Characterization and Role of PKCε and PKCε in Models of Normal and Aberrant Megakaryocytic Differentiation

La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno. In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica. Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina. Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo. È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine. I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.Protein kinases C (PKC) are known to be ubiquitously distributed and to have pleiotropic effects. Isoforms epsilon (PKCε) and delta (PKCδ) are involved in the regulation of cell growth, survival and differentiation; in particular, they have been also investigated for their role in the hematopoiesis and in aberrant processes of differentiation along the erythroid and megakaryocytic lineages. In this PhD thesis, the results of an in vitro study about the role of these two kinases in models of megakaryocytic (MK) differentiation, both normal and pathological, are presented. The observations about PKCε and PKCδ kinetics show how these proteins have a specific modulation during the MK differentiation that results in an opposite pattern of expression and, in the murine model if compared with the human model, also a reciprocal one. In particular, in human megakaryocytopoiesis, PKCε results down-modulated, whereas in mouse its levels increase. Instead, PKCδ shows a high and steady expression in maturing CD34+ MK committed, but it is strongly down-modulated during the latest phases of platelet maturation in the murine model. The study also elucidates the different pathways PKCε and PKCδ work through, being an inhibitory action of PKCε on RhoA during proplatelets (ppt) formation in the mouse model while, in the human MK differentiation, platelets production is regulated by PKCδ through Bcl-xL. In this dissertation it is also demonstrated how in an aberrant megakaryocytopoiesis, as in the pathologic model of primary myeloproliferative neoplasm (PMF), PKCε is strongly deregulated and it results in an altered Bcl-xL expression. A forced down-modulation of this kinase restores a normal MK differentiation and ppt maturation. Therefore, the data presented show that PKCε and PKCδ play a key role in proper megakaryocyte maturation and that PKCε could be a potential new therapeutic target for PMF

Activation and nuclear translocation of PKCε promotes skeletal muscle cell differentiation via HMGA1 downregulation

The role of novel PKCs in skeletal muscle differentiation has recently emerged. PKCθ is the most expressed isoform of PKCs in muscle and it promotes the fusion of myoblasts [1]. Recently, we have demonstrated that PKCε is implicated in myocardiocyte differentiation of bone marrow mesenchymal stem cells [2] but the role of PKCε in skeletal muscle cell regeneration has only recently emerged [3]. We here demonstrate that both nuclear and cytoplasmic fractions of PKCε are up-regulated during in vitro C2C12 cell line and satellite cell differentiation. We also show that PKCε is able to modulate myogenic differentiation genes via a downmodulation of HMGA1 proteins that promotes myogenin accumulation and mature myoblast formation. To study the effects of PKCε on muscle regeneration, we have used the in vivo model of CTX-induced skeletal muscle injury. We show that the upregulation of PKCε also occurs in vivo, particularly in the centro-nucleated regenerating fibers that are derived from the fusion process of the resident satellite cells, suggesting a role for PKCε in human satellite cell-driven muscle repair and substitution, with clinically relevant implications in human muscle pathology

Joint mobility/muscular chain elasticity and motor coordination in a cohort of 9-11 years school children exposed to specifically designed and professionally guided training

Beside the positive role that an active lifestyle plays in the physical and emotional well-being of a child, physically active children have lower risks to encounter injury as adults. However, many groups have reported that only a small population of children in western countries are sufficiently active (1, 2). The aim of this study was to investigate whether joint mobility/muscular elasticity and coordination were related to a merely active lifestyle or could be significantly improved in the presence of a collective, easy-to-perform, but specifically-designed and professionally-guided school program. Specific functional and anthropometric parameters were single-blind tested on 277 children (aged 9-11 years). 148 were randomly assigned to a school-based physical education program specifically designed to increase coordination and elasticity and supervised by professionals, while 129 (control group) continued their usual physical activity at school, with no specific program. The specific program generated a significant improvement of joint mobility and coordination abilities as compared to non-specific physical activity. As a secondary end-point, gender and BMI-related differences emerged during the study, showing that females respond better to a low intensity program, while males benefit of a higher intensity (or a differently designed) program, particularly when belonging to overweight/ obese BMI classes. These results, building up on those from our and other groups, should orient decision-makers in the area of physical exercise for primary school children in favour of specifically designed programs based on demographic and anthropometric data

