title: Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition.
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 (PKCε), 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 PKCε in proplatelet formation. By immunofluorescence we observed that PKCε colocalizes with α/β-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKCε expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKCε 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 PKCε inhibition. Collectively, these data indicate that by regulating RhoA activity, PKCε is a critical mediator of mouse proplatelet formation in vitro.
authors.affiliation: Department of Biomedical, Biotechnological and Translational Sciences (SBiBiT), University of Parma, Parma, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Gobbi
authors.forename: Prisco
authors.initials: P
authors.lastname: Mirandola
authors.forename: Cecilia
authors.initials: C
authors.lastname: Carubbi
authors.forename: Elena
authors.initials: E
authors.lastname: Masselli
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Sykes
authors.forename: Francesca
authors.initials: F
authors.lastname: Ferraro
authors.forename: Antonio
authors.initials: A
authors.lastname: Nouvenne
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Thon
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Italiano
authors.forename: Marco
authors.initials: M
authors.lastname: Vitale
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D005333:Fetus / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D034061:Thrombopoiesis / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D014404:Tubulin; D051744:Protein Kinase C-epsilon; D020742:rhoA GTP-Binding Protein