Die Regulation erythrozytären Überlebens und suizidalen Zelltodes

The life span of erythrocytes is tightly regulated. Therefore, a mechanism is required to remove senescent or damaged erythrocytes without rupture of the cell membrane resulting in the release of hemoglobin which may impair kidney function. The mechanism of suicidal erythrocyte death is called eryptosis and shares similarities with apoptosis of nucleated cells such as exposure of phosphatidylserine at the cell surface, increase in cytosolic Ca2+ concentration, blebbing of the membrane, cell shrinkage and enzymatic degradation of the cytoskeletton. The cell shrinkage of eryptotic cells is mediated by a Ca2+-dependent K+ channel, the Gardos channel. Its activation by an increase in the intracellular Ca2+ concentration results in the efflux of K+, Cl- and osmotically obliged water. Phosphatidylserine-exposing erythrocytes are rapidly engulfed by macrophages equipped with phosphatidylserine receptors and degraded. Excessive eryptosis may lead to anemia, the pathological lack of erythrocytes. The present study was performed to elucidate mechanisms regulating erythrocyte survival and suicidal cell death. First, the functional significance of the Gardos channel for suicidal erythrocyte death and erythrocyte clearance was studied. Furthermore, the protective role of Gardos channels during exposure to hemolytic toxins was elucidated. Both issues were addressed by experiments performed in mice lacking the Ca2+-dependent K+ channel KCa3.1, the Gardos channel, and their wildtype littermates. Using patch-clamp recording, flow cytometry, in vitro hemolysis and a mouse sepsis model, it is shown that Gardos channel activity and Gardos effect delay hemolysis of injured erythrocytes and, thus, prevent the disastrous filtration of released hemoglobin into the renal tubular system. In a further series of experiments, the role of the NO/cGMP pathway, a powerful regulator of the life span of a variety of cells, for erythrocyte survival is investigated. Flow cytometry, Western Blotting, hematological counts, and MRI imaging were used to illustrate by means of a cGKI-deficient mouse model that cGKI is a mediator of erythrocyte survival in vitro and in vivo. Moreover, the participation of the phosphoinositide-dependent kinase PDK1, a key element in the phosphoinositol-3-kinase signalling pathway, which is involved in the regulation of ion channels, transporters, cell volume and cell survival, in the regulation of suicdal erythrocyte death was studied. Experiments performed in hypomorphic mice with some 20% of normal PDK1 acitivity and their wildtype littermates revealed that PDK1 deficiency is associated with decreased Ca2+ entry into erythrocytes and thus with blunted eryptotic effects of oxidative stress, osmotic shock and chloride removal. Finally, the functional significance of host pathogen interactions for suicidal erythrocyte death was investigated. Using flow cytometry, it could be shown that peptidoglycan, a main component of the bacterial cell wall, is a potent stimulus of eryptosis and thereby impairs erythrocyte survival. Peptidoglycan-induced eryptosis may therefore, at least in part, account for anemia observed in patients with bacterial infections.Die Lebensdauer von Erythrozyten ist genau reguliert. Aus diesem Grund ist ein Mechanismus erforderlich, der es erlaubt, alte oder geschädigte Erythrozyten