Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

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L’hématopoïèse est le processus physiologique qui permet le développement de l’ensemble des cellules sanguines matures, leur renouvellement et leur homéostasie tout au long de la vie. L’hématopoïèse est généralement décrite de façon hiérarchique avec, au sommet, les cellules souches hématopoïétiques qui s’autorenouvellent et se différencient en progéniteurs puis en cellules matures. La voie de signalisation NOTCH canonique, contrôle l’activité du facteur de transcription RBPJ. Elle joue un rôle dans le développement des lymphocytes T et la spécification de la différenciation des cellules souches hématopoïétiques normales vers la lignée mégacaryocytaire. Les protéines de la famille OTT1 (OTT1, OTT3 et SHARP) s’expriment de façon ubiquitaire et sont impliquées dans le contrôle de l’activité de RBPJ. Les modalités de régulation de ces activités et l’intégration de signaux provenant d’autres voies de signalisation sont mal caractérisées. L’utilisation d’un modèle de différenciation in vitro de cellules souches hématopoïétiques sur des cellules stromales (OP9) exprimant le ligand NOTCH Delta-like 1 (DL1) ainsi que l’utilisation de modèles murins, nous a permis de montrer un lien entre la voie NOTCH et la voie PI3K/AKT dans le développement mégacaryocytaire. Nos résultats indiquent que la différenciation mégacaryocytaire peut être engagée à partir de progéniteurs myéloïdes engagés dépendant principalement de la voie PI3K/AKT, mais également directement à partir de cellules souches hématopoïétiques pour lesquelles une activation de la voie PI3K/AKT conduit à une synergie avec la voie NOTCH, mais n’est pas essentielle à la spécification mégacaryocytaire. D’autre part, pour comprendre le mécanisme de régulation de la protéine OTT1, j’ai recherché ses partenaires protéiques par crible double hybride chez la levure, et identifié des interactions avec, entre autres, des protéines à activité tyrosine kinase de la famille SRC (dont LYN) et SHARP. La spécificité d’interaction entre OTT1 et LYN a été validée dans un modèle de surexpression ainsi que dans une lignée modélisant la leucémie aigüe mégacaryocytaire. Dans nos modèles, l’interaction avec LYN conduit à la phosphorylation d’OTT1. Les analyses fonctionnelles préliminaires n’ont pas permis à ce jour de mettre en évidence un rôle essentiel de cette interaction dans le développement mégacaryocytaire. / Hematopoiesis is generally described as a hierarchical system, with at the top hematopoietic stem cells which self-renew and differentiate in progenitors, then in mature cells. Canonical Notch signaling controls RBPJ transcriptional activity. It plays a role in T lymphocyte development and stem cell fate. OTT1 family proteins (OTT1, OTT3 and SHARP) are expressed ubiquitously and are implied in control of RBPJ activity. The regulation of these activities and signal integration are all not well characterised. The use of an in vitro model of differentiation for hematopoietic stem cells on OP9 stroma cells expressing the NOTCH Delta-like-1 (DL1) ligand and the use of murine models, allowed us to show a link between NOTCH and PI3K/AKT in megakaryocytic development. Our results indicate that megakaryocytic differentiation can be engaged from myeloid progenitors depending mostly on the PI3K pathway but also from hematopoietic stem cells for which, an activation of PI3K/AKT lead to a synergy with NOTCH, but is not essential for megakaryocytic specification. On the other hand, to understand OTT1’s mechanisms of regulation, I looked for proteic binding partners by the double hybrid screen technique. Among the candidates I identified SHARP and SRC family kinases as LYN. The specific interaction between OTT1 and LYN was validated in a overexpression model and in a cell line modeling acute megakaryoblastic leukemia. In our models, the interaction with LYN lead to the phosphorylation of OTT1. However, the first analysis did not point out an essential role of this interaction in megakaryocytic development.

