scholarly journals SIRT3 Sumoylation Contributes to Chemoresistance in AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3929-3929
Author(s):  
Jiao MA ◽  
Bin Liu ◽  
Dan Yu ◽  
Wayne Tam ◽  
Jianmin Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Superoxide Dismutase ◽  
Hematopoietic Cells ◽  
Mitochondrial Metabolism ◽  
Ros Generation ◽  
Ros Production ◽  
Wild Type ◽  
Low Level ◽  
Mitochondrial Ros ◽  
Anti Oxidant

Abstract Acute myeloid leukemia (AML) is a clonal disease originated from a rare population of malignant hematopoietic cells, called leukemic stem cells (LSCs), which is not only often resistant to standard chemotherapies, but also the major cause of relapse and eventual death of AML patients. The five-year survival of AML keeps as low as 27% for the last few decades. LSCs possess unique metabolism profiles such as higher rates of oxidative phosphorylation, and dependence on fatty acid oxidation for survival, which is distinct from normal hematopoietic cells, and, as a consequence, relatively low level of reactive oxygen species (ROS), a critical regulator for stemness maintenance. Therefore, targeting mitochondrial metabolism, especially ROS, may be a promising strategy to improve chemotherapy outcome for AML. We have previously found in hepatocarcinoma cells that SUMOylation is one of the important post-translational modifications for a variety of cellular proteins, and is capable of regulating the enzymatic activity of some key mitochondrial enzymes involved in the metabolic control, one example of which is SIRT3, a NAD+-dependent protein deacetylase. SIRT3 is reported to influence cellular metabolism and downregulate ROS generation by deacetylating mitochondrial anti-oxidant enzymes. The targets of SIRT3 include superoxide dismutase 2 (SOD2), manganese superoxide dismutase (MnSOD) and isocitrate dehydrogenase 2 (IDH2), which have been shown closely related to leukemogenesis. Since sophisticated regulation of ROS production is required for the maintenance of LSCs, we reproduced SUMOylation of SIRT3, and investigated its role in the mitochondrial metabolism in AML. In fact, SIRT3 SUMOylation at lysine 288 was also found in AML cells. To reveal the consequences of SIRT3 SUMOylation in AML, we constructed a plasmid expressing SIRT3-K288R that fails to be SUMOylated in AML cells. As a result, AML cells expressing SIRT3-K288R protected AML cells from as shown by apoptotic assays and quantitation of activated caspase 3 via reduction of not only total but also mitochondrial ROS production under chemotherapeutic agent-induced cell death comparing to those transfected with vector or overexpressing wild type SIRT3. To further investigate the role of SIRT3 de-SUMOylation in AML, we examined the influence of mitochondrial metabolism and anti-oxidant enzymes by SIRT3-K288R. SIRT3-K288R significantly downregulated the acetylation of mitochondrial anti-oxidant enzymes, such as SOD2, leading to decreased NADP/NADPH ratio and increased GSH/GSSG ratio. SIRT3 de-SUMOylation enhanced OCR but impaired ECAR under both basic and cytarabine treated conditions. We analyzed 18 primary AML samples to evaluate the correlation among SIRT3 SUMOylation, ROS level and chemoresistance. As we expected, low level of SIRT3 SUMOylation correlates with low cellular ROS level in both bulk AML and CD34+CD38- AML stem cells, and less sensitivity to cytarabine. Furthermore, MV4-11 cells bearing control vector, wild type SIRT3 or SIRT3-K288R were engrafted in NSG mice. Cytarabine was administered to the xenografts to evaluate the chemoresistance in these cell line-derived xenograft (CDX) mouse models. Consistent to the in vitro data, SIRT3-K288R reduced total and mitochondrial ROS in vivo, resulted in enhanced leukemogenesis and impaired survival. Taken together, our study showed that SIRT3 can be SUMOylated in AML. De-SUMOylation enhances SIRT3 deacetylase activity, and contribute to the chemoresistance of AML cells via altered mitochondrial metabolism and reduced ROS generation. Thus, SIRT3 and its de-SUMOylase can be utilized as potential therapeutic targets to improve AML chemotherapy. Disclosures No relevant conflicts of interest to declare.

