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).

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.

Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, hoxb2a, hoxc5a, and hoxd3a showed reduction in the thrombocyte counts. I then screened the other 47 hox genes in the zebrafish genome using flow sorting method and found that knockdown of hoxa9a and hoxb1a also resulted in decreased thrombocyte number. Further, I used the dye DiI, which labels only young thrombocytes at specific concentrations and observed that the knockdown of hoxa10b, hoxb2a, hoxc5a, hoxd3a, hoxa9a and hoxb1a, lead to a decrease in young thrombocytes; whereas hoxc11b knockdown lead to increase in number of young thrombocytes. Using bromodeoxyuridine, I also showed that there is increase in release of young thrombocytes into peripheral circulation in hoxc11b knockdown fish which suggests that hoxc11b significantly promotes cell proliferation rather effecting apoptosis. In conclusion, I found six hox genes that are positive regulators and one hox gene is a negative regulator for thrombocyte development.

No description available.

No description available.

L’objectif de notre travail était de déterminer le rôle joué par l’érythropoïétine et la<p>thrombopoïétine, respectivement, dans l’anémie et la thrombocytopénie observées<p>chez des enfants souffrant d’une hémopathie maligne.<p>Par le dosage simultané de la forme soluble du récepteur de la transferrine et de<p>l’érythropoïétine dans le sérum nous avons montré que l’anémie observée chez ces<p>patients est bien la conséquence d’une réduction du nombre de progéniteurs<p>érythropoïétiques (atteinte médullaire centrale) mais que celle-ci n’est pas la<p>conséquence d’une production insuffisante d’érythropoïétine. Nous avons fait la<p>même observation chez des enfants souffrant d’une tumeur solide non<p>hématologique et chez des patients en cours de traitement par chimiothérapie.<p>Chez ces derniers patients, en appliquant un modèle de culture de moelle à long<p>terme, nous avons pu démontrer l’existence d’une altération du microenvironnement<p>médullaire, probablement induite par la chimiothérapie, se<p>traduisant par une réduction de son aptitude à supporter le développement de la<p>lignée érythroïde. Ceci expliquant au moins partiellement l’inadéquation de la<p>réponse érythropoïétique observée chez ces patients en réponse à l’anémie.<p>Dans la dernière partie du travail, nous avons montré que la thrombocytopénie très<p>fréquemment observée chez les patients leucémiques s’accompagne dans la<p>majorité des cas d’une élévation exponentielle de la concentration de<p>thrombopoïétine, excepté dans les cas de leucémies de la lignée myéloïde. Chez ces<p>derniers la concentration de thrombopoïétine est proche des valeurs observées chez<p>des sujets normaux alors qu’elle devrait être 10 à 100 fois plus élevée compte tenu<p>du nombre de plaquettes extrêmement bas. Nous avons pu montrer que ces taux<p>très bas sont la conséquence de la liaison de la thrombopoïétine à un récepteur<p>spécifique et fonctionnel présent à la surface des cellules leucémiques myéloïdes<p>qui, en l’utilisant comme facteur de croissance, (stimulant leur prolifération et<p>retardant leur mort cellulaire) « consomment » la thrombopoïétine présente dans le<p>sérum. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Introdução: A anemia aplástica (AA) adquirida é uma doença grave, caracterizada por pancitopenia e medula óssea hipocelular sem que haja associação com aumento de reticulina ou infiltração anormal na medula. Embora o mecanismo fisiopatológico não esteja totalmente elucidado, atribui-se a uma resposta imunomediada dos linfócitos T no ambiente medular. A trombopoetina (codificada pelo gene THPO) é um hormônio glicoproteico produzido pelo fígado e responsável pelo estímulo de crescimento de megacariócitos, desenvolvimento plaquetário e de demais linhagens e, quando disfuncional, contribui para o desenvolvimento da AA adquirida. Objetivos: Investigar a presença de variantes genéticas no THPO em amostras de sangue periférico e medula óssea de pacientes com AA adquirida (grupo caso) e de indivíduos saudáveis (grupo controle) e verificar a presença de alterações no número de plaquetas durante o seguimento dos pacientes com AA adquirida. Métodos: O gene THPO foi sequenciado em amostras de DNA de medula óssea de 92 pacientes com AA adquirida e no DNA de sangue periférico de 92 controles, cujas amostras haviam sido previamente armazenado no Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). O sequenciamento foi realizado pelo método de Sanger. Realizou-se também a associação entre a presença (ou ausência) de variantes em THPO e o número de plaquetas em 83 pacientes utilizando o teste ANOVA Para outras análises estatísticas, foram utilizados os testes t e qui-quadrado com nível de significância de 5%. Resultados: Foram encontrados três polimorfismos de nucleotídeo único (SNPs) nos pacientes com AA adquirida (rs956732, rs6141 e rs3804618). Os mesmos três SNPs foram observados nos indivíduos do grupo controle (p>0,05). Não houve associação entre o número de plaquetas e a presença de SNPs nos pacientes (p>0,05). Conclusões: Três SNPs foram encontrados em frequências alélicas semelhantes tanto no grupo de pacientes quanto nos controles, sugerindo que a trombopoetina não apresenta alterações genéticas que possam ser associadas à fisiopatologia da AA adquirida nessa coorte. / Introduction: Acquired aplastic anemia (AA) is a severe illness, characterized by pancytopenia and hypocellular bone marrow without increased reticulin or abnormal infiltration of the bone marrow. Although the physiopathological mechanism has not been completely understood, an immune-mediated T-lymphocyte response has been attributed to the bone marrow environment. Thrombopoietin (encoded by THPO), a glycoprotein hormone produced by the liver and responsible for stimulating the growth of megakaryocytes, development of platelets and other lineages that when dysfunctional, contributes to the progress of acquired AA. Objectives: To screen the THPO gene for genetic variants in bone marrow of acquired AA patients and in the peripheral blood of controls, and to verify the correlation between the THPO status and platelet counts in the patients during the treatment. Method: Sanger sequencing of the THPO gene was carried out in 92 acquired AA patients (case group) and 92 controls, in DNA samples previously stored in the Hematology Laboratory of the Ribeirão Preto School of Medicine at the University of São Paulo. The association between the THPO status and the platelet counts was performed in 83 patients through the ANOVA test. The Chi-squared test and t-test were also applied for statistical analysis with a 5% significance level. Results: Three single nucleotide polymorphisms (SNPs) were found in the AA patients (rs956732, rs6141, and rs3804618), as well as in the healthy subjects (p>0,05). No association was verified between the platelet counts and the presence of SNPs in the AA patients (p>0,05). Conclusion: Three SNPs were found in both groups, suggesting that thrombopoietin does not harbor genetic variants that could be etiological for the acquired AA in our cohort.

