The Ion Transporter NKCC1 Links Cell Volume to Cell Mass Regulation by Suppressing mTORC1

Demian, Wael; Persaud, Avinash K.; Jiang, Chunhe; Coyaud, Étienne; Liu, Shixuan; Kapùs, András; Kafri, Ran; Raught, Brian; Rotin, Daniela

doi:10.1016/j.celrep.2019.04.034

Cited by 38 publications

(40 citation statements)

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“…In contrast, treatment of glioma cells with STS66 (60 μM) alone or TMZ plus STS66 for 24 or 48 h significantly reduced glioma cells proliferation and induced G0/G1 arrest, which appears to correlate with NKCC1-mediated reduced K + influx. Consistently, a recent report showed that proliferating cells had increased number of cells in G0/G1 phase (Demian et al, 2019). In summary, in this study, blocking NKCC1 activity with STS66 significantly reduced glioma cells proliferation, which is likely due to reduced intracellular K + and Cl − concentration and prevents cell volume increase during G1 to S-phase of the cell cycle and reduces proliferation.…”

Section: Nkcc1 Protein Activation In Glioma K + (Rb + ) Influx and Prsupporting

confidence: 91%

“…The ratios of p-/t-AKT, p-/t-ERK, and p-/t-p70 were presented in Supplementary Figure S3. It has shown that TMZ stimulates AKT and ERK pathways in human glioma cells (Sun et al, 2012;Bi et al, 2018) but formation of NKCC1 protein and Leu transporter LAT1 complex inhibits AKT/ERK-mTOR1 activation in epithelial cells in response to amino acid-mediated stimulation (Demian et al, 2019). Our data demonstrate that inhibition of NKCC1 protein in glioma cells with BMT or STS66 suppresses AKT and ERK signaling pathways in response to TMZ stress but has no effects on mTORC1 signaling.…”

Section: A C B Dmentioning

confidence: 56%

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Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66

et al. 2020

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Introduction: Na +-K +-2Cl − cotransporter isoform 1 (NKCC1) is important in regulating intracellular K + and Cl − homeostasis and cell volume. In this study, we investigated a role of NKCC1 in regulating glioma K + influx and proliferation in response to apoptosis inducing chemotherapeutic drug temozolomide (TMZ). The efficacy of a new bumetanide (BMT)-derivative NKCC1 inhibitor STS66 [3-(butylamino)-2-phenoxy-5-[(2, 2, 2-trifluoroethylamino) methyl] benzenesulfonamide] in blocking NKCC1 activity was compared with well-established NKCC1 inhibitor BMT. Methods: NKCC1 activity in cultured mouse GL26 and SB28-GFP glioma cells was measured by Rb + (K +) influx. The WNK1-SPAK/OSR1-NKCC1 signaling and AKT/ERK-mTOR signaling protein expression and activation were assessed by immunoblotting. Cell growth was determined by bromodeoxyuridine (BrdU) incorporation assay, MTT proliferation assay, and cell cycle analysis. Impact of STS66 and BMT on cell Rb + influx and growth was measured in glioma cells treated with or without TMZ. Results: Rb + influx assay showed that 10 μM BMT markedly decreased the total Rb + influx and no additional inhibition detected at >10 μM BMT. In contrast, the maximum effects of STS66 on Rb + influx inhibition were at 40-60 μM. Both BMT and STS66 reduced TMZ-mediated NKCC1 activation and protein upregulation. Glioma cell growth can be reduced by STS66. The most robust inhibition of glioma growth, cell cycle, and AKT/ERK signaling was achieved by the TMZ + STS66 treatment. Conclusion: The new BMT-derivative NKCC1 inhibitor STS66 is more effective than BMT in reducing glioma cell growth in part by inhibiting NKCC1-mediated K + influx. TMZ + STS66 combination treatment reduces glioma cell growth via inhibiting cell cycle and AKT-ERK signaling.

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Section: Nkcc1 Protein Activation In Glioma K + (Rb + ) Influx and Prsupporting

confidence: 91%

Section: A C B Dmentioning

confidence: 56%

Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66

et al. 2020

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“…For murine experiments, frozen stocks of wild-type and mutant strains were cultured on CDMN agar plates (C. difficile agar base (Oxoid) supplemented with 7% (v/v) defibrinated horse blood (Lampire Biological Laboratories), 32 mg/L moxalactam (Santa Cruz Biotechnology), 12 mg/L norfloxacin (Sigma-Aldrich) and 500 mg/L cysteine hydrochloride (Fischer) in an anaerobic chamber [27] at 37˚C for 24 hours. Single colonies were picked and grown anaerobically for 16-18 hours at 37˚C to saturation in 5 mL of pre-reduced reinforced Clostridial medium (RCM, Oxoid) for inoculation.…”

Section: Bacterial Growth Conditionsmentioning

confidence: 99%

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis

et al. 2020

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Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

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“…5f ). This widely expressed solute carrier plays a key role in the regulation of ionic balance and cell volume 60 . We also discovered novel oscillating ubiquitin modifications in the MAGE domain of MAGED1, a protein that directly interacts with the core clock protein RORα, to regulate Bmal1, Rev-erbα and E4bp4 gene expression ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology

Hansen

Tanzer

Bruening

et al. 2020

Preprint

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SUMMARYProtein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. We developed a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identified 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells – double the number and quantitative accuracy of data dependent acquisition. Applied to TNF-alpha signaling, the workflow comprehensively captured known sites while adding many novel ones. A first systems-wide investigation of ubiquitination of the circadian cycle uncovered hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting novel connections between metabolism and circadian regulation.

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The Ion Transporter NKCC1 Links Cell Volume to Cell Mass Regulation by Suppressing mTORC1

Cited by 38 publications

References 50 publications

Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66

Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis

Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology

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