Imaging of metabolic activity adaptations to UV stress, drugs and differentiation at cellular resolution in skin and skin equivalents – Implications for oxidative UV damage

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First two unrelated cases of isolated sedoheptulokinase deficiency: A benign disorder?

We present the first two reported unrelated patients with an isolated sedoheptulokinase (SHPK) deficiency. The first patient presented with neonatal cholestasis, hypoglycemia, and anemia, while the second patient presented with congenital arthrogryposis multiplex, multiple contractures, and dysmorphisms. Both patients had elevated excretion of erythritol and sedoheptulose, and each had a homozygous nonsense mutation in SHPK. SHPK is an enzyme that phosphorylates sedoheptulose to sedoheptulose-7-phosphate, which is an important intermediate of the pentose phosphate pathway. It is questionable whether SHPK deficiency is a causal factor for the clinical phenotypes of our patients. This study illustrates the necessity of extensive functional and clinical workup for interpreting a novel variant, including nonsense variants

Sedoheptulose kinase regulates glucose metabolism and immune cell polarization

Die Regulierung von Makrophagen und des zellulären Metabolismus durch verschiedene Arten von Stress ist noch immer nicht zur Gänze verstanden. In dieser Arbeit haben wir versucht, durch gezieltes testen von rund 200 unterschiedlichen Kinasen, neue Regulationswege aufzuzeigen, die eine mögliche Rolle bei der Makrophage Aktivierung spielen. Dieser Screen hat zur Entdeckung einer Sedoheptulose Kinase geführt, die, wie wir in weiterer Folge zeigen konnten, eine wichtige Funktion im Pentose Phosphate Pathway bzw. Glukose Metabolismus inne hält. Diese Kinase wird während der Makrophagen Aktivierung endogen transkriptionell sowie post-translational reguliert. Wir konnten dies im Menschen sowie in Mäusen zeigen. Mittels Gegenregulation durch Überexpression haben wir die Folgen der endogenen Sedoheptulose Kinase Regulation in Makrophagen untersucht. Wir zeigen, dass diese Regulation den Ausgang des Aktivierungsprozesses bestimmt; Makrophagen zeigten eine verminderte NFB Aktivierung, eine mildere Induktion von pro-inflammatorischen Zytokine und keine signifikante Erhöhung von freien Radikalen nach Stimulierung mit bakteriellem Endotoxin. Zusammenfassend haben wir gezeigt, dass eine Sedoheptulose Kinase existiert, die einerseits den zellulären Metabolismus reguliert und andererseits eine fundamentale Rolle in der Immune-Zell-Aktivierung hat. Diese Dualität lässt den Schluss zu, dass metabolische Adaption während der Differenzierung von Makrophagen essentiell ist.Regulation of the innate immune response and the metabolic requirement of cells to deal with environmental stressors is complex and remains poorly understood. Here we screened 199 kinases in endotoxin-stimulated macrophages to identify potential candidates involved in immune regulation. This screen revealed a novel carbohydrate kinase, which we characterized as a mammalian Sedoheptulose kinase (Carkl/Shpk) and as novel regulator of the Pentose Phosphate Pathway. Macrophage activation resulted in Shpk mRNA down-regulation and post-translational modification. This regulation was required to polarize macrophages into effector cells as overexpression of Shpk resulted in improper macrophage activation. Collectively, our data identify the essential function of Shpk in metabolism and for the first time report on an interface fundamental for coordinating bio-energetic adjustments in shaping immune cell function. This functional duality of Shpk underscores the notion that the apparatus involved in the polarization of innate immune cells, is intimately linked to basic cellular metabolism.submitted by Arvand HaschemiAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2010OeBB(VLID)170076

Haschemi A. Sedoheptulose kinase regulates cellular carbohydrate metabolism by sedoheptulose 7-phosphate supply. Biochem Soc Trans (2013

Abstract Dynamic carbon re-routing between catabolic and anabolic metabolism is an essential element of cellular transformation associated with tumour formation and immune cell activation. Such bioenergetic adaptations are important for cellular function and therefore require tight control. Carbohydrate phosphorylation has been proposed as a rate-limiting step of several metabolic networks. The recent identification of a sedoheptulose kinase indicated that free sedoheptulose is a relevant and accessible carbon source in humans. Furthermore, the bioavailability of its phosphorylated form, sedoheptulose 7-phosphate, appears to function as a rheostat for carbon-flux at the interface of glycolysis and the pentose phosphate pathway. In the present paper, we review reports of sedoheptulose metabolism, compare it with glucose metabolism, and discuss the regulation of sedoheptulose kinase as mechanism to achieve bioenergetic reprogramming in cells

Macrophage metabolic regulation in atherosclerotic plaque

International audienceMetabolism plays a key role in controlling immune cell functions. In this review, we will discuss the diversity of plaque resident myeloid cells and will focus on their metabolic demands that could reflect on their particular intraplaque localization. Defining the metabolic configuration of plaque resident myeloid cells according to their topologic distribution could provide answers to key questions regarding their functions and contribution to disease development

Carbon monoxide induced PPARγ SUMOylation and UCP2 block inflammatory gene expression in macrophages.