Abstract P241: Calpain-1 Is Increased in Mitochondria and Contributes to Mitochondrial ROS Generation in High Glucose--Stimulated Endothelial Cells and Endothelial Dysfunction in Mouse Models of Diabetes

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Qing Zhao ◽  
Futian Tang ◽  
Limei Shan ◽  
Inga Cepinskas ◽  
Gedas Cepinskas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
High Glucose ◽  
Aortic Ring ◽  
Ros Generation ◽  
Type I ◽  
Ros Production ◽  
Diabetic Mice ◽  
Calpain Activity ◽  
Over Expression ◽  
Mitochondrial Ros

Objectives: Elevated levels of reactive oxygen species (ROS) are the initial source of endothelial dysfunction in diabetes. Calpain has been implicated in diabetic vascular complications. The present study was to investigate the role of calpain in mitochondrial ROS generation in endothelial cells and vascular dysfunction in diabetic mice. Methods: Endothelial cells cultured from human umbilical vein (HUVEC) were stimulated with high glucose. Calpain activity and protein were determined in mitochondria of HUVEC. Intracellular and mitochondrial ROS generation as well as apoptosis were measured. Type I diabetic OVE 26 mice and type II diabetic db/db mice with calpastatin over-expression (OVE26/CAST and db/db-CAST) were generated, respectively. Type I diabetes was also induced in both wild-type and Tg-CAST mice by injection of streptozocin (STZ). The endothelium-dependent relaxation of aortic ring was measured. Results: High glucose significantly increased calpain-1 protein, calpain activity and ROS generation in mitochondria of HUVEC. Pharmacological inhibition of calpain or over-expression of calpastatin abrogated high glucose-induced intracellular ROS production, mitochondrial ROS generation and apoptosis in HUVEC. Incubation of isolated mitochondria with calpain-1 protein significantly induced its ROS generation and the membrane potential. In diabetic mice, calpain activity was induced in aortic vessels, which correlated with an increase in ROS production and protein tyrosine nitration. Over-expression of calpastatin prevented calpain activity, reduced ROS production and inhibited protein tyrosine nitration in diabetic mice. Aortic ring segments from diabetic mice exhibited a significant reduction in vascular relaxation to acetylcholine, which was reversed by over-expression of calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice. Conclusions: This study has demonstrated a novel role of calpain in mitochondrial ROS generation, which contributes to apoptosis in endothelial cells during hyperglycemia. Thus, over-expression of calpastatin inhibits reduces ROS production and ameliorates endothelium-dependent vascular dysfunction in mouse models of diabetes.

Abstract P311: Blocking Succinate Release Enhances Mitochondrial Reactive Oxygen Species Generation And Worsens Ischemia-reperfusion Injury

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Alexander S Milliken ◽  
Sergiy M Nadtochiy ◽  
Paul S Brookes
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Reactive Oxygen Species Generation ◽  
Ros Generation ◽  
Ros Production ◽  
Succinate Oxidation ◽  
Fluorescence Measurements ◽  
Mitochondrial Ros ◽  
The Impact ◽  
Infarction Heart

Succinate is a metabolite that plays a central role in ischemia-reperfusion (IR) injury,which is relevant to myocardial infarction (heart attack) and stroke. Succinateaccumulates during ischemia and is rapidly consumed at reperfusion driving reactiveoxygen species (ROS) generation at complex-I (Cx-I) and III of the mitochondrial electrontransport chain. This ROS production triggers cell-death, leading to tissue necrosis.Although succinate oxidation has been extensively studied and exploited as a noveltherapeutic target, only 1/3 of the succinate accumulated in ischemia is oxidized atreperfusion, with the remaining 2/3 being released from the cell via monocarboxylatetransporter 1 (MCT1). Extracellular succinate is thought to be pro-inflammatory, and ithas been proposed that preventing succinate release may be therapeutically beneficial.To determine the impact of preventing succinate release on IR injury, we comparedfunctional recovery (i.e. rate x pressure product, RPP) and infarction (i.e. tissue necrosis)of Langendorff perfused mouse hearts treated with an MCT1 inhibitor, AR-C155858,versus vehicle control. This revealed that succinate retention worsens IR injury (i.e.increased infarction and decreased functional recovery) likely due to increased ROS. Totest this hypothesis, we utilized a Langendorff apparatus positioned within aspectrofluorimeter, which permits real-time fluorescence measurements in beatingmouse hearts. Using the mitochondria targeted superoxide probe, MitoSOX red tomeasure ROS production at reperfusion + AR-C155858, demonstrated that succinateretention leads to enhanced mitochondrial ROS generation at the onset of reperfusion.Overall, these results suggest that inhibiting succinate release in the context of IR injurymay not be a viable therapeutic approach, regardless of any downstream anti-inflammatory effects.

