scholarly journals The Role of DOT1L Methyltransferase Activity in Fetal Hematopoiesis

2020 ◽  
Author(s):  
Carrie A. Malcom ◽  
Joanna Piaseka-Srader ◽  
V. Praveen Chakravarthi ◽  
Shaon Borosha ◽  
Anamika Ratri ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mutant Mouse ◽  
Ex Vivo ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Methyltransferase Activity ◽  
Mutant Mouse Line ◽  
Fetal Hematopoiesis

ABSTRACTEarly mammalian erythropoiesis requires the DOT1L methyltransferase. We demonstrated that loss of DOT1L in mutant mice resulted in lethal anemia during midgestation. The molecular mechanisms by which DOT1L regulates embryonic erythropoiesis have not yet been elucidated. In this study, a methyltransferase mutant mouse line (Dot1L-MM) was generated to determine whether the methyltransferase activity of DOT1L is essential for erythropoiesis. Dot1L-MM mice displayed embryonic lethality between embryonic days 10.5 and 13.5, similar to Dot1lL knockout (Dot1L-KO) mice. However, when examined at E10.5, unlike the Dot1L-KO, Dot1L-MM embryos did not exhibit evidence of anemia. In ex vivo hematopoietic differentiation cultures, Dot1L-KO and Dot1L-MM yolk sac (YS) cells both formed reduced numbers of myeloid, and mixed hematopoietic colonies. Erythroid colonies were able to be formed in numbers equal to wildtype embryos. Extensively self-renewing erythroblast (ESRE) cultures were established using YS cells from E10.5 embryos. Dot1L-KO and Dot1L-MM cells expanded significantly less than wild-type cells and exhibited increased cell death. Strikingly, Dot1L-KO and Dot1L-MM cells of YS origin exhibited profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as viability of hematopoietic progenitors. Our results indicate that the methyltransferase activity of DOT1L plays an important role early murine hematopoiesis.

Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Allen M Andres ◽  
Chengqun Huang ◽  
Eric P Ratliff ◽  
Genaro Hernandez ◽  
Pamela Lee ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Wild Type ◽  
Simulated Ischemia ◽  
Selective Targeting ◽  
Cardioprotective Effects ◽  
Mitochondrial Turnover ◽  
First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

scholarly journals X chromosome choice occurs independently of asynchronous replication timing

2005 ◽  
Vol 168 (3) ◽  
pp. 365-373 ◽  
Author(s):  
Joost Gribnau ◽  
Sandra Luikenhuis ◽  
Konrad Hochedlinger ◽  
Kim Monkhorst ◽  
Rudolf Jaenisch
Keyword(s):  
X Chromosome ◽  
Molecular Mechanisms ◽  
Replication Timing ◽  
S Phase ◽  
X Inactivation ◽  
Wild Type ◽  
Gene Deletions ◽  
Asynchronous Replication ◽  
Skewed X Inactivation

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.

PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2393-2393 ◽  
Author(s):  
Rabindranath Bera ◽  
Der-Cherng Liang ◽  
Ming-Chun Chiu ◽  
Ying-Jung Huang ◽  
Sung-Tzu Liang ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Blast Crisis ◽  
K562 Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Megakaryocytic Differentiation ◽  
Deletion Mutations ◽  
Phd Domain

Abstract Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.

Podocalyxin Negatively Regulates Stress Myelopoiesis, Directly Binds Rap-1A and Modulates Its G-CSF-Induced Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1184-1184
Author(s):  
Pan Li ◽  
Rose McGlauflin ◽  
Amanda J Favreau ◽  
Edward Jachimowicz ◽  
Calvin Vary ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Functional Role ◽  
Blood Analysis ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Gm Csf ◽  
Peripheral Blood Analysis ◽  
Specific Deletion

