L’érythropoïèse est le processus de formation des globules rouges. L’anémie demeure à l’heure actuelle un problème de santé publique majeur. Par conséquent, une meilleure compréhension des mécanismes impliqués dans le contrôle de ce processus dans des conditions physiologiques et pathologiques, ainsi que l’établissement de stratégies thérapeutiques ciblées constituent un enjeu de recherche majeur. Le récepteur de la transferrine 1 (CD71/RTf1) est un élément essentiel de l'érythropoïèse, la majorité des travaux de recherche étant focalisés sur son rôle indéniable dans le métabolisme du fer. Cependant, de nouveaux ligands du RTf1 ont été découverts ouvrant de nouvelles perspectives relatives aux fonctionnalités de ce récepteur. Ayant démontré que le RTf1 fixait les immunoglobulines A1 (IgA1), nous nous sommes intéressés au rôle des IgA1 dans l’érythropoïèse. Nous montrons que le RTf1 lié aux polymères d'IgA1 (pIgA1) induit la croissance et une augmentation de la prolifération des érythroblastes en concentration sous-optimale d'érythropoïétine (Epo). De même, l'expression transgénique d’IgA1 humaine (souris alpha1-KI), ou le traitement de souris de type sauvage avec les pIgA1 permettent une récupération accélérée de l’anémie aiguë. L’engagement du RTf1 module la sensibilité à l'Epo, en diminuant le seuil d'activation cellulaire, et en induisant les voies de signalisation MAPK/ERK et phosphatidylinositol-3-kinase (PI3K/AKT). Ces données mettent en évidence un nouveau rôle du RTf1 en tant que régulateur positif de l'érythropoïèse. Parallèlement au RTf1, nous avons identifié un autre récepteur pouvant constituer une cible thérapeutique pour corriger une érythropoïèse inefficace : le récepteur de l’activine de type IIA (ActRIIA). Dans un modèle murin de Beta-thalassémie intermédiaire (Hbbth1/th1), résultant d'une déficience génétique de la chaîne Beta de la globine, nous montrons que l'administration d'une protéine de fusion constituée du domaine extracellulaire de l’ActRIIA lié à un fragment Fc d’IgG de souris (RAP-011), corrige l'anémie, augmente le taux d'hémoglobine et diminue la splénomégalie. Ce traitement favorise l’érythropoïèse splénique et diminue la saturation de la transferrine et l’hémolyse. Fait intéressant, des niveaux élevés de GDF11 (Growth Differentiation Factor 11) sont observés sur des coupes spléniques de souris thalassémiques ainsi que dans le sérum de patients thalassémiques. In vivo, l’inhibition de l’interaction GDF11/ActRIIa par le RAP-011 favorise l’apoptose des érythroblastes précoces par la voie Fas/FasLigand. Ces résultats suggèrent que l’activation constitutive des signaux GDF11/ActRIIA contribue à l’établissement d’une érythropoïèse inefficace caractéristique de la Beta-thalassémie. La neutralisation de cette signalisation inverse ce processus. En conclusion, nos travaux ouvrent de nouvelles perspectives dans la compréhension de l'hématopoïèse normale et pathologique, et pourraient conduire à envisager des traitements innovants pour l'anémie. / Anemia produced by a variety of underlying causes is the most common disorder of the blood, and remains a major global public health problem associated with a poor quality of life for many patients. Thus, better understanding the erythroid process in physiological and pathological conditions, and developing new strategies to boost erythropoiesis appear of great interest. Transferrin receptor 1 (CD71/TfR1) plays an essential role in erythropoiesis, and investigations of TfR1 functions have been focused on their undeniable role in iron metabolism. However, recent data demonstrate that TfR1 is a multi-ligand receptor that participates in a wide array of cellular functions. We have identified TfR1 as a receptor for A1 isotype immunoglobulins (IgA1). In this work, we show that pIgA1s are able through their interaction with the TfR1, to stimulate erythropoiesis by sensitizing erythroblasts to Epo. Likewise, transgenic expression of human IgA1 (Alpha1-KI mice) or treatment of wild-type mice with pIgA1 accelerated recovery from acute anemia. TfR1 engagement by pIgA1 increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These findings unveiled a new role of TfR1 as a signaling competent molecule positively regulating erythropoiesis. In addition to TfR1, our work identifies another receptor as a putative target for correcting ineffective erythropoiesis: the activin receptor IIA (ActRIIA). Indeed, using a mouse model of Beta-thalassemia intermedia (Hbbth1/th1) resulting from a genetic deficiency of Beta-globin chain, we show that administration of a ligand trap (named RAP-011), consisting in a fusion protein between the extracellular domain of ActRIIA and the Fc fragment of a mouse IgG, improves anemia, increases total hemoglobin levels and decreases splenomegaly. In addition, targeting ActRIIa signaling corrects ineffective erythropoiesis in