Alessandra Bertoni

DNA Oxidation as Triggered by H3K9me2 Demethylation Drives Estrogen-Induced Gene Expression

Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine–DNA glycosylase 1 and topoisomeraseIIβ, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.

Early and Late Events Induced by PolyQ-expanded Proteins IDENTIFICATION OF A COMMON PATHOGENIC PROPERTY OF POLYQ-EXPANDED PROTEINS

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24–36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

The endocannabinoid 2-arachidonoylglycerol activates human platelets through non-CB1/CB2 receptors

Summary.  Background:  The endocannabinoid 2‐arachidonoylglycerol (2‐AG) is an endogenous lipid that acts through the activation of G‐protein‐coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2‐AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2‐AG also binds to platelet surface and leads to cell activation. Objective: To investigate the biological consequence of 2‐AG interactions with human platelets and to clarify the role of cannabinoid receptors. Methods: Gel‐filtered platelets were stimulated with 2‐AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA‐2 loaded platelets. Thromboxane A2 (TxA2) generation was evaluated as Thromboxane B2 accumulation with a commercial EIA assay. Results: 2‐AG induced platelet shape change, aggregation and secretion with a dose‐dependent mechanism that required engagement of platelet TxA2 receptors. 2‐AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA2. Indeed 2‐AG was able to induce a robust generation of TxA2 through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2‐AG. Moreover, neither CB1 and CB2 proteins nor CB1/CB2 mRNAs were detected in platelets. Conclusions: 2‐AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non‐CB1/CB2 receptor‐mediated mechanism.

Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops

Histone methylation changes and formation of chromatin loops involving enhancers, promoters and 3′ end regions of genes have been variously associated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A receptor, at the RARE–promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA exposure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethylase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elicits an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nucleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5′ transcription start site and the 3′ end of the genes. The RARE bound-receptor governs the 5′ and 3′ end selection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription.

A diet supplemented with ALA-rich flaxseed prevents cardiomyocyte apoptosis by regulating caveolin-3 expression

AIMS n-3 polyunsaturated fatty acids (PUFAs) induce beneficial effects on the heart, but the mechanisms through which these effects are operated are not completely clarified yet. Among others, cardiac diseases are often associated with increased levels of cytokines, such as tumour necrosis factor-α (TNF), that cause degeneration and death of cardiomyocytes. The present study has been carried out to investigate (i) the potential anti-apoptotic effects induced by the n-3 polyunsaturated α-linolenic acid (ALA) in experimental models of cardiac diseases characterized by high levels of TNF, and (ii) the potential role of caveolin-3 (Cav-3) in the mechanisms involved in this process. METHODS AND RESULTS An ALA-rich flaxseed diet, administered from weaning to hereditary cardiomyopathic hamsters, prevented the onset of myocardial apoptosis associated with high plasma and tissue levels of TNF preserving caveolin-3 expression. To confirm these findings, isolated neonatal mouse cardiomyocytes were exposed to TNF to induce apoptosis. ALA pre-treatment greatly enhanced Cav-3 expression hampering the internalization of the caveolar TNF receptor and, thus, determining the abortion of the apoptotic vs. survival cascade. CONCLUSION This study unveiled the Cav-3 pivotal role in defending cardiomyocytes against the TNF pro-apoptotic action and the ALA capacity to regulate this mechanism preventing cardiac degenerative diseases.

Expression of the endocannabinoid system in the bi-potential HEL cell line: commitment to the megakaryoblastic lineage by 2-arachidonoylglycerol.