The screening of albumin as a key serum component to prevent neutrophil extracellular traps release by selectively inhibiting mitochondrial ROS generation

2020 ◽  
Author(s):  
Yue Zheng ◽  
Yuanfeng Zhu ◽  
Xin Liu ◽  
Hang Zheng ◽  
Yongjun Yang ◽  
...  
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Extracellular Dna ◽  
Organ Injury ◽  
Ros Generation ◽  
Ros Production ◽  
Serum Free ◽  
Extracellular Traps ◽  
Mitochondrial Ros ◽  
Serum Component ◽  
Microbial Defense

Neutrophil extracellular traps (NETs) are extracellular DNA webs released from neutrophils to mediate host anti-microbial defense. As NETs could also induce thrombosis and cause organ injury, their release should be strictly controlled. However, it is not well understood about the intrinsic mechanisms that prevent unfavorable NETs. Herein, an accidental finding of NETs release from human peripheral neutrophils was firstly described in serum free culture, and it was also determined as a conserved effect for serum to prevent NETs. In contrast to canonical NETs induced by phorbol-12-myristate-13-acetate (PMA), NETs formation by serum free culture was rapid and without prevalent NETosis. Next, albumin was screened out as a key serum component that mediated the suppression of NETs. Moreover, NETs induced upon serum or albumin deficiency were independent of the canonical pathway that involves NOX2 activation and cytosol ROS production. Instead, the generation of mitochondrial ROS (mtROS) was upregulated to promote NETs release. Albumin exhibited mtROS scavenging activity and thus inhibited NETs. Serum free culture also induces the release of NET-bound oxidized mtDNA which stimulated IFN-β production. Overall, our research provides new evidences that characterize the NETs production in serum free culture and determine the mechanisms of serum albumin to inhibit NETs.

Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production

2007 ◽  
Vol 293 (3) ◽  
pp. R1159-R1168 ◽  
Author(s):  
Florian L. Muller ◽  
Wook Song ◽  
Youngmok C. Jang ◽  
Yuhong Liu ◽  
Marian Sabia ◽  
...  
Keyword(s):  
Muscle Mass ◽  
Muscle Atrophy ◽  
Common Factor ◽  
Skeletal Muscle Atrophy ◽  
Ros Generation ◽  
Ros Production ◽  
Muscle Mitochondria ◽  
Mitochondrial Ros ◽  
Hindlimb Muscle ◽  
Old Mice

Reactive oxygen species (ROS), especially mitochondrial ROS, are postulated to play a significant role in muscle atrophy. We report a dramatic increase in mitochondrial ROS generation in three conditions associated with muscle atrophy: in aging, in mice lacking CuZn-SOD ( Sod1−/−), and in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). ROS generation in muscle mitochondria is nearly threefold higher in 28- to 32-mo-old than in 10-mo-old mice and is associated with a 30% loss in gastrocnemius mass. In Sod1−/− mice, muscle mitochondrial ROS production is increased >100% in 20-mo compared with 5-mo-old mice along with a >50% loss in muscle mass. ALS G93A mutant mice show a 75% loss of muscle mass during disease progression and up to 12-fold higher muscle mitochondrial ROS generation. In a second ALS mutant model, H46RH48Q mice, ROS production is approximately fourfold higher than in control mice and is associated with a less dramatic loss (30%) in muscle mass. Thus ROS production is strongly correlated with the extent of muscle atrophy in these models. Because each of the models of muscle atrophy studied are associated to some degree with a loss of innervation, we were interested in determining whether denervation plays a role in ROS generation in muscle mitochondria isolated from hindlimb muscle following surgical sciatic nerve transection. Seven days postdenervation, muscle mitochondrial ROS production increased nearly 30-fold. We conclude that enhanced generation of mitochondrial ROS may be a common factor in the mechanism underlying denervation-induced atrophy.

scholarly journals Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cells in vitro