the spleen, reduces hemolysis and transferrin saturation. Interestingly, high levels of Growth Differentiation Factor 11 (GDF11) are detected in spleen sections from Beta-thalassemic mice, as well as in sera from thalassemic patients. In addition, the inactivation of GDF11 promotes terminal erythroblast differentiation. Finally, blockade of the GDF11/ActRIIa signaling, promotes premature apoptosis of early erythroblasts through induction of Fas/FasLigand pathway. Therefore, these results first suggest that constitutive GDF11/ActRIIa signaling pathway may promote ineffective erythropoiesis in Beta-thalassemia intermedia, and secondly, support the use of ActRIIa traps for the treatment of chronic anemia and ineffective erythropoiesis. Altogether, these results open new perspectives in the understanding of normal and pathological hematopoiesis and lead to propose innovative treatments for anemia.

A síndrome de emagrecimento progressivo (SEP) é responsável por elevada morbidade e mortalidade de calitriquídeos mantidos em cativeiro em diferentes instituições. Essa síndrome representa um desafio aos médicos veterinários por suas características ainda pouco esclarecidas e são poucos os estudos multidisciplinares que visam à avaliação dos diferentes sistemas, como os órgãos linfo-hematopoiéticos. O objetivo foi caracterizar a evolução e duração da SEP, associando os dados clínicos, laboratoriais e anatomopatológicos dos órgãos linfo-hematopoiéticos de saguis naturalmente acometidos por SEP no cativeiro. Foram analisadas as fichas clínicas, necroscópicas e os resultados das amostras de sangue e urina de 47 saguis doentes, Callithrix spp., machos e fêmeas, que foram a óbito devido a SEP e eram provenientes de dois criadouros diferentes do estado de São Paulo. Os fragmentos dos órgãos linfo-hematopoiéticos, intestino delgado e do fígado foram processados e avaliados. Os resultados caracterizaram que a SEP acomete calitriquídeos de espécies diferentes, adultos, sem predisposição sexual, mantidos sob condições estáveis de manejo por em média 42 meses e a duração clínica varia de 41 dias a 1 ano e 7 meses. As características clínicas na fase inicial foram predominantemente sinais gastrintestinais e na fase terminal, sinais gastrintestinais e extra-intestinais. A anemia macrocítica normo ou hipocrômica com policromasia, esferocitose, presença de corpúsculos de Heinz e hemoglobinúria foi a alteração hematológica mais frequente. As lesões dos órgãos linfo-hematopoiéticos foram características de anemia hemolítica ou foram inespecíficas e reacionais caracterizadas por hiperplasia ou depleção das células da medula óssea, baço e linfonodo e lesões degenerativas no fígado. Na SEP, a associação clínica, laboratorial e anatomopatológica possibilitou a caracterização da evolução e duração clínica, da anemia e das alterações dos órgãos linfo-hematopoiéticos, cujas lesões foram consideradas secundárias à desnutrição crônica e progressiva decorrente da severa enterite atrófica. / Wasting marmoset syndrome (WMS) causes high morbidity and mortality of marmosets and tamarins kept in captivity in different colonies. WMS challenges the veterinarian due to its unclear and not established features and there are few multidisciplinary studies that carried out an evaluation of different systems, such the lymphohematopoietic system. The aim was described the duration and evolution of illness based on an association of clinical, laboratory and pathological aspects of WMS. Medical record, laboratory data and pathological findings were analyzed of 47 Callithrix spp., males and females, sick marmosets that died due to WMS in two different colonies in São Paulo state. Tissue samples of small intestine, lymphohematopoietc system and liver were histological processed and evaluated. The results showed that WMS affects adult marmosets of different species; there are no sex-related differences, and the marmosets are at least 42 months under similar general management at colony. The clinical duration of WMS is from 41 days to 1 year and 7 months. The clinical features were gastrointestinal symptoms in the beginning and extra-gastrointestinal and gastrointestinal signs in the end. Normochromic or hypochromic macrocytic anemia with polychromasia, spherocytes, Heinz bodies, and hemoglobinuria is the common hematological result. The lymphohematopoietic system lesions were the common findings of hemolytic anemia or unspecific and reacting features such as hyperplasia or depletion of cell numbers of bone marrow, spleen and lymph node, and degenerative lesions of liver. The clinical, laboratory and pathological association allowed the characterization of evolution and duration of the WMS, the anemia and the lesions of lymphohematopoietic organs which lesions were considered secondary to chronic and progressive malnutrition as a result of severe atrophic.

The Imitation Switch (ISWI) nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells (HSPCs) accumulated but their maturation towards erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S-18) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4- OHT-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis.

As síndromes talassêmicas (?- e ?-talassemia) são as desordens mais comuns e frequentes associadas com eritropoese ineficaz. O desbalanço na produção das cadeias ?- e ?-globinas resulta no comprometimento da produção de eritrócitos, em anemia e aumento de progenitores eritroides no sangue periférico. Enquanto os pacientes homozigóticos afetados por essas desordens demonstram alterações características dos parâmetros relacionados a eritropoese, a relação entre grau de anemia, eritropoese alterada e disfunção do metabolismo de ferro ainda não foram investigados nos indivíduos com ?+-talassemia heterozigótica ou ?+-talassêmia. Duzentos e vinte seis indivíduos (75 do gênero feminino e 151 do gênero masculino) foram recrutados e divididos em 5 grupos: Controle (n=28), doadores de sangue regulares (DSR, n=23), ?+-talassemia heterozigótica (TAT, n=14), ?+-thalassemia (traço ?-talassêmico, TBT, n=20) e ?0-talassemia, (?-talassemia maior, BTM, n=27). As amostras foram analisadas para parâmetros hematológicos (Micros ABX 60); ferro sérico, capacidade total de ligação ao ferro e saturação de transferrina por método colorimétrico (Pointe Scientific, Inc., Canton, MI, USA), ferritina e proteína C-reativa ultra sensível por imunoensaio (Immulite 1000); receptor solúvel de transferrina, eritropoetina, fator de diferenciação do crescimento 15 (R&D Systems) e hepcidina (Intrinsic LifeSciences, La Jolla, CA) por ELISA. As razões sTfR/log ferritina e (hepcidina/ferritina)/sTfR foram calculadas para avaliar o metabolismo do ferro. sTfR/log ferritina pode distinguir depleção dos estoques de ferro de eritropoese deficiente de ferro, enquanto (hepcidina/ferritina)/sTfR pode avaliar os estímulos contrários (disponibilidade de ferro e atividade eritropoética) que controlam a síntese de hepcidina e a absorção de ferro, na ausência de estímulos inflamatórios. Foi demonstrado que TAT teve significativa redução da hepcidina e aumento do receptor solúvel de transferrina, com parâmetros hematológicos relativamente normais. Em contraste, todos os parâmetros hematológicos de TBT foram significativamente diferentes do Controle, incluindo aumento dos níveis do receptor solúvel de transferrina, ferritina, eritropoetina e fator de diferenciação do crescimento 15. Essas alterações em ambos os grupos sugerem um balanço alterado entre eritropoese e metabolismo de ferro. Os índices sTfR/log ferritina e (hepcidina/ferritina)/sTfR estão, respectivamente, aumentado e reduzido comparados ao Controle, proporcional a severidade de cada grupo talassêmico. Em conclusão, destacamos que, pela primeira vez, foram descritas alterações no metabolismo de ferro em indivíduos com ?+-talassemia heterozigótica. Esses dados demonstram que, no contexto da saúde pública, são necessários identificação e acompanhamento dos portadores de ?+-talassemia. / The thalassemia syndromes (?- and ?-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of ?- or ?-globin chain production results in impaired red blood cell synthesis, anemia and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis and dysfunctional iron metabolism have not been investigated in both carriers of ?-thalassemia and ?-thalassemia. 226 subjects (75 females and 151 males) were recruited to this study and divided in 5 groups: Control (n=28), repeat blood donors (DSR, n=23), ?+-thalassemia heterozygous carriers (TAT, n=14), ?+-thalassemia (?-thalassemia trait, TBT, n=20) and ?0-thalassemia, (?-thalassemia major, BTM, n=27). Samples were tested for hematological parameters (Micros ABX 60); serum iron, total iron binding capacity, and transferrin saturation by the colorimetric method (Pointe Scientific, Inc., Canton, MI, USA), ferritin and high sensitive C-reactive protein by immunoassay (Immulite 1000); soluble transferrin receptor, erythropoietin and growth differentiation factor 15 (R&D Systems) and hepcidin (Intrinsic LifeSciences, La Jolla, CA) by ELISA. Were calculated the ratios sTfR/log ferritin and (hepcidin/ferritin)/sTfR to evaluate iron metabolism. sTfR/log ferritin can distinguish storage iron depletion from iron-deficient erythropoiesis, while (hepcidin/ferritin)/sTfR can be utilized to explore and quantify the opposing forces (i.e. iron availability and erythropoietic activity) regulating hepcidin synthesis and iron absorption in absence of inflammatory stimuli. We demonstrate that TAT have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, TBT have all hematological parameters significantly different from controls, including increased soluble transferrin receptor, ferritin, erythropoietin and growth differentiation factor 15 levels. These changings in both groups suggest an altered balance between erythropoiesis and iron metabolism. The indexes sTfR/log ferritin and (hepcidin/ferritin)/sTfR are respectively increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we emphasize that, for the first time in the literature, subjects with heterozygous ?+-thalassemia have altered iron metabolism. Our data demonstrate that within the context of public health, identification and monitoring of patients with ?+-thalassemia are needed.

Abstract Adequate oxygen supply is necessary for aerobic cell survival. Cellular oxygen deprivation, also known as hypoxia, leads to various responses that aim to increase cellular oxygen delivery and reduce oxygen consumption. Oxygen homeostasis is mainly regulated by the hypoxia-inducible factor (HIF), which regulates the expression of over 300 genes in response to hypoxia. The stability of HIF is regulated by the HIF prolyl 4-hydroxylases (HIF-P4Hs), enzymes that catalyze the hydroxylation of proline residues in HIFα subunits and target them towards proteasomal degradation. HIF-P4Hs require oxygen as a cosubstrate for the reaction, allowing for hypoxic HIF stabilization and target gene induction at low oxygen concentrations. In this study we investigated the role of HIF-P4H-2 in the regulation of red blood cell production, erythropoiesis. We showed that Tibetans living at high altitude have genetically adapted to their hypoxic environment via mutations in the gene encoding for HIF-P4H-2. The Tibetan HIF-P4H-2 D4E,C127S variant showed enhanced hydroxylation of HIFα at low oxygen concentrations, resulting in reduced HIFα protein stabilization under hypoxia. In other studies we used a genetically modified HIF-P4H-2 hypomorphic mouse line which expresses a reduced amount of wild-type Hif-p4h-2 mRNA in tissues. We showed that these mice develop mild age-dependent erythrocytosis due to splenic extramedullary erythropoiesis, which is independent of serum erythropoietin concentration. In addition, these mice were protected against inflammation-induced anemia, a condition commonly seen in patients with inflammatory diseases. The HIF-P4H-2 hypomorphic mice also had altered basal metabolism in their skeletal muscles, which, together with an increase in mean capillary area, reduced their infarct size after skeletal muscle ischemia-reperfusion injury. These studies suggest that pharmacological HIF-P4H-2 inhibition may provide a novel treatment for EPO-resistant anemias and peripheral artery disease. / Tiivistelmä Riittävä hapensaanti on välttämätöntä aerobisten solujen selviytymiselle. Solun alentunut hapen määrä, toiselta nimeltään hypoksia, johtaa useisiin vasteisiin joiden tarkoituksena on turvata solun hapensaanti ja vähentää hapenkulutusta. Happitasapainoa säätelee hypoksiassa indusoituva tekijä (HIF), joka aktivoi yli 300 geenin luentaa hypoksisissa oloissa. HIFα:n määrää soluissa säätelevät HIF prolyyli-4-hydroksylaasientsyymit (HIF-P4H:t), jotka hydroksyloivat proliini-aminohappotähteitä HIFα-alayksiköissä ja ohjaavat ne proteasomaaliseen hajotukseen. HIF-P4H:t tarvitsevat reaktiossa happea mahdollistaen HIF:n stabilisaation ja kohdegeenien lisääntyneen luennan matalassa hapen osapaineessa. Tässä tutkimuksessa selvitimme HIF-P4H-2-entsyymin roolia punasolujen muodostuksen eli erytropoieesin säätelyssä. Osoitimme, että korkealla vuoristossa asuvat tiibetiläiset ovat geneettisesti sopeutuneet hypoksiseen elinympäristöönsä johtuen HIF-P4H-2-entsyymiä tuottavan geenin mutaatiosta. Tiibetiläisiltä löytynyt HIF-P4H-2D4E,C127S variantti hydroksyloi tehokkaammin HIFα-alayksiköitä matalassa hapen osapaineessa johtaen vähäisempään HIFα-alayksiköiden stabiloitumiseen hypoksiassa. Muissa tutkimuksissamme käytimme geneettisesti muunneltua HIF-P4H-2-hiirikantaa, joka tuottaa alentunutta määrää villityypin Hif-p4h-2 lähetti-RNA:ta kudoksissaan. Näille hiirille kehittyi ikäriippuvaisesti lievä punasoluylimäärä eli erytrosytoosi johtuen pernan kiihtyneestä punasolutuotannosta riippumatta seerumin erytropoietiinikonsentraatiosta. Lisäksi nämä hiiret olivat suojassa tulehduksen aiheuttamalta anemialta, joka on yleinen ilmiö tulehduksellisista sairauksista kärsivillä potilailla. HIF-P4H-2-muuntogeenisten hiirten lihasten energia-aineenvaihdunta oli muuttunut siten, että se yhdessä suurentuneen keskimääräisen kapillaaripinta-alan kanssa pienensi vaurioituneen kudoksen pinta-alaa alaraajaiskemia-altistuksen jälkeen. Nämä tutkimukset osoittavat, että lääkkeellinen HIF-P4H-2-entsyymin estäminen on mahdollinen uusi hoitomuoto erytropoietiinille resistenteissä anemioissa sekä alaraajojen valtimoahtaumataudissa.

Abstract Erythropoiesis is the process of red blood cell production. The main regulator is the erythropoietin (EPO) hormone, which is strongly upregulated in low oxygen concentration (hypoxia) in cells via the hypoxia-inducible transcription factor HIF. The stability of HIF is regulated in an oxygen-dependent manner by three HIF prolyl 4-hydroxylases, all of which are known to participate in the regulation of erythropoiesis. A role in erythropoiesis of a fourth prolyl 4-hydroxylase, P4H-TM, which possesses a transmembrane domain, is not known, but it is able to hydroxylate HIF at least in vitro and in cellulo. The role of P4H-TM in erythropoiesis was studied by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null, Hif-p4h-3 null, and Hif-p4h-2 hypomorph mouse lines. The current study suggests that P4H-TM is involved in the regulation of EPO production, hepcidin expression and erythropoiesis. P4H-TM can thus be a new target for inhibition when designing novel pharmacological treatment strategies for anemia. LOX is required for crosslink formation between lysine residues in fibrillar collagens and elastin. These crosslinks enhance the tensile strength of collagen fibers and provide elasticity to elastic fibers and thus generate important structural support for tissues. LOX is required for normal embryonic development of the cardiovascular and pulmonary systems, and its depletion leads to a generalized elastinopathy and collagenolysis leading to perinatal death of Lox null mice. The development of muscles is a delicate process, which requires coordinated signaling and a homeostatic balance between the muscle and muscle connective tissue. Based on the drastic defects that were found in the present study in the skeletal muscle of Lox null mice, lack of LOX clearly disturbs this balance and increases transforming growth factor β (TGF-β) signaling, which leads to defects in the skeletal muscles. The impaired balance can cause muscle disorders, such as Duchenne Muscular Dystrophy (DMD). Despite the clinical significance, very little is known about the mechanisms controlling this homeostatic balance. The discovery of LOX as a regulating factor during skeletal muscle development will help to clarify the role of extracellular matrix (ECM) in muscle development and in muscle related congenital diseases. / Tiivistelmä Erytropoieesi on fysiologinen prosessi, jossa tuotetaan veren punasoluja ja jonka pääsäätelijänä toimii erytropoietiini (EPO) hormoni. EPO:n geeni ilmentyy voimakkaasti alhaisessa happipitoisuudessa (hypoksia) hypoksia-indusoituvan transkriptiotekijän (HIF) toimesta. HIF-tekijän stabiilisuutta säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4H) hapesta riippuvaisesti, ja niiden tiedetään siten osallistuvan myös erytropoieesin säätelyyn, HIF-P4H-2:n toimiessa pääsäätelijänä. Neljännen transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) roolia