Introdução: A anemia aplástica (AA) adquirida é uma doença grave, caracterizada por pancitopenia e medula óssea hipocelular sem que haja associação com aumento de reticulina ou infiltração anormal na medula. Embora o mecanismo fisiopatológico não esteja totalmente elucidado, atribui-se a uma resposta imunomediada dos linfócitos T no ambiente medular. A trombopoetina (codificada pelo gene THPO) é um hormônio glicoproteico produzido pelo fígado e responsável pelo estímulo de crescimento de megacariócitos, desenvolvimento plaquetário e de demais linhagens e, quando disfuncional, contribui para o desenvolvimento da AA adquirida. Objetivos: Investigar a presença de variantes genéticas no THPO em amostras de sangue periférico e medula óssea de pacientes com AA adquirida (grupo caso) e de indivíduos saudáveis (grupo controle) e verificar a presença de alterações no número de plaquetas durante o seguimento dos pacientes com AA adquirida. Métodos: O gene THPO foi sequenciado em amostras de DNA de medula óssea de 92 pacientes com AA adquirida e no DNA de sangue periférico de 92 controles, cujas amostras haviam sido previamente armazenado no Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). O sequenciamento foi realizado pelo método de Sanger. Realizou-se também a associação entre a presença (ou ausência) de variantes em THPO e o número de plaquetas em 83 pacientes utilizando o teste ANOVA Para outras análises estatísticas, foram utilizados os testes t e qui-quadrado com nível de significância de 5%. Resultados: Foram encontrados três polimorfismos de nucleotídeo único (SNPs) nos pacientes com AA adquirida (rs956732, rs6141 e rs3804618). Os mesmos três SNPs foram observados nos indivíduos do grupo controle (p>0,05). Não houve associação entre o número de plaquetas e a presença de SNPs nos pacientes (p>0,05). Conclusões: Três SNPs foram encontrados em frequências alélicas semelhantes tanto no grupo de pacientes quanto nos controles, sugerindo que a trombopoetina não apresenta alterações genéticas que possam ser associadas à fisiopatologia da AA adquirida nessa coorte. / Introduction: Acquired aplastic anemia (AA) is a severe illness, characterized by pancytopenia and hypocellular bone marrow without increased reticulin or abnormal infiltration of the bone marrow. Although the physiopathological mechanism has not been completely understood, an immune-mediated T-lymphocyte response has been attributed to the bone marrow environment. Thrombopoietin (encoded by THPO), a glycoprotein hormone produced by the liver and responsible for stimulating the growth of megakaryocytes, development of platelets and other lineages that when dysfunctional, contributes to the progress of acquired AA. Objectives: To screen the THPO gene for genetic variants in bone marrow of acquired AA patients and in the peripheral blood of controls, and to verify the correlation between the THPO status and platelet counts in the patients during the treatment. Method: Sanger sequencing of the THPO gene was carried out in 92 acquired AA patients (case group) and 92 controls, in DNA samples previously stored in the Hematology Laboratory of the Ribeirão Preto School of Medicine at the University of São Paulo. The association between the THPO status and the platelet counts was performed in 83 patients through the ANOVA test. The Chi-squared test and t-test were also applied for statistical analysis with a 5% significance level. Results: Three single nucleotide polymorphisms (SNPs) were found in the AA patients (rs956732, rs6141, and rs3804618), as well as in the healthy subjects (p>0,05). No association was verified between the platelet counts and the presence of SNPs in the AA patients (p>0,05). Conclusion: Three SNPs were found in both groups, suggesting that thrombopoietin does not harbor genetic variants that could be etiological for the acquired AA in our cohort.

