Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.Acknowledgements: This work was supported by Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (to MLM-C), Ministerio de Ciencia e Innovación CONSOLIDER-INGENIO 2010 Program Grant CSD2008-00005 (to LAMC); Spanish Ministry of Economy and Competitiveness Grant BFU2013-47531-R, BFU2016-77408-R, PID2019-109055RB-100 (to L.A.M.-C.) (MINECO/FEDER, UE); Asociación Española contra el Cáncer (MLM-C, TC-D), Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Fundacion BBVA UMBRELLA project (to M.L.M.-C.), Ayuda RYC2020-029316-I financiada por MICIN/AEI/10.13039/501100011033 (to TC-D), Plataforma de Investigación Clínica-SCReN (PT17 0017 0020) (to M.I.-L.), programa retos RTC2019-007125-1 (to M.L.M.-C, J.S.), Proyectos Investigacion en Salud DTS20/00138 (to M.L.M.-C., J.S), ERA-Net E-Rare EJP RD Joint Translational Call for Rare Diseases FIGHT-CNNM2 (EJPRD19-040) and from Instituto Carlos III, Spain (REF G95229142) (to L.A.M.-C.), US National Institutes of Health under grant CA217817 (to D.B.), Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thankMINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644) and PhD fellowship fromMINECO (REF BES-2017-080435) awarded to I.G.-R. The collection and storage of patients tissues was supported by the Newcastle Biomedicine Biobank and the European Community’s Seventh Framework Programme (FP7/2001–2013) and Cancer Research UK awards Cancer Research UK grants C18342/A23390; C9380/A18084 and C9380/A26813. Finally, we would like to acknowledge Begoña Rodríguez Iruretagoyena for the technical support provided

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury

Les microvésicules tumorales dans la progression du cancer (Une étude in vitro dans le modèle du cancer de la prostate)

La membrane joue un rôle clé dans un large nombre de processus physiologiques. Lors d une activation cellulaire, l externalisation de PS sur la membrane plasmique est suivie par l émission de microvésicules membranaires (MV). Les MV reflétant le profil antigénique de la cellule et de la stimulation dont elles sont originaires. Le microenvironnement tumoral est enrichi en MV libérées par les cellules qui constitue le tissu cancéreux. En nous focalisant sur l implication des fibroblastes nous proposons un modèle in vitro dans lequel les cellules normales et cancéreuses communiquent entre elles via MV.Le but de cette étude est d élucider un mécanisme, dans lequel les cellules du cancer de la prostate influencent le comportement de cellules normales du stroma, et ces dernières affectent l agressivité des cellules de carcinome, Le tout via la libération mutuelle de MV pour promouvoir ou soutenir la création d une niche favorable au développement de la tumeur.The plasma membrane plays a pivotal role in a large number of physiological processes. After cellular stimulation, externalization of PS in the exoplasmic leaflet of plasma membrane is followed by the shedding of membrane microvesicles (MV) in almost all cell types. MV composition reflect the antigenic profile of cells which they originate from, depending on the stimulus apply. Tumour microenvironment is highly enriched in MV shed from cells infiltrating the tumour tissue. Fibroblasts are associated with tumour cells at all stages of cancer progression. Focusing on fibroblasts implication in cancer, we propose an in vitro model in which cancer and normal cells communicate each other via MV. The aim of this study is to elucidate a mechanism, in which prostate cancer cells influence the behaviour of normal stromal cells that in turn affect the aggressiveness of carcinoma cells by mutual MV shedding, promoting or support the creation of a niche favourable for tumour development.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceItalyFRI

Macrophages contribute to the cyclic activation of adult hair follicle stem cells

Skin epithelial stem cells operate within a complex signaling milieu that orchestrates their lifetime regenerative properties. The question of whether and how immune cells impact on these stem cells within their niche is not well understood. Here we show that skin-resident macrophages decrease in number because of apoptosis before the onset of epithelial hair follicle stem cell activation during the murine hair cycle. This process is linked to distinct gene expression, including Wnt transcription. Interestingly, by mimicking this event through the selective induction of macrophage apoptosis in early telogen, we identify a novel involvement of macrophages in stem cell activation in vivo. Importantly, the macrophage-specific pharmacological inhibition of Wnt production delays hair follicle growth. Thus, perifollicular macrophages contribute to the activation of skin epithelial stem cells as a novel, additional cue that regulates their regenerative activity. This finding may have translational implications for skin repair, inflammatory skin diseases and cancer

FCS Figure Files

FCS Figure File

Resident macrophages express HF-SC stimulatory factors before the onset of anagen.

<p>(A) CD11b⁺Gr1⁻F4/80⁺ macrophages were FACS-isolated from Te and Tl backskin samples. Their mRNAs were purified and used to perform microarray analyses to evaluate changes in gene expression at Tl (P69) versus Te (P44). Histograms show a shortlist of up- and downregulated genes that have been involved in the control of HF-SC activation and apoptosis. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s003" target="_blank">Figure S3 A</a>. (B) Relative mRNA expression of Wnt7b and Wnt10a in FACS-sorted CD11b⁺Gr1⁻F4/80⁺ cells at Te, Tm, Tl, and A; <i>n</i> = 3. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s003" target="_blank">Figure S3A</a>. (C) Immunofluorescence staining of F4/80⁺ perifollicular macrophages (green). (D) Each histogram point represents the mean value of double positive F4/80⁺Wnt7b⁺ and F480⁺Wnt10a⁺ over total F4/80⁺ perifollicular macrophages. 10 fields/section/mouse were analyzed; <i>n</i> = 4. (E) Immunofluorescence of Wnt7b (green)/F4/80 (red), and Wnt10a (green)/F4/80 (red), counterstained with DAPI (blue) of skin sections at Te, Tm, Tl, and A; <i>n</i> = 3. Bar = 50 µm. n.s. non significant, Note: <i>n</i> refers to the number of mice, per point per condition. <i>*p</i>≤0.05. All data used to generate the histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s012" target="_blank">Data S1</a>.</p

Reduction of macrophage numbers in early telogen induces precocious HF growth.

