Timing and Expression Level of Protein Kinase Cε Regulate the Megakaryocytic Differentiation of Human CD34 Cells

Gobbi, Giuliana; Mirandola, Prisco; Sponzilli, Ivonne; Micheloni, Cristina; Malinverno, Chiara; Cocco, Lucio; Vitale, Marco

doi:10.1634/stemcells.2006-0839

Cited by 44 publications

(74 citation statements)

References 35 publications

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“…In agreement with our previous studies in human megakaryocyte coltures, 18 PKCe protein expression increases during the early phases of MK differentiation ( Figure 1A). However, unlike human megakaryopoiesis, mouse PKCe remains highly expressed throughout the entire maturation process, including PLT release (relative densitometric measurements of the samples tested are shown in Figure 1B).…”

Section: Pkc« Levels Are Modulated During Mk Differentiationsupporting

confidence: 93%

“…17 We recently showed that expression of protein kinase Cepsilon (PKCe), a member of the protein kinase C family of serine/ threonine kinases, changes throughout the course of thrombopoietin (TPO)-induced megakaryocyte (MK) differentiation from human CD34 pos cells. 18,19 PKCe has been implicated in both the regulation of cytoskeleton remodeling and the RhoA pathway. 20 Specifically, PKCe, unlike other PKC family members, contains an F-actinbinding region that promotes the formation of F-actin filaments by preventing depolymerization and increasing the rate of actin filament elongation.…”

Section: Introductionmentioning

confidence: 99%

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Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Gobbi

Mirandola

Carubbi

et al. 2013

Blood

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• 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...

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Section: Pkc« Levels Are Modulated During Mk Differentiationsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Gobbi

Mirandola

Carubbi

et al. 2013

Blood

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“…The respective sense and antisense RNA sequences of double-stranded small interfering RNA (siRNA), designed to target human PKCε mRNA (siRNA PKCε ), were synthesized by Silencer siRNA Construction Kit (Ambion, Austin, TX) as previously described (32,33). Nonspecific siRNA duplexes containing the same nucleotides, but in irregular sequence (i.e., scrambled PKCε siRNA), were prepared according to manufacturer's protocol and used as controls.…”

Section: Small Interfering Rna Design and Transfectionmentioning

confidence: 99%

“…Fifty mg proteins from each sample were then migrated in 5% SDS-acrylamide gels and blotted onto nitrocellulose filters. Blotted filters were blocked and incubated with specific primary Abs diluted as previously described (32,33). Specifically, rabbit polyclonal anti-PKCε and anti-b-actin Abs were used at the concentration of 1 mg/ml and diluted 1:5000, respectively.…”

Section: Western Blotmentioning

confidence: 99%

Protein Kinase Cε Regulates Proliferation and Cell Sensitivity to TGF-1β of CD4+ T Lymphocytes: Implications for Hashimoto Thyroiditis

Mirandola

Gobbi

Masselli

et al. 2011

The Journal of Immunology

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We have studied the functional role of protein kinase Cε (PKCε) in the control of human CD4+ T cell proliferation and in their response to TGF-1β. We demonstrate that PKCε sustains CD4+ T cell proliferation triggered in vitro by CD3 stimulation. Transient knockdown of PKCε expression decreases IL-2R chain transcription, and consequently cell surface expression levels of CD25. PKCε silencing in CD4 T cells potentiates the inhibitory effects of TGF-1β, whereas in contrast, the forced expression of PKCε virtually abrogates the inhibitory effects of TGF-1β. Being that PKCε is therefore implicated in the response of CD4 T cells to both CD3-mediated proliferative stimuli and TGF-1β antiproliferative signals, we studied it in Hashimoto thyroiditis (HT), a pathology characterized by abnormal lymphocyte proliferation and activation. When we analyzed CD4 T cells from HT patients, we found a significant increase of PKCε expression, accounting for their enhanced survival, proliferation, and decreased sensitivity to TGF-1β. The increased expression of PKCε in CD4+ T cells of HT patients, which is described for the first time, to our knowledge, in this article, viewed in the perspective of the physiological role of PKCε in normal Th lymphocytes, adds knowledge to the molecular pathophysiology of HT and creates potentially new pharmacological targets for the therapy of this disease.

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“…It is well known that PKCs play an important role in the differentiation of megakaryocytes from cell lines [4][5][6][7][8]. However, the function of specific PKC isoforms, with the exception of " and a, in primary megakaryopoiesis is currently unknown [9][10][11]. A previous report suggests that PKCy mRNA expression is enhanced during megakaryocyte differentiation from progenitor cells, more so than any other PKC isoform [12].…”

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confidence: 99%