Abstract Podocalyxin (PodxL) is a CD34 family member previously identified to mark hematopoietic stem cells (HSCs) and other progenitor cells. Previously, we discovered PodxL as a potent erythropoietin (EPO) response gene and demonstrated to promote egression of immature reticulocytes from bone marrow into circulation. PodxL is upregulated in several cancers, including myeloid and lymphoid leukemia. Herein, we aim to define the functional role of PodxL in hematopoiesis - specifically myelopoiesis - by employing conditional PodxL knock out (KO) mouse models. Hematopoietic-specific deletion was achieved using Cre mice with a Vav1 driver and myeloid-specific deletion was achieved with Lyzm2 - Cre driver. We confirmed the deletion of exons 3-7 at the gene, transcript and protein levels using PCR, RT-qPCR and western blotting, respectively. Peripheral blood analysis revealed no difference in blood cell lineages for either KO mouse strain. At steady state, colony forming unit-granulocyte/macrophage (CFU-GM) assay also showed no difference between the KO strains and wild type. In order to examine the functional role of PodxL during stress myelopoiesis, PodxL-/- ; Vav1-Cre mice were treated with 5-Fluorouracil (5FU), a chemotherapeutic agent induces myeloablation. Notably, during rebound of neutrophils, the PodxL-/- ; Vav1-Cre mice showed a sharp increase in neutrophil counts at day 12.5, which at later time points reverted to normal levels comparable to wild type mice. Previously, our in silico analyses combined with outcomes from truncated EpoR knock-in alleles had revealed that PodxL is a potential STAT5 transcriptional target. Here, we tested if G-CSF induces PodxL expression in hematopoietic progenitors. In vivo, G-CSF significantly induced PodxL expression four fold. We then tested the role of PodxL in G-CSF induced neutrophil formation in vivo. Both KO strains (Podxl-/-;Vav1-Cre and Podxl-/-;Lyzm2-Cre) and wild type were treated with G-CSF (125ug/kg/day) for 5 days. Peripheral blood analysis revealed increased neutrophil and monocyte levels in the PodxL-/-;Vav1-Cremice. In order to then determine a possible role of PodxL at the progenitor level, CFU-GM assays were performed. PodxL-/- ; lyzm2-Cre mice had increased colony forming capabilities but there was no difference in PodxL-/-;Vav1-Cre mice compared to wild type. Our results imply that PodxL is playing a negative regulatory role in stress myelopoiesis. Interestingly, the deletion of PodxL in hematopoietic progenitors (Vav1-Cre) resulted in enhanced migration of neutrophils, whereas deletion of PodxL in myeloid compartment (Lyzm2-Cre) resulted in decreased neutrophil migration. This may be in part due to a compensatory effect by CD34 in the hematopoietic compartment. To dissect the molecular mechanism of PodxL during stress myelopoiesis, upon in vivo G-CSF treatment, bone marrow derived hematopoietic progenitors were isolated and PodxL protein was immunoprecipitated. LC-MS/MS proteomic analysis was performed to identify the interacting partners with PodxL. Rap-1A, a small GTPase and member of the RAS family, was among the top interacting proteins. Rap-1A has been shown to promote adhesion and migration of myeloid cells. The association of PodxL with Rap-1A was further confirmed in hematopoietic progenitors by immunoprecipitation and western blotting. To determine if the interaction of PodxL directly regulates Rap-1A activity, a GTP-Rap-1A activity assay was performed in response to G-CSF, GM-CSF and IL-3. Rap-1A activity was significantly elevated in hematopoietic progenitors upon G-CSF treatment in PodxL-/-:Vav1-Cre mice compared to wild type, followed by IL3; however, GM-CSF did not affect Rap-1A activity. In conclusion, our results indicate an important functional role for PodxL in stress myelopoiesis, a function likely mediated via Rap-1A. A complete understanding of the PodxL/Rap-1A axis may reveal important molecular insights into G-CSF-induced mobilization of neutrophils and provide mechanistic understanding into the pathological role of PodxL in aggressive cancers, including leukemia, which in turn may facilitate identification of novel therapeutic targets in PodxL associated cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of Insulin Receptor Substrate-1 Serine 307 Phosphorylation and Adiponectin in Adipose Tissue Insulin Resistance in Late Pregnancy

Endocrinology ◽  
2007 ◽  
Vol 148 (12) ◽  
pp. 5933-5942 ◽  
Author(s):  
Julio Sevillano ◽  
Javier de Castro ◽  
Carlos Bocos ◽  
Emilio Herrera ◽  
M. Pilar Ramos
Keyword(s):  
Insulin Resistance ◽  
Adipose Tissue ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Late Pregnancy ◽  
Serine Phosphorylation ◽  
Pregnant Rats