<p>(A) The specific uptake of liposomes by macrophages was analyzed by co-immunofluorescence analysis of F4/80⁺ cells (red) and the detection of the liposomal PKH67 label (green) in skin sections after the injection of PKH67-liposomes. Arrows indicate double labeling; <i>n</i> = 2. Bar = 20 µm. (B) P44 mice were injected in the backskin for two alternated days with CL-lipo. Samples were collected for analyses at the time indicated in the diagram. (C) Histograms represent the quantification of F4/80⁺ cells in the backskin after treatment with CL-lipo and Lipo (left). Also shown is the distribution of F4/80⁺ cells in the backskin after treatment with CL-lipo (right). 10 fields/section/mouse were analyzed; <i>n</i> = 4. (D) Hematoxylin–eosin staining of backskin samples isolated after treatment with CL-lipo and Lipo controls. Bar = 250 µm, <i>n</i> = 4. (E) Histomorphometric analysis of HF stages after macrophages reduction. 100 HFs/mouse were analyzed; <i>n</i> = 4. (F) Appearance of the hair coat at T5 (P69), after shaving and treatment with CL-lipo and Lipo controls at T0 (P44). <i>**p</i><0.005; <i>***p</i><0.0005. Note: <i>n</i> refers to the number of mice, per point per condition. All data used to generate the histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s012" target="_blank">Data S1</a>.</p

Reduction of skin macrophages is associated with activation of β-catenin/Wnt signaling.

<p>(A) Immunofluorescence analysis of β-catenin (green) and K5 (red) in backskin sections of mice treated with CL-lipo and Lipo controls; <i>n</i> = 4. *Hair shaft autofluorescence. Bar = 25 µm. (B) Immunofluorescence analysis of CD34 (red) and H2B-GFP signal (green) in T2 backskin sections of TCF/Lef:H2B-GFP transgenic mice treated with CL-lipo and Lipo controls; <i>n</i> = 3. Arrows point to GFP positive cells. Bar = 25 µm. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s011" target="_blank">Figure S11 B</a>. (C) FACS analysis of single cell suspensions of CD34⁺CD49f⁺ HF-SCs (gated) isolated from backskin of mice treated with CL-lipo or Lipo controls at specified time points; <i>n</i> = 2–4. (D) Relative mRNA expression of canonical Wnt/β-catenin target genes and BMP signaling genes in HF-SCs isolated as indicated in (B); <i>n</i> = 2–4. (E) Relative mRNA expression of canonical Wnt/β-catenin target genes and BMP signaling genes in total back-skin samples after treatment with CL-lipo compared to Lipo controls; <i>n</i> = 6. Note: <i>n</i> refers to the number of mice, per point per condition. <i>*p</i>≤0.05. All data used to generate the histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s012" target="_blank">Data S1</a>.</p

Skin-resident macrophages decrease in number before the onset of anagen.

<p>(A) Time course of isolation of backskin samples. The second telogen period was subdivided into three stages followed by anagen. P44, early telogen (Te); P55, mid telogen (Tm); P69, late telogen (Tl); and P82, anagen (A_VI). (B) Fluctuations in the number of skin-resident immune cells during the second hair cycle analyzed by immunofluorescence. Note the decrease in cells expressing the myeloid markers CD11b, Gr1, F4/80 before the onset of anagen. Each histogram point represents the mean value of positive cells per 10× magnification field. 10 fields/section/mouse were analyzed; <i>n</i> = 4. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s001" target="_blank">Figure S1</a>. (C) Expression of F4/80 cells (green) in skin at Te, Tm, Tl, and A_VI stages, counterstained with DAPI (blue). Bar = 100 µm; *hair shaft autofluorescence. (D) Fluctuations in the number of perifollicular macrophages during the second hair cycle analyzed by immunofluorescence. Note the decrease in F4/80⁺ at A_VI. Each histogram point represents the mean value of positive cells per 10× magnification field at 30 µm distance from HF <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002-Paus1" target="_blank">[15]</a>. 10 fields/section/mouse were analyzed; <i>n</i> = 4. (E) FACS analysis of single cell suspensions from total skin samples harvested at Te, Tm, and Tl stages. Histograms show the percentage of F4/80⁺ cells gated from the CD11b⁺Gr1⁻cells (I) and Cd11b⁻Gr1⁺ (II) populations; <i>n</i> = 7–12. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s003" target="_blank">Figure S3A</a>. (F) TUNEL⁺F4/80⁺ cells in Tm. Histograms show the percentage of TUNEL positive cells in the FACS sorted CD11b⁺F4/80⁺Gr1⁻ macrophage population (I) analyzed in cytospin preparations; <i>n</i> = 3. The gating strategy is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s011" target="_blank">Figure S11</a> A. Note: <i>n</i> refers to the number of mice, per point per condition. <i>*p</i>≤0.05; <i>**p</i><0.005; <i>***p</i><0.0005. All data used to generate the histograms can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002002#pbio.1002002.s012" target="_blank">Data S1</a>.</p