PKCε expression is required during proplatelet formation in murine model

Megakaryocytes (MK) remodel their cytoplasm into long proplatelet extensions to generate platelets [1]. We have previously demonstrated that PKCepsilon expression is strictly regulated during megakaryocytopoiesis (MKpoiesis), and its forced expression in the late phases of MK differentiation impairs platelet production [2,3]. However, our preliminary data suggest that PKCepsilon positive platelets may be released around the acute event of myocardial infarction, affecting their aggregation potential. Primary fetal liver (FL) cells isolated from CD1 pregnant mice are the preferential model to study the platelet formation mechanism [4]. Therefore, here we focused on the mouse PKCepsilon positive model to elucidate the role of PKCepsilon in MK maturation. Our data show that not only PKCepsilon expression increases during mouse MK differentiation, but also its forced down-regulation strongly reduces pro-platelet formation. Therefore, PKCepsilon is strongly required for murine proplatelet production. On the basis of these results and other known model systems, we show that PKCepsilon has a relevant role in the completion of platelet release

SARS-CoV-2 omicron (B.1.1.529)-related COVID-19 sequelae in vaccinated and unvaccinated patients with cancer: results from the OnCovid registry

Background COVID-19 sequelae can affect about 15% of patients with cancer who survive the acute phase of SARS-CoV-2 infection and can substantially impair their survival and continuity of oncological care. We aimed to investigate whether previous immunisation affects long-term sequelae in the context of evolving variants of concern of SARS-CoV-2. Methods OnCovid is an active registry that includes patients aged 18 years or older from 37 institutions across Belgium, France, Germany, Italy, Spain, and the UK with a laboratory-confirmed diagnosis of COVID-19 and a history of solid or haematological malignancy, either active or in remission, followed up from COVID-19 diagnosis until death. We evaluated the prevalence of COVID-19 sequelae in patients who survived COVID-19 and underwent a formal clinical reassessment, categorising infection according to the date of diagnosis as the omicron (B.1.1.529) phase from Dec 15, 2021, to Jan 31, 2022; the alpha (B.1.1.7)-delta (B.1.617.2) phase from Dec 1, 2020, to Dec 14, 2021; and the pre-vaccination phase from Feb 27 to Nov 30, 2020. The prevalence of overall COVID-19 sequelae was compared according to SARS-CoV-2 immunisation status and in relation to post-COVID-19 survival and resumption of systemic anticancer therapy. This study is registered with ClinicalTrials.gov, NCT04393974. Findings At the follow-up update on June 20, 2022, 1909 eligible patients, evaluated after a median of 39 days (IQR 24-68) from COVID-19 diagnosis, were included (964 [ 50 center dot 7%] of 1902 patients with sex data were female and 938 [49 center dot 3%] were male). Overall, 317 (16 center dot 6%; 95% CI 14 center dot 8-18 center dot 5) of 1909 patients had at least one sequela from COVID-19 at the first oncological reassessment. The prevalence of COVID-19 sequelae was highest in the prevaccination phase (191 [19 center dot 1%; 95% CI 16 center dot 4-22 center dot 0] of 1000 patients). The prevalence was similar in the alpha-delta phase (110 [16 center dot 8%; 13 center dot 8- 20 center dot 3] of 653 patients, p=0 center dot 24), but significantly lower in the omicron phase (16 [6 center dot 2%; 3 center dot 5-10 center dot 2] of 256 patients, p<0 center dot 0001). In the alpha- delta phase, 84 (18 center dot 3%; 95% CI 14 center dot 6-22 center dot 7) of 458 unvaccinated patients and three (9 center dot 4%; 1 center dot 9- 27 center dot 3) of 32 unvaccinated patients in the omicron phase had sequelae. Patients who received a booster and those who received two vaccine doses had a significantly lower prevalence of overall COVID-19 sequelae than unvaccinated or partially vaccinated patients (ten [7 center dot 4%; 95% CI 3 center dot 5-13 center dot 5] of 136 boosted patients, 18 [9 center dot 8%; 5 center dot 8-15 center dot 5] of 183 patients who had two vaccine doses vs 277 [ 18 center dot 5%; 16 center dot 5-20 center dot 9] of 1489 unvaccinated patients, p=0 center dot 0001), respiratory sequelae (six [4 center dot 4%; 1 center dot 6-9 center dot 6], 11 [6 center dot 0%; 3 center dot 0-10 center dot 7] vs 148 [9 center dot 9%; 8 center dot 4- 11 center dot 6], p= 0 center dot 030), and prolonged fatigue (three [2 center dot 2%; 0 center dot 1-6 center dot 4], ten [5 center dot 4%; 2 center dot 6-10 center dot 0] vs 115 [7 center dot 7%; 6 center dot 3-9 center dot 3], p=0 center dot 037)