Insulin resistance is a hallmark of late pregnancy both in human and rat. Adipose tissue is one of the tissues that most actively contributes to this reduced insulin sensitivity. The aim of the present study was to characterize the molecular mechanisms of insulin resistance in adipose tissue at late pregnancy. To this end, we analyzed the insulin signaling cascade in lumbar adipose tissue of nonpregnant and pregnant (d 20) rats both under basal and insulin-stimulated conditions. We found that the levels of relevant signaling proteins, such as insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase, 3-phosphoinositide-dependent kinase-1, ERK1/2, and phosphatase and tensin homolog (PTEN) did not change at late pregnancy. However, insulin-stimulated tyrosine phosphorylation of both IR and IRS-1 were significantly decreased, coincident with decreased IRS-1/p85 association and impaired phosphorylation of AKR mouse thymoma viral protooncogene (Akt) and ERK1/2. This impaired activation of IRS-1 occurred together with an increase of IRS-1 phosphorylation at serine 307 and a decrease in adiponectin levels. To corroborate the role of IRS-1 in adipose tissue insulin resistance during pregnancy, we treated pregnant rats with the antidiabetic drug englitazone. Englitazone improved glucose tolerance, and this pharmacological reversal of insulin resistance was paralleled by an increase of adiponectin levels in adipose tissue as well as by a reduction of IRS-1 serine phosphorylation. Furthermore, the impaired insulin-stimulated tyrosine phosphorylation of IRS-1 in adipose tissue of pregnant animals could be restored ex vivo by treating isolated adipocytes with adiponectin. Together, our findings support a role for adiponectin and serine phosphorylation of IRS-1 in the modulation of insulin resistance in adipose tissue at late pregnancy.

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

scholarly journals Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Julie Favre ◽  
Emilie Vessieres ◽  
Anne-Laure Guihot ◽  
Coralyne Proux ◽  
Linda Grimaud ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Estrogen Receptor Alpha ◽  
Ex Vivo ◽  
Flow Mediated Dilation ◽  
Wild Type ◽  
Resistance Arteries ◽  
Independent Manner ◽  
Receptor Alpha ◽  
No Bioavailability

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.

Rhodiola and salidroside in the treatment of metabolic disorders

2019 ◽  
Vol 19 (19) ◽  
pp. 1611-1626 ◽  
Author(s):  
Xiang-Li Bai ◽  
Xiu-Ling Deng ◽  
Guang-Jie Wu ◽  
Wen-Jing Li ◽  
Si Jin
Keyword(s):  
Metabolic Disorders ◽  
Energy Homeostasis ◽  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Ex Vivo ◽  
Treatment Strategies ◽  
Ampk Signaling ◽  
Beneficial Effects

Over the past three decades, the knowledge gained about the mechanisms that underpin the potential use of Rhodiola in stress- and ageing-associated disorders has increased, and provided a universal framework for studies that focused on the use of Rhodiola in preventing or curing metabolic diseases. Of particular interest is the emerging role of Rhodiola in the maintenance of energy homeostasis. Moreover, over the last two decades, great efforts have been undertaken to unravel the underlying mechanisms of action of Rhodiola in the treatment of metabolic disorders. Extracts of Rhodiola and salidroside, the most abundant active compound in Rhodiola, are suggested to provide a beneficial effect in mental, behavioral, and metabolic disorders. Both in vivo and ex vivo studies, Rhodiola extracts and salidroside ameliorate metabolic disorders when administered acutely or prior to experimental injury. The mechanism involved includes multi-target effects by modulating various synergistic pathways that control oxidative stress, inflammation, mitochondria, autophagy, and cell death, as well as AMPK signaling that is associated with possible beneficial effects on metabolic disorders. However, evidence-based data supporting the effectiveness of Rhodiola or salidroside in treating metabolic disorders is limited. Therefore, a comprehensive review of available trials showing putative treatment strategies of metabolic disorders that include both clinical effective perspectives and fundamental molecular mechanisms is warranted. This review highlights studies that focus on the potential role of Rhodiola extracts and salidroside in type 2 diabetes and atherosclerosis, the two most common metabolic diseases.

