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Myelodysplastic syndromes (MDS) include a spectrum of stem cell malignancies characterized by ineffective hematopoiesis and predisposition to acute myeloid leukemia (AML) transformation. Patients are predominantly older (greater than 60 years old), with progressive cytopenias resulting from ineffective and cytologically dysplastic hematopoiesis. MDS subtypes are classified by morphologic features and bone marrow blast percentage, as well as cytogenetic pattern, as is the case for deletion 5q MDS. Interstitial deletion of the long arm of chromosome 5, del(5q), is the most common chromosomal abnormality in patients with MDS, and the 5q- syndrome, represents a distinct subset of del(5q) MDS characterized by an isolated deletion, megakaryocyte dysplasia, hypoplastic anemia, and an indolent natural history. MDS risk stratification is most commonly based on the International Prognostic Scoring System (IPSS) with survival outcomes ranging from a few months to many years based on risk factors. There are several therapeutic options for MDS including hematopoietic growth factors, immunosuppressive therapy, azanucleosides, and allogeneic stem cell transplant, however, there is still a need for more effective treatment options, particularly targeted therapeutics. One of the most effective treatments for MDS is selective for del(5q) MDS, and is the second generation immunomodulatory agent, lenalidomide (LEN). LEN is an analog of the known teratogen, thalidomide, and has broad biological effects including selective cytotoxicity to del(5q) clones, activation of T-cells, and expansion of erythroid precursors. In patients with del(5q) MDS, LEN is effective in up to 75% of patients, however, 50% of patients will become resistant within 2-3 years of treatment response. Studies in normal hematopoietic progenitors have shown that LEN induces expansion of the primitive erythroid precursors, which our laboratory has shown is accompanied by sensitization of progenitors to ligand induced erythropoietin receptor (EpoR) signaling. This sensitization is evidenced by increased and prolonged activation of the Signal Transducer and Activator of Transcription 5 (STAT5), compared to Epo stimulation alone. Although EpoR signaling is augmented by LEN, the exact mechanisms by which this is mediated to result in erythroid expansion are not fully characterized. In del(5q) MDS, we have shown that LEN selectively suppresses del(5q) clones via inhibition of the haploinsufficient phosphatases Cdc25c and PP2a, as well as stabilizing the human homolog of the murine double minute-2 protein (MDM2) to decrease expression of the tumor suppressor, p53, however, the mechanisms of action of LEN in non-del(5q) MDS remains elusive. Although most anemic MDS patients have normal or elevated endogenous levels of Epo, as well as comparable levels of progenitor EpoR density relative to healthy individuals, the biologic pathology underlying the impaired EpoR signaling in MDS is poorly defined. Recent reports have shown that membrane microdomains are important for T-cell, c-kit, and integrin signaling, however, there have been no reports on EpoR membrane localization. Lipid rafts are discrete membrane entities that provide platforms by which receptors aggregate and initiate downstream signaling. Furthermore, reports have indicated that there is a decrease in lipid raft density in GM-CSF primed MDS neutrophils, that consequently impaired production of reactive oxygen species (ROS) after fMLP stimulation, suggesting a role of rafts in MDS disease biology. Based on the role of rafts in signaling, and potential role in MDS pathogenesis, we sought to determine whether there was specific membrane localization of EpoR to the raft fractions, and whether disruption of rafts in MDS erythroids could impair EpoR signaling. To address this, we first examined the membrane localization of EpoR on the cell surface. We show here that EpoR translocates to lipid rafts in both erythroid progenitor cell lines as well as primary progenitor cells after stimulation by Epo. Furthermore, we found that Epo stimulation increases the assembly of lipid rafts, as well as the aggregation of rafts on the cell surface. Epo stimulation not only promoted the recruitment of EpoR into the raft fractions, but also downstream signaling intermediates such as Janus kinase 2 (Jak2), STAT5, and Lyn kinase. Moreover, a negative regulator of EpoR signaling, the CD45 tyrosine phosphatase, was redistributed outside of raft fractions after Epo stimulation, potentially enhancing receptor signal competence. Furthermore, disruption of lipid rafts by depletion of membrane cholesterol with MâCD (methyl-β-cyclodextrin) inhibited EpoR signaling in both cell lines and primary bone marrow progenitor cells. Additionally, we found that inhibition of Rho-associated, coiled-coil containing protein kinase (ROCK) and/or Ras-related C3 botulinium toxin substrate 1 (Rac1), blocked the recruitment of the receptor into the raft fractions indicating a critical role of these GTPases, and associated proteins, in the transport and localization of EpoR into raft microdomains. We next asked whether LEN could alter lipid raft assembly in erythroid precursors in the absence of Epo. LEN not only induced raft formation and aggregation but also increased F-actin polymerization. Similar to Epo stimulation, LEN alone was able to induce the recruitment of EpoR, Jak2, and STAT5 into raft fractions. Additionally, CD45 was redistributed outside of raft fractions after LEN treatment. Similarly, inhibition of ROCK blocked LEN induced raft formation and F-actin polymerization, indicating that LEN utilized effectors shared by Epo. Furthermore, LEN was able to increase raft density in raft deficient primary MDS erythroid progenitors. These data demonstrate that LEN may enhance erythroid expansion via induction of EpoR signaling competent raft platforms, to enhance survival and differentiation transcriptional response. Recently, ribosomal protein (RP), S-14, gene (RPS14) haplodeficiency was found to be a key determinant of the hypoplastic anemia in del(5q) MDS. Allelic loss of RPS14 compromises ribosome assembly, thereby causing nucleolar stress and release of free RPs that bind to and promote the degradation of MDM2, the principal negative regulator of p53. As a result, the accumulation of RPs causes lineage restricted stabilization of p53 in erythroid precursors. Our laboratory and colleagues confirmed that cellular p53 expression levels were elevated in del(5q) erythroid precursors, and that LEN decreased expression in responding patients. However, at the time of LEN treatment failure, p53 expression was again elevated at levels exceeding those at baseline. These results suggest that LEN is initially able to reverse p53 accumulation levels and that this action may be a mechanism by which LEN is selectively cytotoxic to del(5q) clones. Subsequent studies showed that LEN inhibits the cereblon E3 ubiquitin ligase complex, the newly discovered target of LEN. Cereblon has been reported to be the principal protein involved in thalidomide induced teratogenicity. Furthermore, the cytotoxic activity of LEN in multiple myeloma is dependent on cereblon. Our laboratory found that LEN inhibits the auto-ubiquitination of MDM2, thereby stabilizing the protein, and promoting ubiquitination of and ultimately the degradation of p53. Additionally, we found that LEN blocked the binding of free ribosomal proteins to MDM2, which are liberated from the nucleosome by ribosomal stress from RPS14 haploinsufficiency, consequently stabilizing the E3-ubiquitin ligase and fostering p53 degradation. In non-del(5q) MDS there is no cytotoxicity of MDS clones by LEN, suggesting an alternative method of erythropoiesis rescue. Although we know that LEN promotes the formation of signaling platforms, and recruitment of EpoR, we wished to determine whether there was an effect of LEN on EpoR expression, as EpoR expression is controlled through ubiquitination and proteasomal degradation. Treatment of erythroid progenitor cell lines and primary erythroid precursors with LEN increased cellular expression of Jak2-associated EpoR in a concentration dependent manner. There was no change in mRNA expression, supporting a post transcriptional mechanism. We then investigated whether receptor up-regulation was limited to EpoR, or included other cytokine receptors. We found that LEN induced expression of another Jak2 associated Type I receptor, IL3-R, but did not alter cellular expression of c-kit, a Type II cytokine receptor. Because Type I cytokine receptor turnover is regulated by a shared E3-ubiquitin ligase, and LEN inhibited both MDM2 and cereblon, we evaluated the effects of LEN on the E3-ubiquitin ligase, Ring Finger Protein-41 (RNF41), which regulates steady state or ligand independent, Jak2 associated Type I receptor internalization. We found that LEN inhibited the ubiquitination activity of RNF41, ultimately stabilizing EpoR membrane residence and increasing expression. In summary, MDS patients display ineffective hematopoiesis likely in part to decreased lipid raft assembly. Stimulation by Epo, or treatment by LEN, not only induced raft formation, but also induced the recruitment of both growth factor receptor, and downstream signaling intermediates into raft fractions to enhance EpoR signal fidelity. We have shown here two methods by which LEN may augment EpoR signaling. First, LEN increases lipid rafts and promotes recruitment of signaling effectors. Second, LEN increases and stabilizes the expression of EpoR through inhibition of the E3 ubiquitin ligase, RNF41. Therefore, we suggest here that LEN may have broad E3 ubiquitin ligase inhibitory effects. These data also indicate that lipid raft upregulation by LEN is mediated through GTPases, suggesting that GTPase activation may also occur via inhibition of specific E3 ubiquitin ligases, a question to be addressed in future studies.

Genetic and epigenetic factors that predispose to ineffective control of subgingival biofilm composition are incompletely understood. The objective of this study was to elucidate how leukocytes impact on the course of periodontitis in Rac-null mice. Models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to alveolar bone loss. Leukocyte margination was differentially impaired during attachment in conditional Rac1-null and during rolling and attachment in Rac2-null mice. The inflammatory responses to subgingival ligatures were altered in Rac-null compared to WT mice. In response to persistent subgingival challenge Rac-null mice had increased alveolar bone loss with resorption patterns characteristic to aggressive periodontitis, partially explained by higher osteoclastic activity in Rac-null mice. This study demonstrates that migratory leukocyte defects are rate limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

Genetic and epigenetic factors that predispose to ineffective control of subgingival biofilm composition are incompletely understood. The objective of this study was to elucidate how leukocytes impact on the course of periodontitis in Rac-null mice. Models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to alveolar bone loss. Leukocyte margination was differentially impaired during attachment in conditional Rac1-null and during rolling and attachment in Rac2-null mice. The inflammatory responses to subgingival ligatures were altered in Rac-null compared to WT mice. In response to persistent subgingival challenge Rac-null mice had increased alveolar bone loss with resorption patterns characteristic to aggressive periodontitis, partially explained by higher osteoclastic activity in Rac-null mice. This study demonstrates that migratory leukocyte defects are rate limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

No description available.

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. [212]-236. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: John J.G. Tesmer. Vita. Includes bibliographical references. Also available from UMI.

Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.

La couche cornée, couche la plus superficielle de l'épiderme, assure une fonction de barrière multifonctionnelle vitale pour l'organisme. Le maintien de cette barrière dépend de la fonctionnalité des cellules sous-jacentes, les kératinocytes granuleux, dernière couche de cellules vivantes de l'épiderme. Les kératinocytes granuleux contiennent dans leur cytoplasme de nombreux organites tubulo-vésiculaires sécrétoires, appelés corps lamellaires (CL). Les CL jouent un rôle majeur dans la formation et le maintien de cette barrière en déversant leur contenu (lipides, lipases, protéases, inhibiteurs de protéases, peptides antimicrobiens,...) à la transition couche cornée - couche granuleuse. Le trafic intracellulaire des CL est donc un processus déterminant dans l'homéostasie de la couche cornée. Cependant, les mécanismes moléculaires impliqués dans la régulation de ce trafic ne sont pas élucidés. Précédemment, au laboratoire, une analyse systématique par spectrométrie de masse des protéines associées aux CL a permis d'identifier plusieurs GTPases de la famille Rab et certains de leurs effecteurs. Ces protéines étant des régulateurs majeurs du trafic vésiculaire dans tous les types cellulaires, elles pourraient jouer un rôle dans le routage des CL et, ainsi, dans la fonctionnalité de la barrière épidermique. L'objectif de ma thèse était d'identifier les GTPases Rab et effecteurs impliqués dans la régulation du trafic intracellulaire des CL. Dans un premier temps, j'ai mis en évidence que la GTPase Rab11a est fortement exprimée dans les kératinocytes granuleux où elle est partiellement associée aux CL. J'ai montré que la déplétion de Rab11a dans un modèle tridimensionnel d'épiderme reconstruit in vitro réalisée grâce à la technique d'interférence à l'ARN, induit une diminution de la densité et de la sécrétion des CL. L'extinction de Rab11a entraîne également une baisse du taux de céramides et de cholestérol, une désorganisation des lipides intercornéocytaires et une augmentation de la perméabilité de la couche cornée. Dans les épidermes reconstruits déplétés, les composants des CL non-sécrétés sont adressés vers une voie de dégradation lysosomale. Dans un deuxième temps, j'ai observé que la perte d'expression de Rab11a induit une anomalie de distribution du moteur moléculaire Myosine-Vb, effecteur majeur de Rab11a. J'ai donc analysé les conséquences de la déplétion de Myosine-Vb dans les épidermes reconstruits et montré qu'elle induit un phénotype comparable à celui observé lors de la déplétion de Rab11a. L'ensemble de ces résultats suggèrent fortement que le complexe bipartite Rab11a-Myosine-Vb constitue un régulateur majeur de la biogenèse des CL et ainsi, de l'homéostasie de la barrière épidermique. Mon travail de thèse est la première étape du décryptage des voies moléculaires impliquées dans la biogenèse des CL et contribue à une meilleure compréhension du rôle du trafic intracellulaire dans la constitution de la barrière épidermique. Il pourrait permettre de caractériser les mécanismes physiopathologiques associés à un défaut de trafic des CL. / The stratum corneum, the most superficial layer of the epidermis, provides a multifunctional protective barrier which is vital for the organism. The maintenance of this barrier is directly dependent on the underlying granular keratinocytes which are the last living cells in the epidermis. The granular keratinocytes contain in their cytoplasm numerous tubulovesicular secretory organelles called lamellar bodies (LB). LB play a major role in the establishment and the maintenance of the epidermal barrier by releasing their content (lipids, lipases, proteases, protease inhibitors, antimicrobial peptides,...) at the junction between the stratum corneum and the stratum granulosum. Because of LB importance in the maintenance of the stratum corneum homeostasis, the regulation of their trafficking deserves further study.Previously, in my laboratory, a proteomic characterization of LB by mass spectrometry has identified several Rab family GTPases and some of their effectors. In any cell type, from yeast to human, Rab GTPases are considered as major regulator of vesicular trafficking. Thus, I postulated that these proteins could play a role in the regulation of LB routing in the cytoplasm of granular keratinocytes. In this context, the aim of my thesis was to determine which Rab GTPases and effectors are involved in this process. In a first step, I demonstrated that Rab11a is strongly expressed in granular keratinocytes where it is associated with LB. I showed that Rab11a silencing using RNA interference technique in an in vitro tridimensional model of reconstructed human epidermis induces a decrease of LB density and secretion in granular keratinocytes. The Rab11a depletion also leads to a decrease of ceramide and cholesterol level and a disorganization of intercorneocyte lipids, generating a defective epidermal barrier. In depleted reconstructed epidermis, there is a missorting of non-secreted LB components, driven to the lysosomal degradation pathway. In a second step, I observed that Rab11a silencing affects distribution of its effector, the molecular motor Myosin-Vb. So, I analyzed the consequences of Myosin-Vb depletion in the model of reconstructed epidermis and I demonstrated that the phenotype obtained is similar that of a Rab11a depleted epidermis. Taken together, these results strongly suggest that the bipartite complex Rab11a-Myosin-Vb is able to regulate the biogenesis of LB in granular keratinocytes. Thus, this molecular complex is a crucial regulator of the epidermal barrier homeostasis. My thesis work is a first step in the deciphering of the molecular pathway involved in LB biogenesis. It is a breakthrough in the comprehension that membrane dynamic in the granular keratinocytes is a major regulator of epidermal barrier. It may contribute to a better understanding of pathophysiological mechanisms related to dysregulated LB trafficking in skin diseases.