice harboring a non-functional Cnnm2 allele (Cnnm2(Δ)), to examine whether CNNM2 compensates for the lost function of CNNM4 in sperm. Cnnm2(+/Δ); Cnnm4(Δ/Δ) mice were infertile, and no obvious histological abnormalities were noted in their testis and epididymis. Their sperm showed normal morphology, but became immotile much more rapidly than those from Cnnm4(Δ/Δ) mice. When capacitation was initiated using serum albumin application, a rapid increase of intracellular Ca(2+) levels was observed in most wild-type sperm, but only about half of sperm from Cnnm4(Δ/Δ) mice exhibited a Ca(2+) response, and the response rate was further reduced in sperm from Cnnm2(+/Δ); Cnnm4(Δ/Δ) mice. Thus, sperm motility and Ca(2+) response were more severely affected in sperm from Cnnm2(+/Δ); Cnnm4(Δ/Δ) mice than in those from Cnnm4(Δ/Δ) mice, implicating CNNM2 in regulating these processes.
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http://dx.doi.org/10.1016/j.bbrc.2016.05.001DOI Listing
June 2016

The Mg2+ transporter CNNM4 regulates sperm Ca2+ homeostasis and is essential for reproduction.

J Cell Sci 2016 05 22;129(9):1940-9. Epub 2016 Mar 22.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Ca(2+) influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg(2+) transporter, is required for Ca(2+) influx during capacitation. We find that Cnnm4-deficient male mice are almost infertile because of sperm dysfunction. Motion analyses show that hyperactivation, a qualitative change in the mode of sperm motility during capacitation, is abrogated in Cnnm4-deficient sperm. In contrast, tyrosine phosphorylation of flagellar proteins, a hallmark of capacitation, is excessively augmented. These seemingly paradoxical phenotypes of Cnnm4-deficient sperm are very similar to those of sperm lacking a functional cation channel of sperm (CatSper) channel, which plays an essential role in Ca(2+) influx during sperm capacitation. Ca(2+) imaging analyses demonstrate that Ca(2+) influx is perturbed in Cnnm4-deficient sperm, and forced Ca(2+) entry into these sperm normalizes the level of tyrosine phosphorylation. Furthermore, we confirm the importance of CNNM4 in sperm by generating germ-cell-specific Cnnm4-deficient mice. These results suggest a new role of CNNM4 in sperm Ca(2+) homeostasis.
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http://dx.doi.org/10.1242/jcs.182220DOI Listing
May 2016

Basolateral sorting of the Mg²⁺ transporter CNNM4 requires interaction with AP-1A and AP-1B.

Biochem Biophys Res Commun 2014 Dec 10;455(3-4):184-9. Epub 2014 Nov 10.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of μ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the μ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with μ1A and μ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.
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http://dx.doi.org/10.1016/j.bbrc.2014.10.138DOI Listing
December 2014

Membrane protein CNNM4-dependent Mg2+ efflux suppresses tumor progression.

J Clin Invest 2014 Dec 27;124(12):5398-410. Epub 2014 Oct 27.

Intracellular Mg(2+) levels are strictly regulated; however, the biological importance of intracellular Mg(2+) levels and the pathways that regulate them remain poorly understood. Here, we determined that intracellular Mg(2+) is important in regulating both energy metabolism and tumor progression. We determined that CNNM4, a membrane protein that stimulates Mg(2+) efflux, binds phosphatase of regenerating liver (PRL), which is frequently overexpressed in malignant human cancers. Biochemical analyses of cultured cells revealed that PRL prevents CNNM4-dependent Mg(2+) efflux and that regulation of intracellular Mg(2+) levels by PRL and CNNM4 is linked to energy metabolism and AMPK/mTOR signaling. Indeed, treatment with the clinically available mTOR inhibitor rapamycin suppressed the growth of cancer cells in which PRL was overexpressed. In ApcΔ(14/+) mice, which spontaneously form benign polyps in the intestine, deletion of Cnnm4 promoted malignant progression of intestinal polyps to adenocarcinomas. IHC analyses of tissues from patients with colon cancer demonstrated an inverse relationship between CNNM4 expression and colon cancer malignancy. Together, these results indicate that CNNM4-dependent Mg(2+) efflux suppresses tumor progression by regulating energy metabolism.
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http://dx.doi.org/10.1172/JCI76614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348944PMC
December 2014

Reconstruction of insulin signal flow from phosphoproteome and metabolome data.

Cell Rep 2014 Aug 14;8(4):1171-83. Epub 2014 Aug 14.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; CREST, Japan Science and Technology Corporation, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data.
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http://dx.doi.org/10.1016/j.celrep.2014.07.021DOI Listing
August 2014

Mg2+-dependent interactions of ATP with the cystathionine-β-synthase (CBS) domains of a magnesium transporter.

J Biol Chem 2014 May 6;289(21):14731-9. Epub 2014 Apr 6.

From the Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan and

Ancient conserved domain protein/cyclin M (CNNM) family proteins are evolutionarily conserved Mg(2+) transporters. However, their biochemical mechanism of action remains unknown. Here, we show the functional importance of the commonly conserved cystathionine-β-synthase (CBS) domains and reveal their unique binding ability to ATP. Deletion mutants of CNNM2 and CNNM4, lacking the CBS domains, are unable to promote Mg(2+) efflux. Furthermore, the substitution of one amino acid residue in the CBS domains of CNNM2, which is associated with human hereditary hypomagnesemia, abrogates Mg(2+) efflux. Binding analyses reveal that the CBS domains of CNNM2 bind directly to ATP and not AMP in a manner dependent on the presence of Mg(2+), which is inhibited in a similar pattern by the disease-associated amino acid substitution. The requirement of Mg(2+) for these interactions is a unique feature among CBS domains, which can be explained by the presence of highly electronegative surface potentials around the ATP binding site on CNNM2. These results demonstrate that the CBS domains play essential roles in Mg(2+) efflux, probably through interactions with ATP. Interactions with ATP, which mostly forms complexes with Mg(2+) in cells, may account for the rapid Mg(2+) transport by CNNM family proteins.
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http://dx.doi.org/10.1074/jbc.M114.551176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031528PMC
May 2014

Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.

PLoS Genet 2013 5;9(12):e1003983. Epub 2013 Dec 5.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.

Transcellular Mg(2+) transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg(2+) extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg(2+) extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg(2+) by exchanging intracellular Mg(2+) with extracellular Na(+). Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg(2+) extrusion activity. These results demonstrate the crucial importance of Mg(2+) extrusion by CNNM4 in organismal and topical regulation of magnesium.
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http://dx.doi.org/10.1371/journal.pgen.1003983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3854942PMC
August 2014
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