by Chuen Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 134-166). / Abstracts in English and Chinese. / ACKNOWLDEGEMENT --- p.ii / PUBLICATIONS --- p.iii-iv / ABBREVIATIONS --- p.v-viii / INDEX FOR FIGURES --- p.ix xii / INDEX FOR TABLES --- p.xiii / ABSTRACT (Chinese and English) --- p.xiv-xvi / TABLE OF CONTENT --- p.xvii / Chapter 1. --- INTRODUCTION --- p.1-37 / Chapter 2. --- OBJECTIVES --- p.38-40 / Chapter 3. --- METHODS AND MATERIALS --- p.41 -70 / Chapter 4. --- RESULTS AND DISCUSSION --- p.71-130 / Chapter 5. --- GENERAL DISCUSSION AND CONCLUSION --- p.131-133 / BIBLIOGRAPHY --- p.134-166

The aim of this project was to investigate the transcriptome of human haematopoietic stem cells (HSCs), megakaryocytes and platelets to gain insights into steady state and accelerated thrombopoiesis that occurs in states of haemostatic demand and in thrombosis by applying these findings to the pathological setting of acute coronary thrombosis. To investigate transcriptional heterogeneity within the human HSC population, single cell RNA sequencing was performed in human bone marrow HSCs. Transcriptionally distinct subpopulations were identified including two megakaryocyte biased subsets with potentially differing functional relevance. Both populations expressed megakaryocyte specific transcripts, one of which also co-expressed common myeloid and megakaryocyte-erythroid progenitor transcripts while the other did not. This study represents the first interrogation of the human bone marrow megakaryocyte transcriptome. Cells were collected from healthy human bone marrow and analysed by low input and single cell RNA sequencing. To identify novel drivers of megakaryocyte maturation, the human bone marrow megakaryocyte transcriptome was compared to that of megakaryocytes cultured from human CD34+ cells, a process known to generate immature megakaryocytes. Transcriptional signatures associated with increasing megakaryocyte ploidy were then investigated. Increasing megakaryocyte ploidy level was found to be associated with an upregulation of transcripts involved in translation and protein processing as well as expression of a number of transmembrane receptors which might have functional relevance. Finally, the pathological setting of acute coronary thrombosis was used as a model for accelerated thrombopoiesis. Megakaryocyte and platelet transcriptomes were compared between patients with acute myocardial infarction (AMI) as well as severe coronary disease and a control group. The transcriptional signature relating to disease compared to control in megakaryocytes included upregulation of platelet activation related transcripts in megakaryocytes isolated from patients with AMI and severe coronary artery disease.

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow (BM) microenvironment in vivo is of considerable interest. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is known as master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions, but has also been shown to regulate a process of differentiation and tissue adaptation in various cell types. The activation of NFAT is dependent on the calcium level within the cell. In resting cells, calcium levels are low and NFAT is cytoplasmic and inactive. A sustained increase in the internal calcium concentration within an external stimuli leads to activation of the calcium-dependent calcineurin, followed by dephosphorylation and nuclear translocation of NFAT. We have previously shown that NFATc2, a member of the NFAT family, is expressed in CD34+ hematopoietic stem cells (HSC). A mouse model harboring NFATc2 deficiency provides the opportunity for in vivo investigation of the role of NFATc2 in hematopoiesis. Our recent observations showed that aged mice lacking the transcription factor NFATc2 develop peripheral blood anemia and thrombocytopenia, BM hypoplasia and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic stem cells ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the BM hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immuno-magnetically purified NFATc2-deficient (KO) HSCs to lethally irradiated wild type (WT) mice, and vice versa. After a post-transplantation period of 6-8 months, peripheral blood, BM as well as spleen and liver of the transplanted animals were analyzed and compared to WT and KO mice transplanted with control cells. Transplantation of NFATc2-deficient HSCs into WT recipients (KO WT) induced similar hematological abnormalities as those occurring in non-transplanted KO mice or in KO mice transplanted with KO cells (KO KO). Compared to WT mice transplanted with WT cells (WT WT), KO WT mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their BM. Likewise, KO WT mice developed clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in WT WT control animals. In addition to the hematopoietic abnormalities, transplantation of NFATc2-deficient HSC also induced osteogenic abnormalities such as BM sclerosis and fibrosis in WT mice. This phenomenon was rather subtle and of incomplete penetrance, but never seen in mice transplanted with WT cells. These data demonstrate for the first time, that the NFATc2 transcription factor directly regulates the intrinsic function of hematopoietic stem cells in vivo. However, the transcriptional targets for NFAT in these cells are yet unknown. In addition to hematopoietic stem cells, NFATc2 has been shown to be expressed in a lineage-specific manner during myeloid differentiation and, notably, is maintained during megakaryopoiesis while it is suppressed during the differentiation of neutrophils. Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary hemostasis, thrombosis and wound healing. The biological role of NFAT in megakaryocytes is unknown. We have recently shown that NFATc2 is not necessary for megakaryocytic differentiation. On the other hand, recent evidence suggests that NFATc2 is required for the transcription of specific megakaryocytic genes. In this study, we showed that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FASLG), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FASLG was detectable as early as four hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT. To investigate the physiological relevance of FASLG expression on megakaryocytes, we performed co-cultures of megakaryocytes with Fas-expressing T-lymphocytes, in which CMK cells were left either unstimulated or pre-stimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FASLG or an antagonistic antibody to Fas during the co-culture period, indicating involvement of the FASLG/Fas apoptosis pathway. These results represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FASLG-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue. Finally, we have identified by RNA sequencing analysis of NFATc2-expressing and -deficient cells, the entire set of genes which is induced by NFATc2 in stimulated megakaryocytes. Functional pathway analysis suggests an involvement of NFATc2 in pro-inflammatory pathways in these cells. The significance of these findings has to be addressed in further studies.

