José L. Sardina

p22phox-dependent NADPH oxidase activity is required for megakaryocytic differentiation.

Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34+ cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22phox-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.

Reactive oxygen species: Are they important for haematopoiesis?

The production of reactive oxygen species (ROS) has traditionally been related to deleterious effects for cells. However, it is now widely accepted that ROS can play an important role in regulating cellular signalling and gene expression. NADPH oxidase ROS production seems to be especially important in this regard. Some lines of evidence suggest that ROS may be important modulators of cell differentiation, including haematopoietic differentiation, in both physiologic and pathologic conditions. Here we shall review how ROS can regulate cell signalling and gene expression. We shall also focus on the importance of ROS for haematopoietic stem cell (HSC) biology and for haematopoietic differentiation. We shall review the involvement of ROS and NADPH oxidases in cancer, and in particular what is known about the relationship between ROS and haematological malignancies. Finally, we shall discuss the use of ROS as cancer therapeutic targets.

Comparison of changes in erythrocyte and platelet phospholipid and fatty acid composition and protein oxidation in chronic obstructive pulmonary disease and asthma

Objective: To analyse and compare the phospholipid and fatty acid composition of total lipids and the occurrence of lipid peroxidation and protein oxidation directly in erythrocytes or platelets from chronic obstructive pulmonary disease (COPD) and asthma patients. Patients: Fifteen consecutive outpatients with COPD (all smokers) and asthma (non-smokers) recruited during a moderate-to-severe (COPD) or moderate (asthma) exacerbation. Fifteen subjects with smoking habits similar to those of COPD patients were studied as a control group. Methods: Phospholipid and total fatty acid compositions were analysed by two-dimensional thin layer chromatography or gas chromatography–mass spectrometry, respectively. The lipid fluorescence of lipid extracts was measured by spectrofluorimetry. Protein carbonyl contents and profiles were measured by immunoblot detection. Results: No differences were found either in erythrocyte or platelet cholesterol or phospholipid levels. Only a decrease in the content of phosphatidylserine + phosphatidylinositol (P < 0.003) was detected in platelets from the asthma patients. In erythrocytes, the fatty acid profile changed in both lung pathologies, especially as regards polyunsaturated fatty acids (decreases in arachidonic and 22:4 fatty acid contents). Other observed changes were: COPD, an increase in palmitic fatty acid; asthma, an increase in oleic and decreases in eicosapentaenoic and 22:6 + 24:1 fatty acids. In platelets, the fatty acid profiles revealed many differences between both lung pathologies: COPD, a decrease in 18:1 and increases in 20:5 and 22:5 + 24:0; asthma, a decrease in 20:4 and increase in 22:6 + 24:1. In COPD vs. asthma patients, fatty acid changes were mainly detected in platelets, especially in 18-carbon species, with decreases in stearic and 18:1 fatty acids in the COPD patients. Protein oxidation levels were increased in both lung pathologies in both erythrocytes and platelets. Conclusions: COPD and asthma are associated with common or specific changes in the lipid composition of erythrocytes and/or platelets. The data point to lipid peroxidation and protein oxidation phenomena in both types of blood cell, although platelets would be more susceptible to stress.

Membrane cholesterol contents modify the protective effects of quercetin and rutin on integrity and cellular viability in oxidized erythrocytes

Flavonoids protect cells damaged by oxidative stress. This, together with other biological activities, is governed by structural features of flavonoids and the nature and physical state of the cell membrane. We have previously proved that membrane cholesterol contents modify the protective power of quercetin and rutin against oxidative stress in erythrocytes. Here we analyzed the lipid asymmetry, the integrity, and cell viability of native and cholesterol-modified erythrocytes exposed to tert-butyl hydroperoxide in presence of both antioxidants. Our results provides clear evidence that quercetin affords better protection than rutin against lipid peroxidation, ROS generation, erythrophagocytosis and cellular instability in oxidized erythrocytes with normal and modified cholesterol contents. Both antioxidants provided a high of protection for the transbilayer aminophospholipid asymmetry, only partly preserving cell morphology in oxidized control and cholesterol-depleted erythrocytes. Cholesterol depletion reduced the protection provided by both antioxidants against phosphatidylserine externalization, erythrophagocytosis and hemolysis, which is in accordance with the lower degree of preservation against lipid peroxidation observed in oxidized cholesterol-depleted erythrocytes. This lower degree of preservation is presumably attributable to the low antioxidant contents in these erythrocyte membranes, or even to a lower efficiency of the antioxidant in a modified lipid environment due to the removal of cholesterol.

