Intestinal Mg2+ accumulation induced by cnnm mutations decreases the body size by suppressing TORC2 signaling in Caenorhabditis elegans.

Mg2+ is a vital ion involved in diverse cellular functions by forming complexes with ATP. Intracellular Mg2+ levels are tightly regulated by the coordinated actions of multiple Mg2+ transporters, such as the Mg2+ efflux transporter, cyclin M (CNNM). Caenorhabditis elegans (C. elegans) worms with mutations in both cnnm-1 and cnnm-3 exhibit excessive Mg2+ accumulation in intestinal cells, leading to various phenotypic abnormalities. In this study, we investigated the mechanism underlying the reduction in body size in cnnm-1; cnnm-3 mutant worms. RNA interference (RNAi) of gtl-1, which encodes a Mg2+-intake channel in intestinal cells, restored the worm body size, confirming that this phenotype is due to excessive Mg2+ accumulation. Moreover, RNAi experiments targeting body size-related genes and analyses of mutant worms revealed that the suppression of the target of rapamycin complex 2 (TORC2) signaling pathway was involved in body size reduction, resulting in downregulated DAF-7 expression in head ASI neurons. As the DAF-7 signaling pathway suppresses dauer formation under stress, cnnm-1; cnnm-3 mutant worms exhibited a greater tendency to form dauer upon induction. Collectively, our results revealed that excessive accumulation of Mg2+ repressed the TORC2 signaling pathway in C. elegans worms and suggest the novel role of the DAF-7 signaling pathway in the regulation of their body size.

Phosphatase-independent role of phosphatase of regenerating liver in cancer progression.

Phosphatase of regenerating liver (PRL) is a family of protein tyrosine phosphatases (PTPs) that are anchored to the plasma membrane by prenylation. They are frequently overexpressed in various types of malignant cancers and their roles in cancer progression have received considerable attention. Mutational analyses of PRLs have shown that their intrinsic phosphatase activity is dispensable for tumor formation induced by PRL overexpression in a lung metastasis model using melanoma cells. Instead, PRLs directly bind to cyclin M (CNNM) Mg2+ exporters in the plasma membrane and potently inhibit their Mg2+ export activity, resulting in an increase in intracellular Mg2+ levels. Experiments using mammalian culture cells, mice, and C. elegans have collectively revealed that dysregulation of Mg2+ levels severely affects ATP and reactive oxygen species (ROS) levels as well as the function of Ca2+ -permeable channels. Moreover, PRL overexpression altered the optimal pH for cell proliferation from normal 7.5 to acidic 6.5, which is typically observed in malignant tumors. Here, we review the phosphatase-independent biological functions of PRLs, focusing on their interactions with CNNM Mg2+ exporters in cancer progression.

PRL stimulates mitotic errors by suppressing kinetochore-localized activation of AMPK during mitosis.

Phosphatase of regenerating liver (PRL) is frequently overexpressed in various malignant cancers and is known to be a driver of malignancy. Here, we demonstrated that PRL overexpression causes mitotic errors that accompany spindle misorientation and aneuploidy, which are intimately associated with cancer progression. Mechanistic analyses of this phenomenon revealed dysregulation of the energy sensor kinase, AMP-activated protein kinase (AMPK), in PRL-induced mitotic errors. Specifically, immunofluorescence analysis showed that levels of phosphorylated AMPK (P-AMPK), an activated form of AMPK, at the kinetochore were reduced by PRL expression. Moreover, artificial activation of AMPK using chemical activators, such as A769662 and AICAR, in PRL-expressing cells restored P-AMPK signals at the kinetochore and normalized spindle orientation. Collectively, these results indicate the crucial importance of the activation of kinetochore-localized AMPK in the normal progression of mitosis, which is specifically perturbed by PRL overexpression.Key words: cancer, AMPK, PRL, kinetochore, mitotic errors.

A novel role for PRL in regulating epithelial cell density by inducing apoptosis at confluence.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-ß pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.

The emerging roles and therapeutic potential of cyclin M/CorC family of Mg2+ transporters.

Cyclin M (CNNM) and its prokaryotic ortholog CorC belong to a family of proteins that function as Mg2+-extruding transporters by stimulating Na+/Mg2+ exchange, and thereby control intracellular Mg2+ levels. The Mg2+-extruding function of CNNM is inhibited by the direct binding of an oncogenic protein, phosphatase of regenerating liver (PRL), and this inhibition is responsible for the PRL-driven malignant progression of cancers. Studies with mouse strains deficient for the CNNM gene family revealed the importance of CNNM4 and CNNM2 in maintaining organismal Mg2+ homeostasis by participating in intestinal Mg2+ absorption and renal reabsorption, respectively. Moreover, CNNM proteins are involved in various diseases, and gene mutations in CNNM2 and CNNM4 cause dominant familial hypomagnesemia and Jalili syndrome, respectively. Genome wide association studies have also revealed the importance of CNNM2 in multiple major diseases, such as hypertension and schizophrenia. Collectively, the molecular and biological characterizations of CNNM/CorC show that they are an intriguing therapeutic target; the current status of drug development targeting these proteins is also discussed.

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

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 Mg2+-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 ß-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.

Identification and mechanistic analysis of an inhibitor of the CorC Mg2+ transporter.