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes can induce osteoblast proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of megakaryocytes with another cell-type of mesenchymal origin - the fibroblast. Our findings implicate the involvement of fibronectin/RGD-binding integrins including á3â1 (VLA-3) and á5â1 (VLA-5) as well as glycoprotein IIb (CD41), all of which are known to be expressed on megakaryocyte membranes. Furthermore, we demonstrate that IL-3 can enhance megakaryocyte-induced osteoblast activation in vitro, as demonstrated in the megakaryocyte-fibroblast model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous.

A new paradigm has evolved in which multiple regulatory interactions between the skeletal and hematopoietic systems have been identified. Previous studies have demonstrated that megakaryocytes (MK) play a dual role in skeletal homeostasis by stimulating osteoblast proliferation and simultaneously inhibiting osteoclast (OC) development. Here we identify a novel regulatory pathway in which the main MK growth factor, thrombopoietin (TPO), directly regulates osteoclastogenesis. To study the role of TPO in OC development, spleen or bone marrow (BM) cells (2x10[exponent]6 cells/ml) or BM macrophages (BMM, 1x10[exponent]5 cells/ml) from C57BL/6 mice , as a source of OC precursors, were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) to induce OC formation. TPO (0.1-1000 ng/ml) and/or primary MK (0-0.5%), derived from C57BL/6 fetal livers, were titrated into these cultures and OC were identified as tartrate resistant acid phosphatase positive (TRAP+) giant cells with >3 nuclei. There was a significant, up to 15-fold reduction in OC formed when MK were added to all OC generating cultures, p < 0.001. Moreover, if OC generating cultures did not contain MK or MK progenitors, TPO treatment significantly enhanced OC formation up to six-fold, p < 0.01. This data demonstrates that MK are responsible for the inhibition of OC formation and that in cultures containing MK or MK progenitors such as BM or spleen cells, that TPO acts indirectly to inhibit OC formation by stimulating megakaryopoiesis, whereas in the absence of MK or MK progenitors TPO directly enhances OC formation. This conclusion is further supported by Real-Time PCR data which demonstrates that OC progenitors express c-mpl, the TPO receptor, albeit at low levels when compared to expression of c-mpl on MK. Finally, we have begun to dissect the c-mpl signaling pathway in OC progenitors. We have found that TPO induces tyrosine phosphorylation of several specific cellular proteins in the JAK/STAT pathway. Thus, TPO acts in a somewhat paradoxical manner by inhibiting OC formation through the stimulation of MK, while simultaneously playing a direct role in enhancing osteoclastogenesis.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 102-114).