erytropoieesissa ei vielä tiedetä, mutta sen tiedetään säätelevän HIF-tekijää. Työssä käytettiin Hif-p4h-2, Hif-p4h-3 ja P4h-tm muuntogeenisiä hiirilinjoja, joiden entsymaattinen aktiivisuus on alentunut tai poistettu. P4H-TM:n osallisuutta erytropoieesin säätelyyn tutkittiin antamalla hiirilinjoille HIF-P4H-entsyymejä inhiboivaa lääkettä. Tutkimuksen tulokset osoittavat ensimmäistä kertaa P4H-TM:n säätelevän EPO-geenin ilmentymistä ja siten erytropoieesia. Ennestään tiedettyjen HIF-P4H entsyymien inhiboinnin lisäksi P4H-TM:n inhibointia voidaan pitää uutena kohteena uusien farmakologisten hoitokeinojen kehityksessä. Lysyylioksidaasi (LOX) katalysoi säikeisten kollageenien välisten sekä elastisten säikeiden välisten poikkisidosten muodostumista. Pokkisidokset antavat vetolujuutta kollageeneille ja joustavuutta elastisille säikeille ja ovat siten tärkeitä kudoksen rakenteelle. LOX:ia tarvitaan sikiön kehityksen aikana mm. hengitys-, sydän- ja verisuonielimistöjen kehityksessä. LOX:in puutos hiirillä aiheuttaa viallisia elastisia- ja kollageenisäikeitä, johtaen poikasten kuolemaan synnytyksen yhteydessä. Lihasten kehitys on tarkoin säädelty prosessi, jossa lihas ja lihaksen sidekudos säätelevät toisiansa. LOX:n suhteen poistogeenisissä Lox-/- sikiöissä löydettiin selviä ongelmia luurankolihasten kehityksessä. LOX:n puutoksen osoitettiin lisäävän transformoivan kasvutekijä beetan (TGF-β) määrää, joka estää luustolihaksia kehittymästä normaalisti. LOX kykenee sitoutumaan TGF-β:aan ja inhiboimaan sen aktiivisuutta ja LOX:n puuttuessa inhibointia ei tapahdu. Tutkimus osoittaa LOX:n olevan keskeinen tekijä lihaksen kehityksessä ja siten auttaa ymmärtämään sidekudoksen merkitystä luurankolihasten kehityksessä ja siihen liittyvissä sairauksissa.

Erythropoietin (Epo) modulates red blood cell production (erythropoiesis) by binding to its receptor and activating STAT5, a Signal Transducer and Activator of Transcription (STAT) protein implicated in both basal and stress erythropoiesis. Epo concentration in serum changes over three orders of magnitude, as it regulates basal erythropoiesis and its acceleration during hypoxic stress. However, it is not known how STAT5 translates the changes in Epo concentration into the required erythropoietic rates. We addressed this question by studying STAT5 phosphorylation, at the single cell level, in developing erythroblasts. We divided erythroid progenitors in tissue into several flow-cytometric subsets and found that each of them exhibited distinct modes of Stat5 activation, based on their developmental stage. STAT5 activation is bistable in mature erythroblasts, resulting in a binary (or digital), low-intensity STAT5 phosphorylation signal (p-Stat5). In early erythroblasts, and in response to stress levels of Epo, the low intensity bistable p-Stat5 signal is superseded by a high-intensity graded, or analog, signal. The gradual shift from high-intensity graded signaling in early erythroblasts to low intensity binary signaling in mature erythroblasts is due to a decline in STAT5 expression with maturation. We were able to convert mature, digital transducing erythroblasts into analog transducers simply by expressing high levels of exogenous STAT5. We found that EpoR-HM mice, expressing a mutant EpoR that lacks STAT5 docking sites, generate the binary, but not the analog, STAT5 signal. Unlike Stat5-null mice, which die perinatally, the EpoR-HM mice are viable but deficient in their response to stress, demonstrating that while binary STAT5 signaling is sufficient to support basal erythropoiesis, analog signaling is required for the stress response. Bistable systems contain a positive loop, which is important for flipping the switch between the two stable ‘on’ or ‘off’ states. We show that bistable activation is the result of an autocatalytic loop in which active STAT5 promotes further STAT5 activation. The isolated STAT5 N-terminal domain, which is not required for STAT5 phosphorylation, enhanced autocatalysis, converting a high intensity graded signal into a high intensity binary response. The N-terminal domain is known to participate in a radical conformational reorientation of STAT5 dimers inherent in STAT5 activation. We propose that the N-terminal domains of active STAT5 dimers facilitate the conformational reorientation of inactive dimers, in a prion-like autocatalytic interaction that underlies bistability and binary signaling. Together, bistable STAT5 activation, combined with a graded response allow erythropoietic rate to faithfully reflect a wide Epo concentration range, while preventing aberrant signaling.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

L’érythropoïèse est le processus de différentiation d’un progéniteur érythroïde multipotent en globules rouges. La différentiation érythroïde est essentiellement contrôlée par le récepteur à l’érythropoïétine (EPOR). Nous avons montré que le récepteur à la transferrine de type 2 (TFR2) est un membre important du complexe formé par l’EPOR. Le TFR2 présente, comme l’EPOR une expression restreinte qui dépend du type cellulaire. Ainsi son expression n’a pu être détectée que dans le foie, l’érythron et l’intestin grêle. Le rôle du TFR2 a été exploré dans les hépatocytes et il a été montré qu’il joue le rôle d’un senseur de fer dans cette lignée et de ce fait contribue à l’homéostasie du fer. Nous avons déterminé le rôle du TFR2 dans les érythroblastes et montré que TFR2 est une protéine escorte de l’EPOR qui contribue à l’érythropoïèse in vitro et in vivo. De plus, nos travaux montrent que le TFR2 est requis pour la production de GDF15 (Growth Differentiation Factor 15) dans les érythroblastes. D’autre part nous avons démontré que la production de GDF15 est augmentée par l’EPO, la déplétion intracellulaire en fer et l’activité transactivatrice de P53. L’inhibition de l’expression de P53, réalisée au cours de l’étude de son rôle dans la production de GDF15, a révélé son implication dans l’érythropoïèse normale. Nous avons mis en évidence l’existence de plusieurs formes du TFR2. Deux d’entre elles résultent de l’utilisation de sites distincts d’initiation de la traduction. Ces deux isoformes sont régulée différemment au cours de la maturation des érythroblastes. La troisième isoforme, appelée TFR2 soluble (sTFR2), est relargée dans le plasma suite au clivage du TFR2. Nous avons montré que la production du sTFR2 est inhibée en présence du ligand de TFR2, la transferrine saturée en fer (holoTF) alors que le TFR2 est stabilisé dans ces mêmes conditions. Les rôles spécifiques des trois formes du TFR2 doivent encore être élucidés. / Erythropoiesis is the differentiation process of a multipotent erythroid progenitor into red blood cells. Erythroid differentiation is primarily controlled by the erythropoietin receptor (EPOR). We showed that the Transferrin receptor 2 (TFR2) is an important member of the EPOR complex. TFR2 has like EPOR a lineage-restricted expression and can solely be detected in the liver, erythron and small intestine. TFR2 function has been explored in hepatocytes where it plays the role of an iron sensor and contributes to iron homeostasis. We determined the role of TFR2 in erythroblasts and showed that TFR2 is an escort protein for EPOR that contributes to optimal erythropoiesis in vitro and in vivo. Moreover we evidenced that TFR2 is absolutely required for the production of Growth differentiation factor 15 (GDF15) in erythroblasts. We further demonstrated that GDF15 production is increased by EPO levels, by intracellular iron depletion as well as by P53 trans-activation activity. The inhibition of P53 expression, realized for the study of its role in GDF15 production, revealed its implication in normal erythropoiesis. We evidenced that TFR2 is expressed under several forms, two of which result from the utilization of distinct translational initiation sites. These two isoforms are differently regulated during erythroid maturation. The third form called soluble TFR2 (sTFR2) is released in the plasma after TFR2 cleavage. We showed that sTFR2 production is inhibited in the presence of TFR2 ligand, iron loaded transferrin (holoTF) whereas cell surface TFR2 expression is stabilized by holoTF. The specific roles of the three forms of TFR2 expressed by erythroblasts remain to be elucidated.