abzubauen, ohne daß die Zellmembran reißt und Hämoglobin freigesetzt wird, was zu akutem Nierenversagen führen könnte. Ein solcher Mechanismus ist der suizidale Erythrozytentod, der Eryptose genannt wird und Ähnlichkeiten zur Apoptose kernhaltiger Zellen aufweist wie zum Beispiel die Externalisierung von Phosphatidylserin auf der Zelloberfläche, die Zunahme der intrazellulären Calciumkonzentration, das Abknopsen von Membranteilen, Zellschrumpfung sowie der enzymatische Abbau des Zytoskeletts. Die Zellschrumpfung eryptotischer Zellen wird durch einen Ca2+-abhängigen K+-Kanal, den Gardos-Kanal, vermittelt. Dessen Aktivierung durch die Erhöhung der intrazellulären Calciumkonzentration führt zum Verlust von K+, Cl- und osmotisch folgendem Wasser. Phosphatidylserin-exponierende Erythrozyten werden zügig von Makrophagen, die über Phosphatidylserinrezeptoren verfügen, phagozytiert und abgebaut. Exzessiv gesteigerte Eryptose kann zu Anämie, dem krankhaften Mangel an roten Blutkörperchen, führen. Ziel der vorliegenden Arbeit war es, Mechanismen aufzuklären, die erythrozytäres Überleben sowie suidzidales Sterben steuern. Zunächst wurde die funktionelle Bedeutung des Gardos-Kanals für den suizidalen Erythrozytentod und die Klärung der roten Blutkörperchen aus dem Blut untersucht. Ferner wurde die protektive Wirkung der Gardos-Kanäle bei Einwirkung hämolytischer Toxine studiert. Beide Fragestellungen wurden durch Experimente an Mäusen, die den Ca2+-abhängigen K+-Kanal KCa3.1 nicht exprimierten, und ihren Wildtyp-Geschwistern angegangen. Durch Messungen mit der Membranfleckklemme, mit dem Durchflußzytometer, durch in vitro Hämolyse und mittels eines Maus-Sepsis-Modells wurde gezeigt, daß die Gardos-Kanal-Aktivität und der Gardos-Effekt die Hämolyse geschädigter Erythrozyten verzögern und dadurch die potentiell schädliche Filtration des hämolytisch freigesetzten Hämoglobins in das Nierentubulssystem unterbinden. In weiteren Experimenten wurde die Rolle des NO/cGMP-Signalweges, eines bedeutsamen Regulators der Lebensdauer verschiedener Zellen, für das erythrozytäre Überleben untersucht. Mittels Durchflußzytometrie, Western Blotting, Blutbildern sowie Kernspintomographie, wurde an einem cGKI-defizienten Maus-Modell gezeigt, daß die cGKI ein Mediator erythrozytären Überlebens in vitro und in vivo ist. Desweiteren wurde die Beteiligung der Phosphoinositid-abhängigen-Kinase PDK1, eines Schlüsselenzyms des Phosphoinositol-3-kinase-Signalweges, der bei der Regulation von Ionenkanälen, Transportern, des Zellvolumens und –überlebens mitwirkt, an der Steuerung suizidalen Erythrozytentodes analysiert. Experimente an hypomorphen Mäusen mit lediglich 20%-iger PDK1-Aktivität und ihren Wildtyp-Geschwistern ergaben, daß PDK1-Defizienz mit vermindertem Calciumeinstrom in Erythrozyten und daher mit abgeschächten eryptotischen Effekten von oxidativem Streß, osmotischem Schock und der Entfernung extrazellulären Chlorids verbunden ist. Schließlich wurde die funktionelle Signifikanz von Wirt-Erregerbeziehungen für suizidalen Erythrozytentod untersucht. Durchflußzytometrisch konnte gezeigt werden, daß Peptidoglykan, ein wesentlicher Bestandteil bakterieller Zellwände, ein potenter Auslöser von Eryptose ist und dadurch erythrozytäres Überleben beeinträchtigt. Zumindest theoretisch könnte daher Peptidoglykan-induzierte Eryptose zur Anämie von Patienten mit bakteriellen Infektionen beitragen