A l’origine de l’hématopoïèse se trouve les cellules souches hématopoïétiques (CSH). Elles constituent un pool de cellules rares présentes dans la moelle osseuse aux niveaux de zones particulières de l’os appelées niche. Les cellules de la niche produisent des cytokines, telles que la thrombopoïétine (TPO), qui régulent les CSH en contrôlant leur quiescence et leur auto-renouvellement. Peu de choses sont connues sur les mécanismes mis en place par la CSH et son environnement pour faire face aux dommages de l’ADN, notamment induits lors de radio- ou chimio-thérapies. Durant cette étude, nous avons mis en évidence un nouveau rôle de la TPO et de son récepteur Mpl dans la réparation de l’ADN des CSH en réponse à des stress génotoxiques. Les CSH déficientes ou haplo-insuffisantes pour Mpl, ou les CSH sauvages et cultivées en absence de TPO, présentent un défaut de réparation et une instabilité génomique. En réponse à l’irradiation, la TPO potentialise l’activation de la voie NF-kB qui permet l’induction du gène précoce Iex-1. La TPO est également l’activateur majeur de la voie ERK dans les CSH. IEX-1 et pERK forment un complexe tripartite avec DNA-PK, une protéine clé de la voie Non Homologous End Joining (NHEJ). La DNA-PK est fortement activée par la TPO, ce qui augmente la fidélité et l’efficacité de la voie NHEJ et permet d’améliorer l’intégrité génomique des CSH. Par ailleurs, nous montrons qu’une simple injection de TPO ou de son agoniste Romiplostim, avant irradiation ou injection de doxorubicine, limite la mutagénèse des CSH et leur perte de fonction associée. Cet effet est spécifique de la TPO, d’autres cytokines comme le SCF et le Flt3-L, n’ont aucun effet sur la réparation. Ces résultats montrent que la TPO contrôle directement les voies de signalisation aboutissant à la réparation de l’ADN des CSH. Ils ouvrent des perspectives nouvelles pour l’utilisation des agonistes de la TPO comme adjuvant protecteur avant radio- ou chimiothérapie pour minimiser les risques de développement de leucémies aigües myéloïdes secondaires. L’expression de Mpl étant haploinsuffisante pour la fonction de réparation de l’ADN, ces résultats suggèrent que Mpl pourrait être tumeur suppresseur en réponse aux traitements chimio-ou radio-thérapeutiques. / Hematopoietic stem cells (HSC) are at the beginning of hematopoeisis. They constitute a pool of rare cells in bone marrow in specifics zones of bones called niche. Niche’s cells produce cytokines, like thrombopoietin (TPO). These cytokines regulates HSC by controlling quiescence and self-renewal. Few are known about mechanism used by HSC and its environment to prevent DNA damage, and especially those induced by radio- or chemo-therapies. In this study, we discover a new role of TPO and its receptor Mpl in DNA repair of HSC in response to genotoxic stress. HSC without Mpl, or wild type HSC cultured without TPO, show an important defect of DNA repair and genomic instability. In response to irradiation, TPO increases activation of NF-KB pathway that increases induction of IEX-1 early gene. TPO is also the major activator of ERK pathway in HSC. IEX-1 and p-ERK can form a tripartite complex with DNA-PK, a key protein of Non homologous end joining pathway (NHEJ). DNA-PK is fully activated by TPO which increase fidelity and efficacy of NHEJ pathway leading to better genomic integrity of HSC. We also show in this study that a simple injection of TPO or Romiplostim before irradiation or Doxorubicin injection, decrease mutagenesis of HSC and their loss of function associated. This effect of TPO is specific of TPO because other cytokines like SCF or Flt3-L have no effect on the DNA repair. These results show that TPO can directly control signaling pathway leading to repair of HSC’s DNA and open new avenues for TPO agonist using. They can be used to protect HSC before radio- or chemo-therapies and to minimize development of secondary acute myeloid leukemia. Expression of Mpl being haplo-insufficient for DNA repair functions, this result suggests that Mpl could be a tumor suppressor in response to radio- or chemo-therapies treatments.