2016 ◽  
Vol 473 (22) ◽  
pp. 4255-4270 ◽  
Author(s):  
Alaa Shafie ◽  
Mingzhan Xue ◽  
Guy Barker ◽  
Daniel Zehnder ◽  
Paul J. Thornalley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Renal Failure ◽  
Embryonic Stem Cells ◽  
Copy Number ◽  
Embryonic Stem ◽  
Vascular Complications ◽  
Wild Type ◽  
Gene Trapping ◽  
Low Level ◽  
Glyoxalase 1

Glyoxalase 1 (Glo1) is a cytoplasmic enzyme with a cytoprotective function linked to metabolism of the cytotoxic side product of glycolysis, methylglyoxal (MG). It prevents dicarbonyl stress — the abnormal accumulation of reactive dicarbonyl metabolites, increasing protein and DNA damage. Increased Glo1 expression delays ageing and suppresses carcinogenesis, insulin resistance, cardiovascular disease and vascular complications of diabetes and renal failure. Surprisingly, gene trapping by the International Mouse Knockout Consortium (IMKC) to generate putative Glo1 knockout mice produced a mouse line with the phenotype characterised as normal and healthy. Here, we show that gene trapping mutation was successful, but the presence of Glo1 gene duplication, probably in the embryonic stem cells (ESCs) before gene trapping, maintained wild-type levels of Glo1 expression and activity and sustained the healthy phenotype. In further investigation of the consequences of dicarbonyl stress in ESCs, we found that prolonged exposure of mouse ESCs in culture to high concentrations of MG and/or hypoxia led to low-level increase in Glo1 copy number. In clinical translation, we found a high prevalence of low-level GLO1 copy number increase in renal failure where there is severe dicarbonyl stress. In conclusion, the IMKC Glo1 mutant mouse is not deficient in Glo1 expression through duplication of the Glo1 wild-type allele. Dicarbonyl stress and/or hypoxia induces low-level copy number alternation in ESCs. Similar processes may drive rare GLO1 duplication in health and disease.

scholarly journals TGF-β1 induces senescence of bone marrow mesenchymal stem cells via increase of mitochondrial ROS production

2014 ◽  
Vol 14 (1) ◽  
pp. 21 ◽  
Author(s):  
Junfang Wu ◽  
Jie Niu ◽  
Xiaopeng Li ◽  
Xianwei Wang ◽  
Zhikun Guo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Ros Production ◽  
Mitochondrial Ros ◽  
Marrow Mesenchymal Stem Cells ◽  
Tgf Β1

Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Xiaoling Zhang ◽  
June Li ◽  
Daniel P. Sejas ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Premature Senescence ◽  
Wild Type ◽  
Cell Cycle Profile ◽  
Cycle Arrest ◽  
Associated Proteins

Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc–/– BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin–) ScaI– c-kit+ cells and more primitive Lin– ScaI+ c-kit+ cells in Fancc–/– BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc–/– LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc–/– LTBMC cells stained intensely for SA-β-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21WAF1/CIP1. Taken together, these results suggest that reoxygenation induces premature senescence in Fancc–/– BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.

Abstract 210: Thioredoxin-2 Inhibits Mitochondrial Ros Generation And Ask1 Activity To Maintain Cardiac Function

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Wang Min ◽  
Qunhua Huang
Keyword(s):  
Heart Failure ◽  
Cardiac Function ◽  
Dilated Cardiomyopathy ◽  
Mitochondrial Dysfunction ◽  
Ros Generation ◽  
Contractile Dysfunction ◽  
Ros Production ◽  
Atp Production ◽  
Mitochondrial Ros ◽  
Cellular Apoptosis