Beneficial effect of aurothiomalate on murine malaria

<p>Abstract</p> <p>Background</p> <p>Premature death of <it>Plasmodium</it>-infected erythrocytes is considered to favourably influence the clinical course of malaria. Aurothiomalate has previously been shown to trigger erythrocyte death or eryptosis, which is characterized by cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing cells are rapidly cleared from circulating blood. The present study thus tested whether sodium aurothiomalate influences the intraerythrocytic parasite development <it>in vitro </it>and the clinical course of murine malaria <it>in vivo</it>.</p> <p>Methods</p> <p>Human erythrocytes were infected with <it>Plasmodium falciparum </it>BinH <it>in vitro </it>and mice were infected (intraperitoneal injection of 1 × 10⁶parasitized murine erythrocytes) with <it>Plasmodium berghei </it>ANKA <it>in vivo</it>.</p> <p>Results</p> <p>Exposure to aurothiomalate significantly decreased the <it>in vitro </it>parasitemia of <it>P. falciparum</it>-infected human erythrocytes without influencing the intraerythrocytic DNA/RNA content. Administration of sodium aurothiomalate <it>in vivo </it>(daily 10 mg/kg b.w. s.c. from the 8^thday of infection) enhanced the percentage of phosphatidylserine-exposing infected and noninfected erythrocytes in blood. All nontreated mice died within 30 days of infection. Aurothiomalate-treatment delayed the lethal course of malaria leading to survival of more than 50% of the mice 30 days after infection.</p> <p>Conclusions</p> <p>Sodium aurothiomalate influences the survival of <it>Plasmodium berghei</it>-infected mice, an effect only partially explained by stimulation of eryptosis.</p

Azathioprine favourably influences the course of malaria

<p>Abstract</p> <p>Background</p> <p>Azathioprine triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Eryptosis may accelerate the clearance of <it>Plasmodium</it>-infected erythrocytes. The present study thus explored whether azathioprine influences eryptosis of <it>Plasmodium</it>-infected erythrocytes, development of parasitaemia and thus the course of malaria.</p> <p>Methods</p> <p>Human erythrocytes were infected <it>in vitro </it>with <it>Plasmodium falciparum (P. falciparum) </it>(strain BinH) in the absence and presence of azathioprine (0.001 – 10 μM), parasitaemia determined utilizing Syto16, phosphatidylserine exposure estimated from annexin V-binding and cell volume from forward scatter in FACS analysis. Mice were infected with <it>Plasmodium berghei (P. berghei) </it>ANKA by injecting parasitized murine erythrocytes (1 × 10⁶) intraperitoneally. Where indicated azathioprine (5 mg/kg b.w.) was administered subcutaneously from the eighth day of infection.</p> <p>Results</p> <p><it>In vitro </it>infection of human erythrocytes with <it>P. falciparum </it>increased annexin V-binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content (≥ 1 μM) and <it>in vitro </it>parasitaemia (≥ 1 μM). Administration of azathioprine significantly decreased the parasitaemia of circulating erythrocytes and increased the survival of <it>P. berghei</it>-infected mice (from 0% to 77% 22 days after infection).</p> <p>Conclusion</p> <p>Azathioprine inhibits intraerythrocytic growth of <it>P. falciparum</it>, enhances suicidal death of infected erythrocytes, decreases parasitaemia and fosters host survival during malaria.</p

Anti-malarial effect of gum arabic

<p>Abstract</p> <p>Background</p> <p>Gum Arabic (GA), a nonabsorbable nutrient from the exudate of <it>Acacia senegal</it>, exerts a powerful immunomodulatory effect on dendritic cells, antigen-presenting cells involved in the initiation of both innate and adaptive immunity. On the other hand GA degradation delivers short chain fatty acids, which in turn have been shown to foster the expression of foetal haemoglobin in erythrocytes. Increased levels of erythrocyte foetal haemoglobin are known to impede the intraerythrocytic growth of <it>Plasmodium </it>and thus confer some protection against malaria. The present study tested whether gum arabic may influence the clinical course of malaria.</p> <p>Methods</p> <p>Human erythrocytes were <it>in vitro </it>infected with <it>Plasmodium falciparum </it>in the absence and presence of butyrate and mice were <it>in vivo </it>infected with <it>Plasmodium berghei </it>ANKA by injecting parasitized murine erythrocytes (1 × 10⁶) intraperitoneally. Half of the mice received gum arabic (10% in drinking water starting 10 days before the day of infection).</p> <p>Results</p> <p>According to the <it>in vitro </it>experiments butyrate significantly blunted parasitaemia only at concentrations much higher (3 mM) than those encountered <it>in vivo </it>following GA ingestion (<1 μM). According to the <it>in vivo </it>experiments the administration of gum arabic slightly but significantly decreased the parasitaemia and significantly extended the life span of infected mice.</p> <p>Discussion</p> <p>GA moderately influences the parasitaemia and survival of <it>Plasmodium-</it>infected mice. The underlying mechanism remained, however, elusive.</p> <p>Conclusions</p> <p>Gum arabic favourably influences the course of murine malaria.</p

Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU145 prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Recently we have reported membrane androgen receptors-induced apoptotic regression of prostate cancer cells regulated by Rho/ROCK/actin signaling. In the present study we explored the specificity of these receptors and we analyzed downstream effectors controlling survival and apoptosis in hormone refractory DU145-prostate cancer cells stimulated with membrane androgen receptor-selective agonists.</p> <p>Results</p> <p>Using membrane impermeable conjugates of serum albumin covalently linked to testosterone, we show here down-regulation of the activity of pro-survival gene products, namely PI-3K/Akt and NF-κB, in DU145 cells. Testosterone-albumin conjugates further induced FasL expression. A FasL blocking peptide abrogated membrane androgen receptors-dependent apoptosis. In addition, testosterone-albumin conjugates increased caspase-3 and Bad protein activity. The actin cytoskeleton drug cytochalasin B and the ROCK inhibitor Y-27632 inhibited FasL induction and caspase-3 activation, indicating that the newly identified Rho/Rock/actin signaling may regulate the downstream pro-apoptotic effectors in DU145 cells. Finally, other steroids or steroid-albumin conjugates did not interfere with these receptors indicating testosterone specificity.</p> <p>Conclusion</p> <p>Collectively, our results provide novel mechanistic insights pointing to specific pro-apoptotic molecules controlling membrane androgen receptors-induced apoptotic regression of prostate cancer cells and corroborate previously published observations on the potential use of membrane androgen receptor-agonists as novel anti-tumor agents in prostate cancer.</p

Up-regulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB/Akt and PIKfyve

Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive T308A/S473APKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant S318APIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (Ile), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of T308A/S473APKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of S318APIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of Ile following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells

Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo

<p>Abstract</p> <p>Background</p> <p>Membrane androgen receptors (mAR) have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer.</p> <p>Results</p> <p>Using fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a K_Dof 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, <it>in vivo </it>studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors.</p> <p>Conclusion</p> <p>Our results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses <it>in vitro </it>and extensive reduction of tumor incidence <it>in vivo</it>.</p

Phosphate Homeostasis, Inflammation and the Regulation of FGF-23

Fibroblast growth factor 23 (FGF23) is released primarily from osteoblasts/osteocytes in bone. In cooperation with the transmembrane protein Klotho, FGF23 is a powerful inhibitor of 1α 25OH Vitamin D Hydroxylase (Cyp27b1) and thus of the formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). As 1,25(OH)2D3 up-regulates intestinal calcium and phosphate absorption, the downregulation of 1,25(OH)2D3 synthesis counteracts phosphate excess and tissue calcification. FGF23 also directly inhibits renal phosphate reabsorption. Other actions of FGF23 include triggering of cardiac hypertrophy. FGF23 formation and/or release are stimulated by 1,25(OH)2D3, phosphate excess, Ca2+, PTH, leptin, catecholamines, mineralocorticoids, volume depletion, lithium, high fat diet, iron deficiency, TNFα and TGFß2. The stimulating effect of 1,25(OH)2D3 on FGF23 expression is dependent on RAC1/PAK1 induced actin-polymerisation. Intracellular signaling involved in the stimulation of FGF23 release also includes increases in the cytosolic Ca2+ concentration ([Ca2+]i) following intracellular Ca2+ release and store-operated Ca2+ entry (SOCE). SOCE is accomplished by the Ca2+ release-activated calcium channel protein 1 (Orai1) and its stimulator stromal interaction molecule 1 (STIM1). Expression of Orai1, SOCE and FGF23-formation are up-regulated by the proinflammatory transcription factor NFκB. The present brief review describes the cellular mechanisms involved in FGF23 regulation and its sensitivity to both phosphate metabolism and inflammation. The case is made that up-regulation of FGF23 by inflammatory mediators and signaling may amplify inflammation by inhibiting formation of the anti-inflammatory 1,25(OH)2D3