nPKC Epsilon Negatively Regulates Platelet Functional Responses

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2855-2855
Author(s):  
Yamini Saraswathy Bynagari ◽  
Bela Nagy ◽  
Kamala Bhavaraju ◽  
Donna Woulfe ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Dense Granule ◽  
Dependent Manner ◽  
P2y1 Receptor ◽  
Wild Type ◽  
Functional Responses ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(𝛉), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 509-509 ◽  
Author(s):  
Safak Yalcin ◽  
Sathish Kumar Mungamuri ◽  
Dragan Marinkovic ◽  
Xin Zhang ◽  
Wei Tong ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Reactive oxygen species (ROS) are toxic byproducts of oxidative metabolism implicated in many debilitating human disorders including hematological malignancies and aging. ROS are also generated by growth factors and cytokine stimulation and play critical functions in normal cellular signaling. However, not much is known of how ROS impact physiological processes in normal and diseased states. We and others have recently shown critical functions for box (O) family of forkhead transcription factors (Fox)O in the regulation of physiological ROS in primitive hematopoietic cells. In particular, FoxO3 has emerged as the principal FoxO whose regulation of ROS is essential for the maintenance of hematopoietic stem cell pool. Although FoxO3’s activity is constitutively repressed by several oncoproteins that play critical roles in myeloproliferative disorders the role of FoxO3 in the regulation of primitive hematopoietic progenitors remains elusive. FoxO’s function is restrained by AKT serine threonine protein kinase. AKT supports growth, survival and proliferation by promoting inhibition of FoxO and activation of the mammalian target of rapamycin (mTOR) and its downstream target p70 S6 Kinase (S6K) through phosphorylation. We demonstrate that loss of FoxO3 leads to a myeloproliferative-like syndrome characterized by leukocytosis, splenomegaly, enhanced generation of primitive progenitors including colony-forming-unit-spleen (CFU-S) in hematopoietic organs and hypersensitivity of hematopoietic progenitor cells to cytokines in FoxO3 null mice. These findings were intriguing since we had not found FoxO3 null hematopoietic stem cells to exhibit enhanced cycling in vivo or to generate excessive hematopoietic progenitors ex vivo (Yalcin et al., JBC, 2008). To investigate the mechanism of enhanced myeloproliferation, we interrogated cytokine-mediated activation of signaling pathways in freshly isolated FoxO3 null versus wild type bone marrow cells enriched for hematopoietic progenitors. To our surprise we found that stimulation with cytokines including IL-3 led to hyperphosphorylation of AKT, mTOR and S6K but not STAT5 proteins in FoxO3 null as compared to wild type cytokine-starved hematopoietic progenitors. In agreement with these results, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of FoxO3 null- but not wild type-derived CFU-Sd12 in lethally irradiated hosts. These unexpected results suggested that AKT/mTOR signaling pathway is specifically overactivated as part of a feedback loop mechanism and mediates enhanced generation of FoxO3 null primitive multipotential hematopoietic progenitors in vivo. We further showed that phosphorylation of AKT/mTOR/S6K is highly sensitive to ROS scavenger N-Acetyl-Cysteine (NAC) in vivo and ex vivo in both wild type and FoxO3 null primitive hematopoietic progenitors indicating that ROS are involved in cytokine signaling in primary hematopoietic progenitor cells. Interestingly, in vivo administration of NAC normalized the number of FoxO3 null-derived CFU-Sd12 in lethally irradiated hosts without any impact on wild type CFU-Sd12 strongly suggesting that ROS mediate specifically enhanced generation of primitive hematopoietic progenitors in FoxO3 null mice. In this context, we were surprised to find similar levels of ROS concentrations in FoxO3 mutant as compared to control hematopoietic progenitors. Thus, we asked whether the increase in FoxO3 null primitive hematopoietic progenitor compartment is due to an increase sensitivity of cytokine signaling to ROS as opposed to increased ROS build up per se in these cells. In search for a mechanism we found the expression of Lnk, a negative regulator of cytokine signaling, to be highly reduced in FoxO3 null primitive hematopoietic progenitor cells. We further demonstrated that retroviral reintroduction of Lnk but not vector control in FoxO3 null primitive bone marrow cells reduced significantly the number of FoxO3 null-derived CFU-Sd12in vivo. Collectively, these results suggest that reduced expression of Lnk hypersensitizes FoxO3-deficient hematopoietic progenitors to ROS generated by cytokine signaling leading to myeloproliferation. These cumulative findings uncover a mechanism by which deregulation of cellular sensitivity to physiological ROS leads to hematopoietic malignancies specifically in disorders in which FoxO play a role.

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