The role of the endocannabinoid system in haematopoietic cells is not completely understood. We investigated whether human erythroleukemia (HEL) cells were able to bind, metabolise and transport the main endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). We also investigated whether AEA or 2-AG could modulate HEL differentiation. Although able to internalise both endocannabinoids, HEL cells had the machinery to metabolise 2-AG only, since they were devoid of the enzymes needed to synthesise and degrade AEA. Nonetheless, the intracellular transport of exogenous AEA might be required to activate the vanilloid receptors, with yet unknown implications for vascular biology. On the contrary, 2-AG appeared to play a role in lineage determination. Indeed, 2-AG itself drove HEL cells towards megakaryocytic differentiation, as it enhanced expression of β3 integrin subunit, a megakaryocyte/platelet surface antigen, and glycoprotein VI, a late marker of megakaryocytes; in parallel, it reduced the amount of messenger RNA encoding for glycophorin A, a marker of erythroid phenotype. All these effects were mediated by activation of CB2 cannabinoid receptors that triggered an extracellular signal-regulated kinase-dependent signalling cascade. In addition, classical inducers of megakaryocyte differentiation reduced 2-AG synthesis (although they did not affect the binding efficiency of CB2 receptors), suggesting that levels of this endocannabinoid may be critical for committing HEL cells towards the megakaryocytic lineage.

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.

The phytoestrogen 8-prenylnaringenin inhibits agonist-dependent activation of human platelets

BACKGROUND Phytoestrogens are plant-derived polyphenolic compounds that exert beneficial effects on human health, mostly related to their estrogen mimetic activity. In particular a strong correlation between phytoestrogens intake and a lower risk of cardiovascular diseases has been reported. The flavanone 8-prenylnaringenin, extracted from hop flowers, has been identified as a novel phytoestrogen, unique with respect to estrogen receptors specificity and potency. However, to date no investigations on the 8-prenylnaringenin role in modulating platelet function have been undertaken. METHODS We evaluated the effect of 8-prenylnaringenin on platelet aggregation, intracellular calcium mobilization and protein phosphorylation triggered by thrombin and collagen, and platelet adhesion and dense granule secretion triggered by collagen. RESULTS 8-Prenylnaringenin inhibited platelet aggregation induced by different agonists and platelet adhesion to collagen matrix. 8-Prenylnaringenin directly increased intracellular cAMP and cGMP levels and thus promoted VASP phosphorylation. However, these molecular events were not responsible for the inhibitory action of 8-prenylnaringenin on platelets. Moreover, 8-prenylnaringenin inhibited the phosphorylation of Pyk2, Akt, and ERK1/2. Finally, 8-prenylnaringenin suppressed the mobilization of calcium and the secretion of dense granules. All these effects were independent of estrogen receptors recruitment. CONCLUSIONS 8-Prenylnaringenin exerted anti-aggregatory and anti-adhesive effects on human platelets, independently of estrogen receptors, acting as an inhibitor of multiple proteins essential for the morphological and biochemical transformations that occur during platelet activation and aggregation. GENERAL SIGNIFICANCE 8-Prenylnaringenin may represent a useful tool in the therapy and prevention of vascular diseases associated with platelet aggregation, such as atherosclerosis, myocardial infarction, coronary artery disease, and thrombosis.

Dehydroepiandrosterone-sulfate inhibits thrombin-induced platelet aggregation.

Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, are the most abundant steroids circulating in human blood. DHEA stimulates endothelial cells to release high amounts of nitric oxide in the circulation. Nitric oxide activates guanylyl cyclase in platelets thus decreasing the responsiveness of these cells to physiological agonists. However, the impact of DHEA-S and DHEA on platelet function and their possible role in modulating the response of human platelets to physiological agonists were not yet investigated. Here, DHEA-S, but not DHEA, inhibited in vitro thrombin-dependent platelet aggregation in a dose-dependent manner. DHEA-S exerted this effect by decreasing thrombin-dependent dense granule secretion, and so impairing the positive feed-back loop provided by ADP. Furthermore, DHEA-S inhibited thrombin-dependent activation of Akt, ERK1/2, and p38 MAP kinase. Although both DHEA-S and DHEA directly activated in platelets the inhibitory cGMP/PGK/VASP pathway, these events were not responsible for the inhibitory action of DHEA-S in platelets. In addition DHEA-S acted in synergism with nitric oxide in inhibiting platelet aggregation. In conclusion DHEA-S inhibited platelet activation caused by a mild stimulus without completely hampering platelet functionality and thus DHEA-S may participate in the physiological mechanisms that maintain circulating platelets in a resting state. The role played by DHEA-S could be relevant mainly when the functionality of the vascular endothelium is compromised.