Background: Increasing evidence indicates that mitochondrial-derived reactive oxygen species (ROS) and cellular apoptosis contribute to the pathogenesis of cardiac dysfunction. Mitochondrial thioredoxin (Trx2) is a key protein regulating cellular redox and survival, However, but its role in normal cardiac growth has not been determined. Methods and Results: We have generated cardiac-specific Trx2 knockout mice (Trx2-cKO) to determine the physiological importance of the Trx2 system in the heart. Trx2-cKO mice developed a spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, fibrosis, reduced ventricular wall thickness, and progressive contractile dysfunction, resulting in death due to heart failure by 4 months of age. Cardiac changes in Trx2-cKO mice were accompanied by disruption of mitochondrial integrity and function, as evident by alterations in mitochondrial number, ultrastructure, membrane potential and ATP production. Increases in ASK1 signaling and ROS production preceded mitochondrial damage, cellular apoptosis and contractile dysfunction in both Trx2-cKO hearts and isolated cardiomyocytes. Moreover, deletion of ASK1 attenuates ROS production, mitochondrial dysfunction and cellular apoptosis in Trx2-deficient cardiomyocytes. These data indicate that ASK1 is a major target of Trx2 and that activation of ASK1 is causally associated with mitochondrial dysfunction, ROS production and cellular apoptosis. We also detected reduced Trx2 expression and increased ASK1 activity in human hearts from patients with cardiomyopathy, suggesting that this mechanism is clinically important. Conclusion: Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial ROS production and ASK1-dependent apoptosis. These results suggest that pharmacological inhibition of ASK1 may provide a therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure.

Thrombopoietin (TPO) Regulates HIF-1α Level through Generation of Mitochondrial Reactive Oxygen Species (ROS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3145-3145
Author(s):  
Kozue Yoshida ◽  
Keita Kirito ◽  
Kenneth Kaushansky ◽  
Norio Komatsu
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mitochondrial Function ◽  
Leukemic Cell ◽  
Complex Iii ◽  
Ros Generation ◽  
Ros Production ◽  
Hematopoietic Stem ◽  
Respiratory Complexes ◽  
Mitochondrial Respiratory

Abstract Hypoxia inducible factor (HIF)-1 is a master transcriptional regulator for adaptation of cells to hypoxia. In addition to hypoxic responses, HIF-1 also plays an important role in the development of hematopoietic stem cells. Genetic deletion of β subunit of HIF-1 causes impairment of hematopoiesis. Culture of hematopoietic stem cells under hypoxic condition induces elevation of HIF-1α , another subunit of HIF-1, and subsequently enhances the growth of these cells. In our previous work we found that thrombopoietin (TPO), an important and non-redundant cytokine required for normal stem cell development, induces HIF-1α elevation in the TPO-dependent human leukemic cell line UT-7/TPO and in Sca-1+/c-kit+/Gr-1- cells (Kirito, K. et.al. Blood 2005). Under normoxic conditions HIF-1α is hydroxylated on proline residues by prolyl hydroxylase (PHD), which leads to its recognition by the von Hippel-Lindau tumor suppressor protein (pVHL), leading to degradation of HIF-1α . Hypoxia inhibits PHD function, blocking ubiquitination of HIF-1α , stabilizing the protein. We found that TPO controls stability of HIF-1α even under normoxic conditions. However, the mechanism by which TPO controls the stability of the protein remains unclear. Recently, several groups have reported that mitochondrial ROS play crucial roles in stabilization of HIF-1α in response to hypoxia. Disruption of mitochondrial function, either by interfering RNA against complex III of the mitochondrial electron transport chain or genetic elimination of cytochrome c, completely abolished the hypoxia-induced HIF-1α response. Based on these findings we hypothesized that ROS might be involved in TPO-induced HIF-1α elevation. To examine our hypothesis, we first tested whether TPO induced ROS production in UT-7/TPO cells using 2′, 7′-dichlorofluorescein diacetate, a redox sensitive fluorescence dye, and found that the hormone clearly induced ROS production in these cells. Next, we analyzed whether TPO-induced ROS generation is required for accumulation of HIF-1α . Pre-treatment of UT-7/TPO cells with the ROS scavenger catalase completely blocked HIF-1α elevation after TPO treatment. Furthermore, diphenylene iodinium (DPI), an inhibitor for ROS generating flavoenzymes including mitochondrial respiratory complexes, also inhibited the effects of TPO on HIF-1α levels. These results indicate that TPO induced HIF-1α activation is mediated by ROS production. To study the molecular pathway(s) by which TPO affects ROS, we tested the effects of ROS blockade on several known TPO-responsive signaling molecules; neither DPI nor catalase affected the activation of JAK2, STAT5, p38-MAPK or p42/p44-ERK induced by TPO, although AKT activation was blocked. Moreover, LY294002, an inhibitor of PI3-kinase and its activation of AKT also blocked of the HIF-1α response to TPO. Finally, inhibition of mitochondrial function in UT-7/TPO cells with rotenone or oligomycin also inhibited TPO-dependent accumulation of HIF-1α without affecting Jak2 activation. In conclusion, we found that TPO regulates HIF-1α levels through activation of ROS generation within mitochondrial respiratory complexes. We speculate that TPO mimics hypoxia by induction of ROS generation at mitochondria and subsequent elevation of HIF-1α , and regulates important genes for metabolisms and survival of hematopoietic stem cells.