Carbon monoxide (CO) dampens pro-inflammatory responses in a peroxisome proliferator-activated receptor-γ (PPARγ) and p38 mitogen-activated protein kinase (MAPK) dependent manner. Previously, we demonstrated that CO inhibits lipopolysaccharide (LPS)-induced expression of the proinflammatory early growth response-1 (Egr-1) transcription factor in macrophages via activation of PPARγ. Here, we further characterize the molecular mechanisms by which CO modulates the activity of PPARγ and Egr-1 repression. We demonstrate that CO enhances SUMOylation of PPARγ which we find was attributed to mitochondrial ROS generation. Ectopic expression of a SUMOylation-defective PPARγ-K365R mutant partially abolished CO-mediated suppression of LPS-induced Egr-1 promoter activity. Expression of a PPARγ-K77R mutant did not impair the effect of CO. In addition to PPARγ SUMOylation, CO-activated p38 MAPK was responsible for Egr-1 repression. Blocking both CO-induced PPARγ SUMOylation and p38 activation, completely reversed the effects of CO on inflammatory gene expression. In primary macrophages isolated form C57/BL6 male mice, we identify mitochondrial ROS formation by CO as the upstream trigger for the observed effects on Egr-1 in part through uncoupling protein 2 (UCP2). Macrophages derived from bone marrow isolated from Ucp2 gene Knock-Out C57/BL6 mice (Ucp2(-/-)), produced significantly less ROS with CO exposure versus wild-type macrophages. Moreover, absence of UCP2 resulted in a complete loss of CO mediated Egr-1 repression. Collectively, these results indentify p38 activation, PPARγ-SUMOylation and ROS formation via UCP2 as a cooperative system by which CO impacts the inflammatory response

CO increases ROS levels via UCP2 to inhibit Egr-1 expression.

<p>(A) Bone-marrow derived macrophages from wild-type (open bars) or <i>Ucp2</i>^−/− (filled bars) mice were treated with CO (250 ppm) and ROS formation was measured with MitoSOX at indicated times by FACS analysis. MitoSOX is specific for mitochondria-derived ROS. Left, the basal effect of UCP2 loss was compared to control cells. Right, we show the effect of CO on ROS formation in <i>Ucp2</i>^−/− and wild-type macrophages. (C) Bone-marrow derived macrophages from wild-type or <i>Ucp2</i>^−/− mice were pretreated with 250 ppm CO or Air for 3 hours, prior to incubation with LPS (10 ng/ml) for indicated times. Egr-1 protein expression was analyzed by western blotting and β-actin was used as a loading control. (D) The bar graph represents densitometry analysis of western blots after 1 hr LPS stimulation in the presence or absence of CO. Depicted western blot is one of three representative experiments. Bar graphs indicate mean fold change ± SEM of three independent experiments (^*P<0.05 and ^**P<0.001).</p

CO treatment enhances SUMOylation of PPARγ.

<p>(<b>A</b>) 293FT cells were transfected in 10 cm dishes with mixtures of expression plasmids, consisting of 2.5 µg HA-PPARγ, 5.5 µg myc-SUMO-1, and 2.0 µg of PIAS1 as indicated. 24 hours after transfection, cells were incubated for 12–18 hours with Rosiglitazone and/or CO (250 ppm) followed by immunoprecipitation of PPARγ (left panel) or SUMO-1 (right panel) from cell lysates and immunoblotting for HA-tag. The western blots are representative of three independent experiments. (<b>B</b>) 293FT cells transfected with the expression plasmids for HA-PPARγ, myc-SUMO-1 and PIAS1 were incubated for 12–18 hours with or without CO (250 ppm) in the presence or absence of N-Acetylcysteine (10 mM). SUMOylated PPARγ was calculated as the ratio of SUMOylated PPARγ versus total PPARγ form pull-down assays. Shown are mean +/− SEM densitometry values of three independent experiments.</p

Natural killer T cell dysfunction in CD39-null mice protects against concanavalin A-induced hepatitis

Concanavalin A (Con A)–induced injury is an established natural killer T (NKT) cell–mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A–stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5’-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A–induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-γ when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. CONCLUSION: CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A–induced hepatitis. This study illustrates a further role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury