Orientadores: Maria de Fatima Sonati, Carolina Lanaro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T03:33:21Z (GMT). No. of bitstreams: 1 Zaccariotto_TaniaRegina_D.pdf: 2817876 bytes, checksum: 398eb33fe8d04a2605c7871ddf95d10b (MD5) Previous issue date: 2009 / Resumo: As fosfatidilinositol-fosfato quinases (PIPKs) são uma família de enzimas lipídio quinases responsáveis pela produção do segundo mensageiro PI4,5P2 (fosfatidilinositol 4,5 bifosfato), que tem um importante papel regulatório em uma variedade de processos celulares, inclusive na expressão gênica. As PIPKs são classificadas em 3 subfamílias -PIPK I (isoformas a, P e y), PIPK II (a, P e y) e PIPK III - que apresentam distintas funções e localização celular. Recentemente, em estudo desenvolvido em nosso laboratório, o gene da PIPKIIa apresentou-se diferencialmente expresso em reticulócitos de dois irmãos com Doença da Hb H. Sua maior expressão, bem como do gene da globina P, foi encontrada no paciente com concentração de Hb H mais elevada, sugerindo uma relação entre a PIPKIIa e a produção de globinas, particularmente da globina p. No presente trabalho, avaliamos, por PCR Quantitativa em Tempo Real (qRT-PCR), os perfis de expressão dos genes das PIPKs (I e II, isoformas a, P, y, e III) durante a eritropoese em cultura de células CD34+ do sangue periférico de 11 indivíduos sadios e de 6 pacientes com hemoglobinopatias (2 a-talassêmicos, 2 P-talassêmicos e 2 com Anemia Falciforme), comparando-os com os perfis de expressão dos genes das globinas a, P e y, nos dias 7, 10 e 13 da cultura eritróide. Na cultura de células dos indivíduos controles, os resultados revelaram que a expressão de todos os genes PIPKs aumentam durante a diferenciação eritróide e que o perfil de expressão do gene da PIPKIIa coincide com o perfil dos genes de globinas. Nas culturas de células dos pacientes, a expressão do gene da PIPKIIa tornou-se maior durante a diferenciação, enquanto os genes das outras PIPKs apresentaram resultados heterogêneos. O gene PIPKIIa esteve altamente expresso na cultura de células de um dos pacientes a-talassêmicos, enquanto na cultura de um dos P-talassêmicos apresentou expressão reduzida em relação ao controle. Os resultados também foram diferentes entre as culturas de células dos pacientes falciformes. Este é o primeiro estudo sobre o perfil de expressão dos genes dessas PIP quinases durante a eritropoese humana in vitro. Um padrão de normalidade foi estabelecido. Embora os resultados tenham sido heterogêneos entre os pacientes, o que enfatiza a complexidade dos sistemas regulatórios que atuam na formação da hemoglobina, eles fortalecem a hipótese da existência de uma relação entre PIPKIIa e produção de globina ß / Abstract: Phosphatidylinositol-phosphate-kinases (PIPKs), a family of lipid kinases, produce the second messenger PI4,5P2 (phosphatidylinositol 4,5-biphosphate), which regulates various cellular activities, incluing the gene expression. PIPKs are divided into three subfamilies [PIPK I (a, p, y), PIPK II (a, p, y) and PIPK III], which are functionally distinct and located in different subcellular compartments. In a recent study in our laboratory, the PIPKIIa gene was differentially expressed in reticulocytes from two siblings with Hb H disease. Expression of both the PIPKIIa and p globin genes were higher in the patient with the higher Hb H level, suggesting a relationship between PIPKIIa and the production of globins, particularly P-globin. The aim of this study was to determine the gene expression profiles of PIPKs (I and II - with their isoforms - and III) during erythropoiesis in peripheral blood haematopoietic CD34+ cell culture from eleven healthy volunteers and six patients with haemoglobinopathies (2 with a-thalassemia, 2 with P-thalassemia and 2 with sickle cell anaemia) using quantitative real time PCR (qRT-PCR) and to compare these profiles with the gene expression profiles of a, P and y globins on the 7th, 10th and 13th days of culture. The results for the cell cultures from healthy individuals showed that the expression of PIPKs increase during erythroid differentiation and that the PIPKIIa and globin expression profiles are similar. Expression of the PIPKIIa gene increased in the patients' cell cultures during erythroid differentiation, whereas expression of the other PIPK genes varied. PIPKIIa was overexpressed in the cell culture of an a-thalassemic patient, while its expression was reduced in the culture of a P-thalassemic patient. The results also differed between the cultures from sickle cell patients. This is the first study of the gene expression profiles of PIPKs during in