Dissertations / Theses on the topic 'KLFs factor'

Krüppel like factors (KLFs) are a family of 17 proteins whose main function is gene regulation by binding to DNA elements in the promoters of various genes. KLF transcription factors recognize CACCC-elements and act as activators or repressors of the gene expression. Among the 17 family members, KLF1, KLF2, and KLF4 share high homology to each other. KLF1 is the founding member of the family and is an erythroid-specific protein. KLF2 is expressed in erythroid, endothelial, and other cells. KLF4 is expressed in endothelial, smooth muscle, and other cells. In this thesis, the functions of these KLFs were reviewed in the context of subjects related to erythropoiesis and cardiovascular development. A mouse model lacking KLF1, KLF2, and KLF4 was used to investigate whether these genes have overlapping functions in regulating the embryonic β-globin genes during early embryogenesis. Quantitative RT-PCR assays were used to measure the expression level of Ey- and βh1- globin mRNA at embryonic day 9.5 (E9.5). It was found that KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos express significantly decreased amounts of Ey- and βh1-globin genes when compared to WT and KLF4-/- embryos. There were no significant changes in the levels of Ey- and βh1-globin mRNA between KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos. It was demonstrated here that KLF1 does not regulate KLF2 in mouse erythroid cells at E10.5.

Obesity is a disease characterised by an excess of white adipose tissue. It leads to increased risk of several associated disorders including cardiovascular disease and type 2 diabetes mellitus. The number of people who are obese is increasing worldwide and as such much research is conducted on obesity and associated disorders. The use of animal models in the study of obesity allows understanding of involvement of specific genes in fat formation and related processes. This can be used to identify targets for prevention or treatment of obesity. Kriippel—like factors are a family of transcription factors that have been implicated in a number of roles, one of which is metabolism. The Klf3'/' mouse has decreased adipose tissue but the metabolic implications of this had not been determined. Klf8 has high homology to Klf3 in the zinc finger region and was suspected to have a role in metabolism and fat formation. A metabolic role for Klf8 had not been investigated. In this thesis, the metabolic profiles of Klf3— and Klf8—deficient mice were examined. Klf3'/' male mice were found to be resistant to diet-induced weight gain. They maintained lower WAT mass and smaller adipocytes. In addition, these mice had reduced hepatic steatosis and improved glucose tolerance and circulating hormone levels. Klf88W male and Klf8+/g’ female mice had no clear phenotype suggesting Klf8 either has roles in areas other than metabolism, or that functional redundancy occurs, thus compensating for the decreased Klf8. These studies show that Klf3 could potentially be targeted for treatments of obesity as mice lacking this protein are lean, resistant to diet-induced obesity and appear metabolically healthy.

Krüppel-like factors (KLFs) are a family of 3 Cys2/His2 zinc finger transcription factors with a diverse set of roles in cellular differentiation, cell cycle regulation, tumor suppression, erythropoiesis, angiogenesis, and other processes. During embryonic development, KLF2 has a role in vessel maturation. Adult conditional KLF4 knockout mouse embryos have thickened arterial intima follow vascular injury. Breeding KLF2+/- and KLF4+/- mice resulted in the generation of KLF2/KLF4 double knockout (DKO) embryos. KLF2/KLF4 DKO embryos died by E10.5 with cranial bleeding. Using immunohistochemistry, embryo whole-mounts were examined for differences in gross vascularization between wild-type (WT), KLF2-/- and KLF2/KLF4 (DKO embryonic day 9.5 (E9.5) embryos. No obvious gross capillary abnormalities were noted in E9.5 KLF2/KLF4 DKOs, although the posterior cardinal vein appeared to narrow rostral to caudal in KLF2-/- and KLF2/KLF4 DKO embryos. Light and electronic microscopy were employed to investigate potential structural and ultrastructural phenotypes in KLF2/KLF4 DKO embryos. Microscopy confirmed hemorrhaging near and endothelial breaks in the primary head vein (PHV) in E9.5 KLF2/KLF4 DKOs (n=8) and E10.5 KLF2-/-KLF4+/- embryos (n=1). Electron micrographs illustrated a disrupted endothelium in KLF2/KLF4 DKOs with endothelial cells having filopodia-like projections. Surprisingly, KLF2-/- embryos had the presence of wider medial PHV endothelial gaps compared to WT at the electron micrograph level. Density counts revealed a 15% reduction in midline cranial mesenchyme at the level of hemorrhaging in KLF2/KLF4 DKOs compared to KLF2-/- (n=3). An in-situ hybridization localized KLF2 RNA expression to the endothelium of the PHV. A quantitative reverse transcriptase polymerase chain reaction assay revealed that the eNOS expression is synergistically regulated by KLF2 and KLF4, as a shared downstream target. It is proposed that KLF2 and KLF4 share in the regulation of multiple gene targets, leading to early death by E10.5.

Krüppel-like factors KLF1 and KLF2 are closely related transcription factors with three zinc finger domains in their carboxy-termini. KLF1 (erythroid Krüppel-like factor, or EKLF) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β- globin genes. KLF1 and KLF2 have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double knockout (KO) mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, and double KO embryos are more anemic and die sooner than either single KO. We have shown that KLF1 and KLF2 positively regulate the human ϵ- (embryonic) and γ-globin (fetal) genes during embryonic erythropoiesis. Chromatin immunoprecipitation assays (ChIP) show that KLF1 and KLF2 bind to the promoters of the human ϵ- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region (LCR) in mouse embryonic erythroid cells. ChIP assays show that KLF1 but not KLF2 ablation results in abnormal histone modifications in the β-globin locus in mouse embryonic erythroid cells. H3K9Ac and H3K4me3, which correlate with open chromatin and active transcription, are both reduced in KLF1-/- primitive erythroid cells. Human CD34+ hematopoietic stem cells obtained from umbilical cord blood were in vitro differentiated along the erythroid lineage. ChIP assays indicate that both KLF1 and KLF2 bind to the promoter of γ-globin gene in this fetal erythroid model. KLF1 knockdown in these cells affects mainly adult β- globin gene expression. However, the decrease in β- globin gene expression in KLF1 knockdown also affects the ratio of γ- to β- globin in these cells. H3K9Ac and H3K4me3 were decreased only at the β- globin gene which coincides with lower recruitment of RNA polymerase II and its active form, RNA polymerase II phospho-serine 2. In conclusion, we showed using mouse primitive erythroid cells and cord blood definitive cells that KLF1 and KLF2 coordinate the regulation of the mouse and human β- globin genes by direct binding to the promoters and LCR in the β- globin locus. In conclusion, cord blood hematopoietic cells could serve as a complimentary system in addition to the transgenic mouse models to study the regulation of γ- globin gene expression.

Hemoglobinopathies are some of the most common monogenic disorders in the world, affecting millions of people and representing a growing burden on health systems worldwide. Although the pathophysiology of sickle cell anemia and beta-thalassemia, two of the most common hemoglobinopathies, have been the focus of much research over the last century, patients affected by these diseases still lack a widely applicable and easily available cure. Sickle cell anemia and beta-thalassemia are caused by defects in the structure and production of the beta-globin chains that, along with the alpha-globin chains make up the heterotetrameric hemoglobin molecule. Studies geared towards re-expression of the silenced fetal gamma-globin gene in adult erythroid cells as a therapeutic strategy to alleviate the symptoms of beta-globin deficiencies have met with some success for the treatment of sickle cell anemia but not for beta-thalassemia. A better understanding of normal gamma-globin gene regulation will undoubtedly advance the development of more effective therapeutic strategies. Because many of the potential targets that may be modulated to achieve gamma-globin re-expression also have functions in erythroid cells other than regulating the gamma-globin gene, it is imperative to understand their role in all aspects of erythropoiesis before they are used for therapy. The current study focuses on the role of two Krüppel-like transcription factors, KLF1 and KLF2, which have known roles in the processes of primitive and definitive erythropoiesis as well as globin gene regulation. The regulation of primitive erythropoiesis by KLF1 and KLF2 is studied using the mouse as a model system because it is not possible to study primitive erythropoiesis in humans. Previous studies have shown that KLF1 and KLF2 are essential for and have overlapping roles in primitive erythropoiesis. Simultaneous ablation of KLF1 and KLF2 results in a severely anemic embryonic phenotype that is not evident in KLF1 or KLF2 single knockout embryos. In this study, we show that this anemia is caused by a paucity of blood cells, and exacerbated by diminished beta-like globin gene expression. The anemia phenotype is dose-dependent, and interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining both normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. It was discovered that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation, through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous deficiency of KLF1 and KLF2, than with loss of a single factor. The regulation of human beta-like globin gene expression is studied using a recently developed in vitro system for the production of erythroid cells from umbilical cord blood hematopoietic precursor cells, representing a more “fetal” model of globin gene expression. Previous studies have shown that KLF1 binds to the promoters of the gamma- and beta-globin genes, while KLF2 binds to the promoter of the gamma-globin gene in cord blood-derived erythroid cells. Studies using transgenic mice carrying the entire human beta-globin locus had indicated that KLF1 and KLF2 positively regulate gamma-globin expression in mouse embryonic erythroid cells. We demonstrate in this study that KLF1 appears to have dual roles in the regulation of gamma-globin expression in human cord blood-derived definitive erythroid cells. Partial depletion of KLF1 causes elevated gamma-globin expression, while nearly complete depletion of KLF1 results in a down-regulation of gamma-globin expression. Of particular interest was the observation that KLF2 positively regulates gamma-globin expression in cord blood-derived erythroid cells. Surprisingly, KLF2 also positively regulates beta-globin expression in these cells. If regulation of gamma-globin by KLF2 proves to be a direct effect, KLF2 will join a very small group of factors known to directly activate gamma-globin expression.

