A Mechanistic examination of the inhibitory role of cyclic AMP signaling on megakaryocytic differentiation
Rubinstein, Jeremy Devin, Department of Pathology, University of Virginia
Goldfarb, Adam, Department of Pathology, University of Virginia
Megakaryopoiesis is the biological process by which platelet precursor cells are produced from multipotent progenitor cells in the adult bone marrow. The differentiation and survival of these cells is tightly regulated by multiple factors, such as levels of the hormone thrombopoietin (TPO) and the coordinated activity, in a time and context dependent manner, of a number of transcription factors. While many of the regulatory pathways controlling this process have been elucidated, the mechanism by which cyclic AMP (cAMP) signaling impairs megakaryopoiesis in a lineage specific fashion remains poorly understood. This is of potential clinical relevance as the phosphodiesterase inhibitor anagrelide is used to lower platelet counts in patients with myeloproliferative disorders. Despite longstanding use, the mechanism by which this drug inhibits megakaryocytic differentiation is unknown. In the current study, the effect of cAMP signaling on megakaryopoiesis was evaluated using primary human progenitor cells. Using the adenylyl cyclase agonist forskolin to induce cAMP signaling, results presented here demonstrate that the inhibition of megakaryocytic differentiation occurs in a protein kinase A (PKA) dependent manner. Taking advantage of this system to screen for downstream effectors, we identified the transcription factor E2A as a key target in a novel repressive signaling pathway. Specifically, forskolin acting through PKA induced the downregulation of E2A, and enforced expression of E2A overrode the inhibitory effects of forskolin on megakaryopoiesis. The dependency of megakaryopoiesis on critical thresholds of E2A expression was confirmed in vivo in haploinsufficient mice and ex vivo using shRNA knockdown in human progenitor cells. CDKN1A, encoding the protein p21, has previously been shown to be an E2A target gene. Here were showed that forskolin impaired the normal upregulation of p21 seen during megakaryopoiesis, likely due to the downregulation of E2A. Similarly to E2A, p21 knockdown impaired megakaryopoiesis while its enforced expression overrode the effects of FSK. These results thus implicate the E2A-CDKN1A transcriptional axis in the control of megakaryopoiesis and reveal the lineage-selective inhibition of this axis as a likely mechanistic basis for the inhibitory effects of cAMP signaling. Additionally, this pathway may in part explain the anti-megakaryocytic properties of anagrelide.
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PHD (Doctor of Philosophy)
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