PKCε is a regulator of hypertrophic differentiation of chondrocytes in osteoarthritis

OBJECTIVE: Osteoarthritis (OA) is a common and highly debilitating degenerative disease whose complex pathogenesis and the multiplicity of the molecular processes involved, hinder its complete understanding. Protein Kinase C (PKC) novel isozyme PKCε recently proved to be an interesting molecule for further investigations as it can represent an intriguing, new actor in the acquisition of a OA phenotype by the chondrocyte. DESIGN: PKCε was modulated in primary chondrocytes from human OA patient knee cartilage samples by means of short hairpin RNA (ShRNA) and the expression of cartilage specific markers observed at mRNA and protein level. The involvement of Histone deacetylases (HDACs) signaling pathway was also investigated through the use of specific inhibitors MS-275 and Inhibitor VIII. RESULTS: PKCε loss induces up-regulation of Runt-domain transcription factor (RUNX2), Metalloproteinase 13 (MMP13) and Collagen X (COL10) as well as an enhanced calcium deposition in OA chondrocyte cultures. In parallel, PKCε knock-down also leads to SOX9 and Collagen II (COL2) down-modulation and to a lower deposition of glycosaminoglycans (GAGs) in the extracellular matrix (ECM). This novel regulatory role of PKCε over cartilage hypertrophic phenotype is exerted via an HDAC-mediated pathway, as HDAC2 and HDAC4 expression is modulated by PKCε. HDAC2 and HDAC4, in turn, are at least in part responsible for the modulation of the master transcription factors RUNX2 and SOX9, key regulators of chondrocyte phenotype. CONCLUSIONS: PKCε prevents the phenotypic progression of the OA chondrocyte, acting on cartilage specific markers through the modulation of the transcription factors SOX9 and RUNX2. The loss of PKCε enhances, in fact, the OA hypertrophic phenotype, with clear implications in the pathophysiology of the disease

Protein kinase C (PKC)ε and human CD4 T cell proliferation

T-lymphocytes contain up to eight different PKC isotypes and PKCθ has become the most interesting isotype for T-cell activation, proliferation, and transforming growth factor (TGF)-1β signalling [1]. However, It has been suggested that also PKCε may have a role in inflammation and immune-mediated disorders [2]. Thus, we have analyzed the ability of PKCε to control human CD4+ T cell proliferation and their sensitivity to TGF-1β. We demonstrate a nonredundant role of PKCε in CD4+ T cell proliferation triggered in vitro by CD3 stimulation. PKCε sustains NF-kB and, consequently, IL-2 receptor chains transcription and CD25 cell surface expression levels. Moreover, PKCε silencing potentiates the inhibitory effects of TGF-1β, affecting Smad2 phosphorylation levels. Finally, assuming that PKCε could be involved in CD4+ T cell mediated-autoimmune diseases, we have isolated CD4 T cells from Hashimoto Thyroiditis (HT) patients an autoimmune disorder characterized by reduced serum concentration of TGF-1β and TReg cell subsets with defective suppressive functions [3,4]. In HT CD4+ T cells we found a significant increase of PKCε expression, accounting for their decreased sensitivity to TGF-1β. The potentially new roles of PKCε in the pathophysiology of HT and Th/Treg polarization are discussed

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

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