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Gobbi G , Mirandola P , Carubbi C , Masselli E , Sykes SM , Ferraro F , Nouvenne A , Thon JN , Italiano JE Jr , Vitale M
Proplatelet generation in the mouse requires PKCepsilon-dependent RhoA inhibition
Blood. 2013 Aug 15;122(7) :1305-11
PMID: 23838351    PMCID: PMC3952532     URL: https://www.ncbi.nlm.nih.gov/pubmed/23838351
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Abstract
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 (PKCepsilon), 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 PKCepsilon in proplatelet formation. By immunofluorescence we observed that PKCepsilon colocalizes with alpha/beta-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKCepsilon expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKCepsilon 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 PKCepsilon inhibition. Collectively, these data indicate that by regulating RhoA activity, PKCepsilon is a critical mediator of mouse proplatelet formation in vitro.
Notes
Gobbi, Giuliana Mirandola, Prisco Carubbi, Cecilia Masselli, Elena Sykes, Stephen M Ferraro, Francesca Nouvenne, Antonio Thon, Jonathan N Italiano, Joseph E Jr Vitale, Marco eng Research Support, Non-U.S. Gov't Blood. 2013 Aug 15;122(7):1305-11. doi: 10.1182/blood-2013-04-490599. Epub 2013 Jul 9.