HIF-1 Prevents Hematopoietic Cells from Cell Damage by Overproduction of Mitochondrial ROS after Cytokine Stimulation through Induction of PDK-1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2435-2435 ◽  
Author(s):  
Keita Kirito ◽  
Kozue Yoshida ◽  
Yongzhen Hu ◽  
Qiao Qiao ◽  
Kumi Sakoe ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Pyruvate Dehydrogenase ◽  
Hematopoietic Cells ◽  
Feedback Mechanism ◽  
Dependent Manner ◽  
Ros Production ◽  
Ros Scavenging ◽  
Mitochondrial Ros ◽  
Primary Mouse ◽  
Cytokine Stimulation

Abstract Reactive oxygen species (ROS) play numerous functions in both physiological and pathological hematopoiesis. Although ROS are generated in multiple cellular compartments and by multiple enzymes, the majority is derived from mitochondria coupled with glucose metabolism. To detoxify the cytotoxicity of ROS, cells have several ROS scavenging mechanisms. In addition, recent studies have revealed that hypoxia inducible factor-1 (HIF-1) contributes to the reduction of mitochondrial ROS production through the induction of pyruvate dehydrogenase kinase-1 (PDK-1) in response to hypoxia. PDK-1 phosphorylates and inactivates pyruvate dehydrogenase (PDH) E1α, which converts pyruvate to acetylcoenzyme A. Thus, the induction of PDK-1 by HIF-1 blocks the entry of pyruvate into the tricarboxylic acid (TCA) cycle and subsequently suppresses mitochondrial ROS production under hypoxic conditions. In addition to hypoxia, mitochondrial ROS production is also enhanced by cytokine stimulation. Previously, we showed that thrombopoietin (TPO) induced the generation of mitochondrial ROS and that mitochondrial ROS induced by TPO promoted the elevation of HIF-1α, a subunit of HIF-1, both in UT-7/TPO cells and in primary mouse progenitor cells. Based on these observations, we speculated that TPO-activated HIF-1 works as a feedback mechanism to block overproduction of ROS, which may be harmful to hematopoietic cells, by controlling PDK-1 expression. To investigate this notion, we first analyzed ROS production kinetics after TPO stimulation using UT-7/TPO cells. ROS production was gradually increased, peaking at 48 hr, and then decreasing by 72 hr after TPO stimulation. PDK-1 expression increased 48 hr after TPO treatment before ROS dropped. Phosphorylation of PDH-E1α was enhanced by TPO in the same fashion. To confirm that PDK-1 induction by TPO contributes to reduction of ROS, we treated UT-7/TPO cells with a PDK-1 inhibitor, dichloroacetate (DCA). As expected, DCA blocked the phosphorylation of PDH-E1α and induced sustained ROS production. Furthermore, DCA treatment resulted in increased apoptotic cell ratio after TPO stimulation. These results support our hypothesis that PDK-1 works to prevent overproduction of ROS after TPO stimulation and also suggest that sustained production of ROS after cytokine stimulation might be toxic to hematopoietic cells. Next, we analyzed whether HIF-1 is required for this process. Echinomycin, a specific inhibitor of HIF-1, blocked PDK-1 elevation by TPO in a dose-dependent manner. For further study, we established a stable HIF-1α knockdown system in UT-7/TPO cells using siRNA. TPO failed to induce PDK-1 expression in these clones. Concomitantly, ROS levels in HIF-1α knockdown cells remained high, compared to parental cells stimulated with TPO for 72 hr. We also found sustained activation of p38 MAPK and JNK in HIF-1α knockdown clones after TPO stimulation. Taken together, our observations suggest that TPO-induced activation of HIF-1 and subsequent induction of PDK-1 is an important mechanism to prevent overproduction of mitochondrial ROS, a secondary product of glucose metabolism. This feedback mechanism may be critical for protecting hematopoietic cells from DNA damage by ROS production after cytokine stimulation.

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