vitro human erythroid differentiation. We identified a standard pattern of gene expression in the healthy group. Although the results varied between patients, our findings strengthen the hypothesis of a relationship between PIPKIIa and production of P-globin / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Abstract SWAP-70 originally identified as a signaling protein exclusively expressed in B-cells has been recently described in other cells of the hematopoietic system, such as mast cells and dendritic cells. Here we describe a novel role of SWAP-70 in hematopoiesis, specifically in regulation of erythropoiesis. SWAP-70 protein expression is detected at the stage of the hematopoietic stem cell (HSC). Its expression persists throughout several stages of multipotent and myeloid progenitors. In erythroid development SWAP-70 is found from early committed to erythroid lineage precursors, burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E); however its expression declines with erythroid maturation and it is lastly detectable at the basophilic erythroblast stage. The protein’s deficiency leads to 3-fold increase in HSC numbers in the bone marrow (BM). The lack of SWAP-70 does not affect intermediate myeloid progenitors and the first erythroid committed progenitor, BFU-E. Hematopoietic tissues (BM and spleen) of Swap-70-/- mice carry 2-times less CFU-Es, thus SWAP-70 appears to be important at this stage. Swap-70-/- mice have the same frequencies of later erythroid progenitors, Ter-119+ erythroblasts, in the BM but fewer in the spleen. BM and splenic Ter-119+ erythroid Swap-70-/- compartment (basophilic, polychromatic and orthochromatic erythroblasts) exhibit an altered profile that is characterized by the delayed maturation of cells at the polychromatic stage. SWAP-70 deficiency is not critical for steady state erythropoiesis and does not influence blood homeostasis. Yet SWAP-70 is essential for proper stress response in conditions of anemia. Swap-70-/- mice have normal steady state hematocrite level but fail to restore it after induced anemia, thus showing sluggish blunted response to erythropoietic stress. In resting conditions Swap-70-/- early erythroid progenitors (CFU-Es) exhibit aberrant preactivation of the integrin VLA-4, which supports homotypic and heterotypic interaction within the erythroid niche, and are hyperadhesive to fibronectin. Similarly, Swap-70-/- basophilic erythroblasts are hyperadhesive to splenic tissue. Based on our data and our initial observations we propose a novel function of SWAP-70 in the c-kit signaling pathway and integrin-mediated, i.e. VLA-4, interactions that are important for HSC and erythroid progenitor maintanence and differentiation. Better understanding of mechanisms governing red blood cell development and homeostasis is of high relevance in the context of treatment of anemia, a very common blood disorder, which leads to a wide range of clinical complications and is the most common cancer-associated morbidity.

The cell division cycles of differentiating cells are coordinated so as to generate sufficient numbers of mature cells. The cell cycle may also regulate the process of differentiation, in ways that are not well understood. We previously discovered that during erythropoiesis, the cell cycle is synchronized with a specific developmental switch, where erythroid progenitors known as colony-forming-unit-erythroid (CFU-e) transition from a self-renewal state to a state of erythroid terminal differentiation (ETD). This switch takes place during a single cell cycle S phase and is dependent on S-phase progression. My work shows that this S phase is unusual, in that it is shorter than S phase in preceding cycles, as a result of a global increase in replication fork speed. I found that the CDK inhibitor, p57KIP2, negatively regulates replication fork speed in self-renewing CFU-e, and its down-regulation at the switch to ETD results in S-phase shortening. p57KIP2-mediated inhibition of CDK2 is essential for CFU-e self-renewal. It exerts this effect by reducing replication stress and also reducing the probability of transition from CFU-e to ETD, promoting CFU-e self-renewal instead. CDK2 inhibiting drugs that mimic the action of p57KIP2 stimulate erythropoiesis both in vitro and in vivo, through expansion of the CFU-e pool. In addition to p57KIP2, E2f4 also regulates S-phase shortening and the efficiency of the CFU-e to ETD transition. Overall, my work shows that S-phase speed regulates a key erythroid cell fate decision, and suggests a possible translational application of CDK2 inhibiting drugs in the stimulation of erythropoiesis.

Congenital sideroblastic anemias (CSA) are inherited diseases resulting from defects in heme biosynthesis, mitochondrial iron-sulfur cluster (ISC) assembly, or mitochondrial translation. CSAs are characterized by pathological iron deposits in the mitochondria of bone marrow erythroblasts. Recently the Fleming Lab at Boston Children’s Hospital has reported mutations in HSPA9, a chaperone involved in ISC assembly, as a cause of nonsyndromic CSA. Here we identified a CSA patient harboring two variants in HSCB, encoding a binding partner of HSPA9: a paternally inherited promoter variant (c-134C>A) and a maternally inherited frameshift variant (T87fs) predicted to result in a truncated protein. To better understand the pathophysiology of these variants, we investigated HSCB protein expression and function in patient-derived skin fibroblasts. Patient fibroblasts show evidence of decreased HSCB protein levels. shRNA targeting HSCB was employed to specifically suppress HSCB expression in the K562 erythroid-like cell line model. shRNA-infected K562 cells presented with perturbed iron homeostasis, a shift to glycolytic energy production, and diminished hemoglobinization. Targeted deletion of murine Hscb is embryonic lethal prior day E7.0. Tissue-specific lox-Cre transgenic lines, including Vav-, EpoR- and Mx-Cre demonstrate that Hscb is essential for hematopoiesis and erythropoiesis. Mutant mice present with hematopoietic defects similar to the index patient. Vav-Cre animals die prior to post-natal day 9 with decreased red cell counts, white cell counts, and decreased hemoglobin compared to wild-type animals. Floxed-null EpoR-Cre animals die before embryonic day 13. To excise Hscb specifically in the hematopoietic compartment of adult animals, conditional Mx-Cre animals were generated through bone marrow transplantation and temporally induced with polyinosinic-polycytidylic acid treatment. The animals died 22 days post-injection with decreased red blood cells, white blood cells, hemoglobin, and an overall decline in hematopoiesis of the bone marrow. These data demonstrate that HSCB is required for erythropoiesis and hematopoiesis and that the patient mutations are a pathogenic cause of CSA.

L’érythropoïèse est un processus extrêmement prolifératif, et qui doit donc être très étroitement régulé. L’érythropoïétine (EPO) est l’un des facteurs absolument nécessaires à l’érythropoïèse. Cependant, dans la moelle osseuse, la quantité d'EPO circulante est sous-optimale et la capacité des érythroblastes à survivre dépend donc de leur sensibilité à l'EPO. Les facteurs modulant la réponse à l'EPO au cours de l'érythropoïèse sont encore largement inconnus.Nous avons donc voulu explorer plusieurs facteurs pouvant potentiellement être impliqués dans la régulation de l’érythropoïèse et plus précisément dans la réponse à l’EPO : tout d’abord, la transferrine ainsi que ses récepteurs (TfR), la transferrine et le TfR1 étant également essentiels à l’érythropoïèse, ainsi qu’un phytoestrogène provenant d’une plante nommée Curcuma comosa, les oestrogènes étant eux aussi connus pour favoriser l’érythropoïèse.Concernant la transferrine, nous avons voulu principalement explorer son rôle sur la signalisation, ayant récemment montré au laboratoire que le TfR1, essentiellement connu pour son rôle dans l’endocytose du fer, est également capable d’entraîner une signalisation.Nous avons montré que la transferrine potentialise la stimulation induite par l’EPO des voies ERK, AKT et STAT5. Cet effet est conservé même en l’absence d’endocytose du TfR1. Aucune coopération n’a été trouvée entre la transferrine et le stem cell factor (SCF).Nous avons également observé qu’en l’absence du TfR2, il existe une augmentation de l’expression de l’EPO-R et de la signalisation induite par l’EPO, sans impact de la transferrine dans ce contexte. Par ailleurs, nous avons montré que le Curcuma comosa améliore la prolifération et la différenciation des progéniteurs érythroïdes précoces, par un mécanisme de potentialisation de la signalisation induite par l’EPO impliquant le récepteur aux oestrogènes ER-α.En conclusion, la transferrine et ses récepteurs, ainsi qu’un phytoestrogène et l’ER-α, sont impliqués dans la régulation de l’érythropoïèse via leur action sur la signalisation induite par l’EPO. L’approfondissement