Cyclic AMP Signaling Inhibits Megakaryocytic Differentiation by Targeting Transcription Factor 3 (E2A) Cyclin-dependent Kinase Inhibitor 1A (CDKN1A) Transcriptional Axis

Rubinstein, Jeremy D.; Elagib, Kamaleldin E.; Goldfarb, Adam N.

doi:10.1074/jbc.m112.366476

Cited by 7 publications

(9 citation statements)

References 47 publications

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“…It has been recently reported that anagrelide inhibits megakaryopoiesis through its PDEIII inhibitory activity, by stimulating cAMP/PKA signaling and subsequently targeting the transcription factor E2A (TCF3), down-regulating its mRNA levels [25]. Nevertheless, both our microarray dataset and our Q-PCR analysis showed no evidence that anagrelide reduced TCF3 mRNA levels at the concentration tested.…”

Section: Discussioncontrasting

confidence: 72%

The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis

Ahluwalia

Butcher

Donovan

et al. 2015

Journal of Thrombosis and Haemostasis

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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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Section: Discussioncontrasting

confidence: 72%

The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis

Ahluwalia

Butcher

Donovan

et al. 2015

Journal of Thrombosis and Haemostasis

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“…Neither 100 gM PKI nor 500 lM Rp-cAMP were able to revert anagrelide-induced inhibition of megakaryocyte maturation (Fig. 4A), while 100 gM PKI was able to partially revert the effect of 20 lM db-cAMP (data not shown), in accordance with recent data showing that cAMP exerts its inhibitory effect on the megakaryocyte lineage through a PKA-dependent mechanism [13]. Similar results were obtained when the addition of 500 gM anagrelide was delayed until day 4 of culture and also when lower 250 gM anagrelide concentrations or higher PKI doses, up to 1 lM, were used (data not shown).…”

Section: Role Of Camp Blockade In Anagrelide-induced Inhibition Of Mesupporting

confidence: 90%

“…found under these conditions (data not shown), whereas 500 gM anagrelide has been reported to repress the E2A/ p21 loop when added from the beginning of culture [13].…”

Section: Gene Expression Pattern Of Molecules Involved In Proplateletmentioning

confidence: 76%

“…), indicating that anagrelide‐induced inhibition of PPF is not due to decreased expression of these key molecular regulators of thrombopoiesis. In addition, no change in gene expression of the megakaryocytic regulator E2A, which is modulated by cAMP signaling, was found under these conditions (data not shown), whereas 500 η m anagrelide has been reported to repress the E2A/p21 loop when added from the beginning of culture .…”

Section: Resultsmentioning

confidence: 99%

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Anagrelide platelet‐lowering effect is due to inhibition of both megakaryocyte maturation and proplatelet formation: insight into potential mechanisms

Espasandin

Glembotsky

Grodzielski

et al. 2015

Journal of Thrombosis and Haemostasis

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To cite this article: Espasandin YR, Glembotsky AC, Grodzielski M, Lev PR, Goette NP, Molinas FC, Marta RF, Heller PG. Anagrelide platelet-lowering effect is due to inhibition of both megakaryocyte maturation and proplatelet formation: insight into potential mechanisms. J Thromb Haemost 2015; 13: 631-42. effect of anagrelide results from impaired megakaryocyte maturation and reduced PPF, both of which are deregu-lated in essential thrombocythemia. These effects seem unrelated to PDE3 inhibition, which is responsible for anagrelide 0 s cardiovascular side-effects and antiplatelet activity. Further work in this field may lead to the potential development of drugs to treat thrombocytosis in myeloproliferative disorders with an improved pharmaco-logic profile.

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“…Production of lentiviral particles by transient cotransfections of HEK293T cells was carried out using the calcium phosphate method, as previously described (59). Spinoculation of cells, puromycin selection of transduced CD34 + cells, and analysis of GFP + transduced primary progenitors were performed as previously described (59,60).…”

Section: Methodsmentioning

confidence: 99%

Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response

Richardson¹,

Delehanty²,

Bullock³

et al. 2013

J. Clin. Invest.

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The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

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Cyclic AMP Signaling Inhibits Megakaryocytic Differentiation by Targeting Transcription Factor 3 (E2A) Cyclin-dependent Kinase Inhibitor 1A (CDKN1A) Transcriptional Axis

Cited by 7 publications

References 47 publications

The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis

The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis

Anagrelide platelet‐lowering effect is due to inhibition of both megakaryocyte maturation and proplatelet formation: insight into potential mechanisms

Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response

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