Differential roles of cAMP and cGMP in megakaryocyte maturation and platelet biogenesis

Abstract

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulate the activity of protein kinase A (PKA) and protein kinase G (PKG), respectively. This process helps maintain circulating platelets in a resting state. Here we studied the role of cAMP and cGMP in the regulation of megakaryocyte (MK) differentiation and platelet formation. Cultured, platelet-producing MKs were differentiated from fetal livers harvested from 13.5 days postcoital mouse embryos. MK development was accompanied by a dramatic increase in cAMP production and expression of soluble guanylate cyclase, PKG, and PKA as well as their downstream targets vasodilator-stimulated phosphoprotein (VASP) and MENA. Stimulation of prostaglandin E1 receptor/adenylyl cyclase or soluble guanylate cyclase/PKG in cultured MKs increased VASP phosphorylation, indicating that these components share a common signaling pathway. To dissect out the role of cyclic nucleotides in MK differentiation, cAMP/PKA and cGMP/PKG signaling were alternately blocked in cultured MKs. Down-regulation of cAMP pathway effectors decreased MK numbers and ploidy. Notably, cGMP levels increased at the beginning of MK development and returned to basal levels in parallel with MK maturation. However, inhibition of cGMP pathway effectors had no effect on MK development. In addition, platelet release from mature MKs was enhanced by cGMP and inhibited by cAMP. Our data suggest that cAMP plays an important role in MK differentiation, while cAMP and cGMP have opposite effects on platelet production. Identifying the signaling pathways that underpin MK development and proplatelet formation will provide greater insights into thrombopoiesis and may potentially yield useful therapeutic targets.

Thrombocytosis as a Response to High Interleukin-6 Levels in cGMP-Dependent Protein Kinase I Mutant Mice

“…In line with our present findings, Begonja et al 16 have recently shown that the cGMP/cGKI pathway has no effect on megakaryocyte development in vitro and does not affect megakaryocyte proliferation or polyploidization. These authors further showed that a pharmacological stimulation of the cGMP pathway enhanced release of platelet-like particles from fetal liver cells-derived megakaryocytes, 16 suggesting that the cGMP/cGKI pathway might play a role in platelet biogenesis. However, here we did not observe any effects of endogenous cGKI on platelet production under physiological conditions in different cGKI mutant mouse lines.…”

“…Using a conditional approach, we targeted cGKI specifically in the megakaryocyte/ platelet lineage and confirmed that megakaryocyte cGKI does not intrinsically affect megakaryocyte maturation or platelet production. In line with our present findings, Begonja et al 16 have recently shown that the cGMP/cGKI pathway has no effect on megakaryocyte development in vitro and does not affect megakaryocyte proliferation or polyploidization. These authors further showed that a pharmacological stimulation of the cGMP pathway enhanced release of platelet-like particles from fetal liver cells-derived megakaryocytes, 16 suggesting that the cGMP/cGKI pathway might play a role in platelet biogenesis.…”

“…: first, pharmacological overstimulation of cGMP/cGKI pathway might indeed enhance platelet formation in vitro as suggested by Begonja et al 16 ; however, intrinsic megakaryocyte cGMP/ cGKI signaling should not be overstimulated under physiological conditions in vivo. Thus, we conclude from our in vivo data that the physiological activity of megakaryocyte cGMP/cGKI pathway is not essential for thrombopoiesis.…”

“…Recent in vitro findings indicate that platelet release from fetal liver cells-derived megakaryocytes is enhanced by high cGMP, but blocked by high cAMP levels in response to prostaglandin I 2 or agonists that directly activate adenylyl cyclase. 16 As in many other cell types, the rate of cGMP production through soluble guanylyl cyclase activity and its degradation via cGMP-hydrolyzing phospodiesterases (PDEs) determine the amplitude, duration, and spatiotemporal distribution of an intraplatelet cGMP signal. 17 Of the 3 known platelet PDE isoforms, 2 isoforms, PDE-2 and PDE-3, may maintain an intensive cross-talk between nitric oxide (NO)/soluble guanylyl cyclase/cGMP and prostaglandin I 2 /adenylyl cyclase/ cAMP because these PDEs limit, with different affinities for their substrates and with different catalytic rates, the levels of both cyclic nucleotides, producing either inactive 5´-AMP or 5´-GMP.…”

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

“…Indeed, Oevermann et al 29 showed that MRP4 expression is increased in the megakaryocyte lineage during hematopoietic stem cell differentiation. Moreover, Begonja et al 31 reported evidence of a dominant role of cyclic nucleotides in megakaryocyte differentiation. However, because platelet counts were only slightly increased in MRP4 2/2 mice, MRP4…”