The CorC/CNNM family of Na+-dependent Mg2+ 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 Mg2+ 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.

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

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/ß-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/ß-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.

Structural basis for the Mg2+ recognition and regulation of the CorC Mg2+ transporter.

The CNNM/CorC family proteins are Mg2+ 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 Mg2+ and genetic disorders. Here, we determined the crystal structure of the Mg2+-bound CorC TM domain dimer. Each protomer has a single Mg2+ binding site with a fully dehydrated Mg2+ ion. The residues at the Mg2+ 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 Mg2+ export activity of CorC.

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

Dietary magnesium (Mg2+) 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 Mg2+ feeding also enhances long-term memory in Drosophila. Normal and Mg2+-enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg2+-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex 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 Mg2+. We propose that regulated neuronal Mg2+ efflux is critical for normal and Mg2+-enhanced memory.

The proverbial saying 'you are what you eat' perfectly summarizes the concept that our diet can influence both our mental and physical health. We know that foods that are good for the heart, such as nuts, oily fish and berries, are also good for the brain. We know too that vitamins and minerals are essential for overall good health. But is there any evidence that increasing your intake of specific vitamins or minerals could help boost your brain power? While it might sound almost too good to be true, there is some evidence that this is the case for at least one mineral, magnesium. Studies in rodents have shown that adding magnesium supplements to food improves how well the animals perform on memory tasks. Both young and old animals benefit from additional magnesium. Even elderly rodents with a condition similar to Alzheimer's disease show less memory loss when given magnesium supplements. But what about other species? Wu et al. now show that magnesium supplements also boost memory performance in fruit flies. One group of flies was fed with standard cornmeal for several days, while the other group received cornmeal supplemented with magnesium. Both groups were then trained to associate an odor with a food reward. Flies that had received the extra magnesium showed better memory for the odor when tested 24 hours after training. Wu et al. show that magnesium improves memory in the flies via a different mechanism to that reported previously for rodents. In rodents, magnesium increased levels of a receptor protein for a brain chemical called glutamate. In fruit flies, by contrast, the memory boost depended on a protein that transports magnesium out of neurons. Mutant flies that lacked this transporter showed memory impairments. Unlike normal flies, those without the transporter showed no memory improvement after eating magnesium-enriched food. The results suggest that the transporter may help adjust magnesium levels inside brain cells in response to neural activity. Humans produce four variants of this magnesium transporter, each encoded by a different gene. One of these transporters has already been implicated in brain development. The findings of Wu et al. suggest that the transporters may also act in the adult brain to influence cognition. Further studies are needed to test whether targeting the magnesium transporter could ultimately hold promise for treating memory impairments.

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

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 Ca2+ 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.

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

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 pseudo-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.

Excessive Mg2+ Impairs Intestinal Homeostasis by Enhanced Production of Adenosine Triphosphate and Reactive Oxygen Species.

Aims: Mg2+ is fundamental for life, and its shortage severely impairs vital functions. However, whether excessive Mg2+ has beneficial or adverse effects has remained unknown. To clarify this issue, we analyzed the effect of suppressing the functions of Cyclin M (CNNM) Mg2+ efflux transporters in various experimental systems. Results: Investigation of short-lived Caenorhabditis elegans worms mutated for CNNM genes revealed reactive oxygen species (ROS) augmentation in intestinal cells, coincidently with high levels of Mg2+. Knockdown of gtl-1, encoding Mg2+-incorporating channel into intestinal cells, reduced ROS levels and restored life span, confirming the causative role of excessive Mg2+. 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 Mg2+ stimulates adenosine triphosphate overproduction and accelerates mitochondrial electron transport, whose suppression shut down ROS generation. Innovation and Conclusion: These results provide definitive evidence that excessive Mg2+ drives overproduction of ROS by affecting energy metabolism, implying the crucial importance of the tight regulation of intracellular Mg2+ levels.

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

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.

Cnnm4 deficiency suppresses Ca2+ signaling and promotes cell proliferation in the colon epithelia.

CNNM4 is a Mg2+ transporter highly expressed in the colon epithelia. Its importance in regulating intracellular Mg2+ 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 Ca2+ influx. Indeed, Ca2+-imaging analyses using the organoid culture reveal that Ca2+ 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 Mg2+ and Ca2+ in the colon epithelia, which is crucial for maintaining the dynamic homeostasis of the epithelial tissue.

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

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.

The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity.

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 Mg2+ 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-ß-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 Mg2+ 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 Mg2+ efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg2+ transport and associated diseases.

Phosphatase of regenerating liver maintains cellular magnesium homeostasis.

Phosphatase of regenerating liver (PRL) is highly expressed in malignant cancers and promotes cancer progression. Recent studies have suggested its functional relationship with Mg2+, but the importance and molecular details of this relationship remain unknown. Here, we report that PRL expression is regulated by Mg2+ and PRL protects cells from apoptosis under Mg2+-depleted conditions. When cultured cells were subjected to Mg2+ 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 Mg2+ depletion. Imaging analyses with a fluorescent probe for Mg2+ showed that PRL knockdown severely reduced intracellular Mg2+ levels, indicating a role for PRL in maintaining intracellular Mg2+ We also examined the mechanism of augmented expression of PRL proteins and found that PRL mRNA transcription was stimulated by Mg2+ 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 Mg2+ homeostasis.