Comparative antioxidant capacities of quercetin and butylated hydroxyanisole in cholesterol-modified erythrocytes damaged by tert-butylhydroperoxide.

Phenolic compounds are potent antioxidants that scavenge reactive oxygen species (ROS), protecting the cells against oxidative damage. Their antioxidant capacities are governed by their structural features and the nature and physical state of the cell membrane. Our study compares the protective effects of butylated hydroxyanisole (BHA) and quercetin against the cellular injury induced by oxidative stress, and the influence of membrane cholesterol contents in their antioxidant capacities, analyzing the structural changes and cellular stability of native and cholesterol-modified erythrocytes exposed to tert-butylhydroperoxide in presence of each antioxidant. The data provide clear evidence that BHA affords better protection than quercetin against ROS generation, lipid peroxidation and lipid and GSH losses in oxidized erythrocytes. However, cellular integrity and stability are better protected by quercetin owing to the hemolytic effect of BHA. Both antioxidants suppress the alterations in membrane fluidity with similar efficiency, reducing methemoglobin formation in all oxidized erythrocytes. Membrane cholesterol depletion decreases the protection against the oxidative damage provided by both antioxidants. This lower preservation may be due to low antioxidant contents, a lower antioxidant capacity, or even to an increased oxidative damage in this membrane type as a consequence of environment modifications after cholesterol depletion.

APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts.

Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease.

The guanine-exchange factor Ric8a binds to the Ca2+ sensor NCS-1 to regulate synapse number and neurotransmitter release

ABSTRACT The conserved Ca2+-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and G&agr;s regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2–Ric8a–G&agr;s, which diverges downstream. These mechanisms expose the Frq2–Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

Rolipram and SP600125 suppress the early increase in PTP1B expression during cerulein-induced pancreatitis in rats.

Objectives: To analyze the expression modulation of pancreatic protein tyrosine phosphatase (PTP)1B during the development of cerulein (Cer)-induced acute pancreatitis (AP) and the effect of inhibition of type 4 phosphodiesterase and c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 on its expression levels. Methods: Acute pancreatitis was induced in rats by subcutaneous injections of 20 &mgr;g Cer per kilogram body weight at hourly intervals, and the animals were killed at 2, 4, or 9 hours after the first injection. Neutropenia was induced with vinblastine sulfate. Phosphodiesterase and the mitogen-activated protein kinases were inhibited with rolipram and SP600125, respectively, before the induction of AP. Results: Protein tyrosine phosphatase 1B increases its expression at the levels of both protein and messenger RNA during the early phase of Cer-induced AP. The increase in protein expression persisted along the development of the disease, and neutrophil infiltration seemed to play a central role. Rolipram and SP600125 pretreatments mostly suppressed the increase in the expression of PTP1B during the early phase of AP. Conclusions: Cerulein-induced AP is associated with an increase in the expression of PTP1B in its early phase. An increase in cyclic adenosine monophosphate levels in inflammatory cells and the inhibition of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 are able to suppress the increase in PTP1B protein level.

PTPN13 regulates cellular signalling and β-catenin function during megakaryocytic differentiation

PTPN13 is a high-molecular weight intracellular phosphatase with several isoforms that exhibits a highly modular structure. Although in recent years different roles have been described for PTPN13, we are still far from understanding its function in cell biology. Here we show that PTPN13 expression is activated during megakaryocytic differentiation at the protein and mRNA level. Our results show that the upregulation of PTPN13 inhibits megakaryocytic differentiation, while PTPN13 silencing triggers differentiation. The ability of PTPN13 to alter megakaryocytic differentiation can be explained by its capacity to regulate ERK and STAT signalling. Interestingly, the silencing of β-catenin produced the same effect as PTPN13 downregulation. We demonstrate that both proteins coimmunoprecipitate and colocalise. Moreover, we provide evidence showing that PTPN13 can regulate β-catenin phosphorylation, stability and transcriptional activity. Therefore, the ability of PTPN13 to control megakaryocytic differentiation must be intimately linked to the regulation of β-catenin function. Moreover, our results show for the first time that PTPN13 is stabilised upon Wnt signalling, which makes PTPN13 an important player in canonical Wnt signalling. Our results show that PTPN13 behaves as an important regulator of megakaryocytic differentiation in cell lines and also in murine haematopoietic progenitors. This importance can be explained by the ability of PTPN13 to regulate cellular signalling, and especially through the regulation of β-catenin stability and function. Our results hold true for different megakaryocytic cell lines and also for haematopoietic progenitors, suggesting that these two proteins may play a relevant role during in vivo megakaryopoiesis.