The oestrogen receptor GPER is expressed in human haematopoietic stem cells but not in mature megakaryocytes

Megakaryocytes are bone-marrow precursor cells that differentiate to produce blood platelets via intermediate cytoplasmic extensions known as proplatelets (Patel et al, 2005). Recent advances in the understanding of megakaryocyte differentiation and platelet formation have been mostly obtained by biological studies of cultured cells. Although platelet formation from megakaryocytes has been actively studied, the molecular mechanisms of this process are still incompletely understood. Growing evidence has accrued that sex hormones may play a crucial role during megakaryopoiesis (Kostyak & Naik, 2007). Accordingly, it has been shown that high levels of oestrogens and conventional hormone replacement therapies increase the number of megakaryocytes in mice (Perry et al,2000) and in postmenopausal women (Bord et al, 2000). Nagata et al (2003) have also shown that oestradiol can be synthesised by murine megakaryocytes and that oestradiol positively affects proplatelet formation. In addition, Bord et al (2004) reported that oestrogens can promote megakaryocyte proliferation and maturation, thereby modulating the expression of the classical oestrogen receptors (ER)a and ERb. We have previously demonstrated that oestrogens can potentiate platelet aggregation through a rapid and reversible ERb-mediated signalling (Moro et al, 2005) and that membrane lipid rafts coordinate this pathway (Reineri et al, 2007). Megakaryocytes and platelets are known to express ERb (Khetawat et al, 2000). Genomic effects of oestrogens in megakaryocytes have been suggested to contribute to gender differences in platelet function. However, oestrogens can also induce rapid, non-genomic effects through interaction with classical ERs, localised on the plasma membrane, and through a member of the 7-transmembrane G protein-coupled receptor family, GPR30 (Cheskis et al, 2007), now known as G proteincoupled oestrogen receptor 1 (GPER). Despite the potential importance of GPER in megakaryocyte differentiation and maturation, its exact role in this process has not yet been determined. In this context, we sought to evaluate whether the expression of GPER could change during megakaryocytic differentiation. CD34 cells were isolated from human cord blood and cultured in StemSpan Serum-Free Expansion Medium medium (Stem-Cell Technologies, Vancouver, BC, Canada) supplemented with 10 ng/ml thrombopoietin, interleukin (IL)6 and IL11 (all from PeproTech EC Ltd, London, UK) at 37 C in 5% CO2 to induce megakaryocyte differentiation. After 13 days of culture cells were harvested, cytospun on glass coverslips, and stained with goat polyclonal anti-CD61 (Santa Cruz, Heidelberg, Germany) and secondary antibody conjugated with Alexa Fluor-488 (Invitrogen, Milan, Italy). Nuclear counterstaining was performed with Hoechst 33258. Specimens were mounted in Mowiol-488. Conventional fluorescence microscopy was performed through an Axioscope 2 Plus microscope (Carl Zeiss, Göttingen, Germany), using a 63/1Æ25 or a 100/1Æ30 Plan Neofluar oil-immersion objective. For each specimen, at least 100 cells were evaluated. The percentage of mature megakaryocytes positive for CD61 staining and with polyploid nuclei at the end of culture was 70 ± 15% (Fig 1). To evaluate GPER expression during megakaryocyte differentiation three independent experiments were performed, each using a different pool of three cord blood samples (a total of nine cord blood samples was used), to eliminate any biological variability. GPER expression was assessed in cells on days 0 (CD34), 3, 7 of differentiation, as well as in mature megakaryocytes isolated at day 13 by CD61 immunomagnetic beads technique (Miltenyi-Biotec, Bergisch Gladbach, Germany). Total RNA was extracted with the RNAqueous kit (Ambion Inc, Foster City, CA, USA) and reverse transcription reactions were