de ces données pourrait ouvrir de nouvelles pistes thérapeutiques dans le traitement de l’anémie. / Erythropoiesis is an extremely proliferative process and must be very closely regulated. Erythropoietin (EPO) is one of the major factors necessary for erythropoiesis. However, in the bone marrow, the amount of circulating EPO is suboptimal and the ability of erythroblasts to survive therefore depends on their sensitivity to EPO. The factors modulating the response to EPO during erythropoiesis are still largely unknown. We therefore wanted to explore several factors that could potentially be involved in the regulation of erythropoiesis and more specifically in the response to EPO: first, transferrin and its receptors (TfR), transferrin and TfR1 being also essential for erythropoiesis, as well as a phytoestrogen from a plant called Curcuma comosa, as estrogens are also known to promote erythropoiesis. Regarding transferrin, we mainly wanted to explore its role on signaling, having recently shown in the laboratory that TfR1, essentially known for its role in iron endocytosis, is a signaling-competent receptor. We have shown that transferrin potentiates EPO-induced stimulation of the ERK, AKT and STAT5 pathways. This effect is maintained even in the absence of TfR1 endocytosis. No cooperation was found between transferrin and stem cell factor (SCF). We also observed that in the absence of TfR2, there is an increase in EPO-R expression and EPO-induced signaling, without any impact of transferrin in this context.In addition, we have shown that Curcuma comosa improves the proliferation and differentiation of early erythroid progenitors through a mechanism involving the ER-α estrogen receptor, able to potentiate EPO-induced signaling. In conclusion, transferrin and its receptors, as well as a phytoestrogen and ER-α, are involved in the regulation of erythropoiesis through their action on EPO-induced signaling. Further investigation of these data could provide new therapeutic strategies in the treatment of anemia.

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Sickle cell anemia, caused by a point mutation that affects the HBB gene, is one of the most common human genetic disorders world-wide and has a high morbidity and mortality. A single FDA approved drug, hydroxyurea, is available for its ability to induce fetal hemoglobin expression, a major modulator of disease severity. Not every patient responds to treatment and additional HbF-inducing drugs are needed. In this thesis, I outline an induced pluripotent stem cell-based approach to the study of sickle cell disease (SCD). In the lab, we are currently building a library of SCD-induced pluripotent stem cell (iPSC) lines from a cohort of SCD patients with different genetic backgrounds and fetal hemoglobin levels. Utilizing a directed-differentiation approach, iPSC can give rise to hematopoietic progenitors that are similar to megakaryocyte-erythroid progenitors and can be further specified to become cells of either lineage. I examined the hypothesis that an iPSC-based system would be capable of producing fully functional erythroid cells and also recapitulate the variation in fetal hemoglobin levels seen in SCD patients. Directed-differentiation of iPSCs produced erythroid-lineage cells that were responsive to oxygen levels and erythropoietin, and were capable of further maturation and increased hemoglobin production. A humanized mouse model demonstrated the ability of these cells to localize to the bone marrow, contribute to the peripheral blood, and survive in vivo for over two weeks. The maturation capability of SCD-specific iPSC-derived erythroid lineage cells was correlated with hemoglobin expression and compared to control cells. Characterization of in vitro and in vivo differences between control and SCD-specific iPSC-derived erythroid-lineage cells demonstrated variation amongst individuals, similar to the variation seen in patients. Both of these patient-specific iPSC-based in vitro and in vivo models allow for the examination of the effect of genetic variability on fetal hemoglobin expression and also for the modeling of patient-specific responses to drug treatment. This information will facilitate better clinical treatment of the disease.

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