Summary. Background: Anagrelide is a selective inhibitor of megakaryocytopoiesis used to treat thrombocytosis in patients with chronic myeloproliferative disorders. The effectiveness of anagrelide in lowering platelet counts is firmly established, but its primary mechanism of action remains elusive. Objectives and Methods: Here, we have evaluated whether anagrelide interferes with the major signal transduction cascades stimulated by thrombopoietin in the hematopoietic cell line UT-7/mpl and in cultured CD34 + -derived human hematopoietic cells. In addition, we have used quantitative mRNA expression analysis to assess whether the drug affects the levels of known transcription factors that control megakaryocytopoiesis. Results: In UT-7/ mpl cells, anagrelide (1 lM) did not interfere with MPLmediated signaling as monitored by its lack of effect on JAK2 phosphorylation. Similarly, the drug did not affect the phosphorylation of STAT3, ERK1/2 or AKT in either UT-7/mpl cells or primary hematopoietic cells. In contrast, during thrombopoietin-induced megakaryocytic differentiation of normal hematopoietic cultures, anagrelide (0.3 lM) reduced the rise in the mRNA levels of the transcription factors GATA-1 and FOG-1 as well as those of the downstream genes encoding FLI-1, NF-E2, glycoprotein IIb and MPL. However, the drug showed no effect on GATA-2 or RUNX-1 mRNA expression. Furthermore, anagrelide did not diminish the rise in GATA-1 and FOG-1 expression during erythropoietin-stimulated erythroid differentiation. Cilostamide, an exclusive and equipotent phosphodiesterase III (PDEIII) inhibitor, did not alter the expression of these genes. Conclusions: Anagrelide suppresses megakaryocytopoiesis by reducing the expression levels of GATA-1 and FOG-1 via a PDEIII-independent mechanism that is differentiation context-specific and does not involve inhibition of MPL-mediated early signal transduction events.
BackgroundIn obesity, phenotypic switches occur in macrophage populations such that the predominantly M2-polarised anti-inflammatory state seen in lean individuals changes to a predominantly M1-polarised pro-inflammatory state in those who are obese. However, the mechanisms by which these phenotypic shifts occur have not yet been fully elucidated.ResultsThe effects of oxLDL (1-40 μg/ml; 24 h) on several parameters relevant to the Unfolded Protein Response (UPR)-mediated lipotoxic effects of oxLDL (disruption of ER Ca2+ handling; activation of the UPR transcription factor XBP-1; upregulation of the UPR target genes BiP and CHOP; apoptosis; cell viability) were investigated in human primary monocyte-derived macrophages, and also in monocyte-macrophages derived from the THP-1 monocytic cell line. A consistent pattern was observed: M2-polarised macrophages were more sensitive to the lipotoxic effects of oxLDL than either non-polarised macrophages or non-differentiated monocytic cells. Specifically, M2-polarised macrophages were the only cell type to undergo significantly increased apoptosis (Primary cells: 1.23 ± 0.01 basal; THP-1-derived: 1.97 ± 0.12 basal; P < 0.05 in both cases) and decreased cell viability (Primary cells: 0.79 ± 0.04 basal; THP-1-derived: 0.67 ± 0.02 basal; P < 0.05 in both cases) when exposed to oxLDL levels similar to those seen in overweight individuals (ie. 1 μg/ml).ConclusionsWe propose that the enhanced susceptibility of M2-polarised macrophages to lipotoxicity seen in the present in vitro study could, over time, contribute to the phenotypic shift seen in obese individuals in vivo. This is because a higher degree of oxLDL-induced lipotoxic cell death within M2 macrophages could contribute to a decrease in numbers of M2 cells, and thus a relative increase in proportion of non-M2 cells, within macrophage populations. Given the pro-inflammatory characteristics of a predominantly M1-polarised state, the data presented here may constitute a useful contribution to our understanding of the origin of the pro-inflammatory nature of obesity, and of the pathogenesis of obesity-associated inflammatory disorders such as Type 2 Diabetes and atherosclerosis.
Obesity is a chronic and relapsing public health problem with an extensive list of associated comorbidities. The worldwide prevalence of obesity has nearly tripled over the last five decades and continues to pose a serious threat to wider society and the wellbeing of future generations. The pathogenesis of obesity is complex but diet plays a key role in the onset and progression of the disease. The human diet has changed drastically across the globe, with an estimate that approximately 72% of the calories consumed today come from foods that were not part of our ancestral diets and are not compatible with our metabolism. Additionally, multiple nutrient-independent factors, e.g., cost, accessibility, behaviours, culture, education, work commitments, knowledge and societal set-up, influence our food choices and eating patterns. Much research has been focused on ‘what to eat’ or ‘how much to eat’ to reduce the obesity burden, but increasingly evidence indicates that ‘when to eat’ is fundamental to human metabolism. Aligning feeding patterns to the 24-h circadian clock that regulates a wide range of physiological and behavioural processes has multiple health-promoting effects with anti-obesity being a major part. This article explores the current understanding of the interactions between the body clocks, bioactive dietary components and the less appreciated role of meal timings in energy homeostasis and obesity.
Histamine stimulated inositol phosphate formation by human skin fibroblasts. The effect of histamine was reduced but still readily apparent in the absence of extracellular Ca2+. Histamine caused a transient increase in intracellular free Ca2+ as detected by indo-1 and fura-2 fluorescence studies on cell populations and on individual cells. Similar increases were observed in the absence of extracellular Ca2+, indicating that the effect was primarily due to mobilization of Ca2+ from intracellular stores, presumably by inositol trisphosphate (IP3). The effects of histamine on phosphoinositide metabolism and intracellular Ca2+ were inhibited by pretreatment of the cells with phorbol esters, suggesting that the histamine receptor in fibroblasts is subject to feedback regulation by protein kinase C. Histamine inhibited the incorporation of [3H]-thymidine into DNA. The effects of histamine on inositol phosphate formation, intracellular Ca2+, and thymidine incorporation were blocked by the H1 receptor antagonist mepyramine. Our results indicate that human skin fibroblasts have H1 receptors coupled to the formation of inositol phosphates and mobilization of intracellular Ca2+. We suggest that this H1 receptor also mediates a block of the cell cycle and that histamine may play a physiological role in the regulation of fibroblast proliferation.
To cite this article: Ahluwalia M, Butcher L, Donovan H, Killick-Cole C, Jones PM, Erusalimsky JD. The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis. J Thromb Haemost 2015; 13: 1103-12.Summary. Background: Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years, the molecular mechanism accounting for this activity is still unclear. Objectives and Methods: To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions, using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. Results: We identified 328 annotated genes differentially regulated by anagrelide, including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3, whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4, a known TRIB3 inducer. Notably, immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2a, which is an upstream regulator of ATF4, and increased ATF4 protein levels. Furthermore, salubrinal, an inhibitor of eIF2a dephosphorylation, increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. Conclusions: These findings link signaling through eIF2a/ATF4 to the antimegakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis.
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