Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Abstract

• PKCe, regulating RhoA activity, is a critical mediator of proplatelet formation. • PKCe shut down results inRhoA expression levels that are incompatible with normal platelet generation.During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kinase Cepsilon (PKC«), a member of the protein kinase C family of serine/threonine-kinases, expression varies during human megakaryocyte differentiation and modulates megakaryocyte maturation and platelet release. Here we used an in vitro model of murine platelet production to investigate a potential role for PKC« in proplatelet formation. By immunofluorescence we observed that PKC« colocalizes with a/b-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKC« expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKC« inhibition resulted in lower proplatelet numbers and larger diameter platelets in culture as well as persistent RhoA activation. Finally, we demonstrate that pharmacological inhibition of RhoA is capable of reversing the proplatelet defects mediated by PKC« inhibition. Collectively, these data indicate that by regulating RhoA activity, PKC« is a critical mediator of mouse proplatelet formation in vitro. (Blood. 2013;122(7):1305-1311) IntroductionDefective platelet numbers can compromise proper wound healing and cause bleeding, whereas excessive platelet production or activation can lead to thrombosis. As a result, platelet homeostasis is a tightly regulated process. The process of generating thousands of platelets from a single megakaryocyte is characterized by several morphologically distinct stages that involve extensive cytoskeletal remodeling. In particular, megakaryoctyes, following polyploidization and prior to platelet release, form elongated cellular processes called proplatelets (proPLT).1 Perturbations in proPLT formation lead to alterations in platelet morphology and thrombocytopenia, 2,3 emphasizing the importance of this step. The marginal microtubule band, actin-based cytoskeleton, and spectrin-based membrane skeleton are all prominent components of the cytoskeletal remodeling that occurs during platelet formation. 4 Specifically, dynamic tubulin polymerization is required for the proper platelet morphology, assembly of the marginal microtubule coil, proPLT extensions and microtubule band formation.4-8 Various mutations in genes that regulate actin polymerization and turnover cause thrombocytopenia due to altered formation and morphology of proPLT exten...

Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation

“…These effects led to the rearrangement of the actin and microtublin cytoskeletons, which was a known key determinant for PPF, platelet formation, and platelet release. 25,37 Although both NE and EPI stimulate platelet activation, the underlying mechanism is unclear. Here, we discovered that NEand EPI-induced platelet activation was at least partially attributed to ERK1/2 signaling.…”

“…To uncover the underlying mechanisms, we investigated the influences of NE and EPI on the activity of RhoA GTPase, which was reported to regulate the rearrangement of the MK cytoskeleton during PPF. 25,26 Both the NE and EPI treatments led to a significant increase in the Ser188 phosphorylation of RhoA ( Figure 6D-E). Ser188 phosphorylation negatively regulates the activity of RhoA 27,28 ; thus, our results indicated that NE and EPI stimulated PPF via the downregulation of RhoA activity.…”

Proteasome function is required for platelet production

“…RhoA-dependent signaling has been linked to the production of proplatelets (17,18). Therefore, we treated human megakaryocytes with Y27632, a selective inhibitor of the Rho-associated protein kinase p160ROCK, or with C3 transferase, a direct RhoA inhibitor.…”

Human thrombopoiesis depends on Protein kinase C /protein kinase C functional couple

“…32 Furthermore, Kostyak and colleagues have shown that, in a mouse model, PKCδ down-regulation reinforces MK differentiation and platelet production. 34 Since it is well documented that PKCε and PKCδ have opposite expression and function in mouse versus human platelets, 43 we hypothesized that high levels of PKCδ are necessary for adequate human MK differentiation and platelet release.…”

“…[28][29][30] We previously demonstrated that PKCε has a key role in human megakaryocytopoiesis in vitro 27,31 and platelet function in vivo, 3 as well as in proPLT production in the murine model. 32 Specifically, PKCε levels increase in the early phase of in vitro human megakaryocytic (MK) differentiation and decrease in the late phase before platelets release, 31 and a forced PKCε overexpression prevents MK full maturation, 31 while its down-regulation increases MK differentiation. 33 Additionally, more recently we have demonstrated that primary MK from myelofibrotic (PMF) patients express higher levels of PKCε than those from healthy donors (HD), and that PKCε inhibition in PMF restores a bona fide normal MK differentiation.…”