Erythroid Krüppel-like factor (EKLF or KLF1) is a transcription factor with roles in embryonic and adult erythropoiesis. KLF1 knockout mouse embryos die due to severe anemia. Dominant human mutations in KLF1 can cause hereditary persistence of fetal hemoglobin. We show that KLF1 positively regulates β-globin and Bcl11A gene expression using KLF1 knockdown in in vitro-differentiated CD34+ human umbilical cord blood cells. -globin expression appears dependent on KLF1; it is increased with modest KLF1 knockdown but not in cells with low KLF1. KLF2 mRNA amounts are usually increased in KLF1 knockdown. KLF1 knockdown in CD34+ cells results in reduced colony forming ability. Interestingly, the expression of certain proliferation and cell cycle genes is reduced due to KLF1 knockout in mouse or knockdown in human erythroid cells. In conclusion, KLF1 is an important regulator of the β-globin locus and has roles in proliferation and cell cycle.

Breast cancer is the major cause of cancer death among women worldwide. Understanding the mechanisms underlying breast cancer progression remains urgent for developing effective treatment strategies to eliminate breast cancer mortality. Our recent studies have demonstrated that Kruppel-like transcriptional factor 8 (KLF8) plays a critical role for breast cancer progression. Other studies have shown that Epithelial stromal interaction 1 (EPSTI1), a recently identified stromal fibroblast-induced gene in non-invasive breast cancer cells and epidermal growth factor receptor (EGFR) are highly overexpressed in aggressively invasive breast carcinomas including triple negative breast cancers. In this thesis project, we demonstrate high co-overexpression of KLF8 with EPSTI1 as well as EGFR in invasive breast cancer cells and patient tumors. We also show that KLF8 upregulates the expression of EPSTI1 by directly binding and activating the EPSTI1 gene promoter, and KLF8 upregulates the expression of EGFR not only by directly activating the EGFR gene promoter but also by preventing EGFR translation from microRNA141-dependent inhibition. Genetic, signaling and animal cancer model analyses indicate that downstream of KLF8, EPSTI1 promotes the tumor invasion and metastasis by activating NF-?B through binding valosin containing protein (VCP) and subsequent degradation of I?B?, whereas EGFR promotes tumor growth and metastasis via activation of ERK. Taken together, these data identify EPSTI1 and EGFR as novel KLF8 targets in breast cancer and suggest that KLF8 may be targeted for new effective treatment of breast cancer.
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

Les maladies chroniques du foie associées à l’obésité (NAFLD, Non-Alcoholic Fatty Liver Disease) sont un problème de santé publique mondial. Ces complications sont l’évolution d’un foie sain vers la stéatose hépatique (accumulation de lipides dans les hépatocytes) puis vers la stéatohépatite (NASH) caractérisée par une importante inflammation et une souffrance hépatocytaire. Ce stade peut ensuite évoluer vers des complications plus sévères telles que la cirrhose et le carcinome hépatocellulaire. Mieux comprendre les mécanismes physiopathologiques qui sous-tendent la transition stéatose/NASH constitue un enjeu majeur pour l’identification de nouvelles cibles thérapeutiques.L’horloge circadienne coordonne la plupart des fonctions physiologiques, dont les fonctions hépatiques au cours du cycle jour/nuit. Elle est composée d’une horloge centrale située dans les noyaux supra-chiasmatiques de l’hypothalamus et d’horloges périphériques dans toutes les autres cellules de l’organisme. Les altérations de l’horloge circadienne associées à des changements du mode de vie (travail en heure décalées, jet lags chroniques, prises alimentaires irrégulières, composition des régimes alimentaires, etc.), constituent des facteurs de risque pour le développement de nombreuses pathologies dont le syndrome métabolique. De nombreux et récents éléments laissent présumer que l’altération de l’horloge circadienne jouerait un rôle important dans la pathogenèse des NAFLD. Le facteur de transcription Krüppel like Factor 10 (Klf10) est directement régulé par l’horloge circadienne dans le foie où il régule l’expression de nombreux gènes impliqués dans homéostasie glucido-lipidique. KLF10 joue également un rôle dans la régulation de réponses inflammatoires chroniques ainsi que de la mort et de la prolifération cellulaire. Ces données nous ont donc conduits à étudier l’implication du cycle circadien et de KLF10 sur le développement des complications hépatiques. Nos travaux ont permis de mettre en évidence que la stéatose et l’inflammation hépatiques suivent un rythme circadien à la différence de la souffrance hépatique dans un modèle murin de stéatohépatite (régime déficient en méthionine et choline (MCD)). Cela est associé à des altérations de l’oscillation des gènes horloges dans le foie mais aussi le rein, pouvant suggérer une modification généralisée du système circadien dans ce modèle. De plus, l’expression hépatique de Klf10 perd sa rythmicité avec le développement des complications hépatiques. L’absence de Klf10 chez la souris est associée à une forte augmentation de la souffrance hépatique sans impacter le développement de la stéatose et l’inflammation sous régime MCD. Nous avons montré que l’expression hépatique de Fsp27 gagne de la rythmicité sous régime MCD, augmente avec la sévérité des NAFLDs, favorise la souffrance hépatocytaire, et que ce gène est surexprimé chez les souris déficientes pour Klf10 sous régime MCD. Le rôle protecteur de KLF10 semble spécifique des hépatocytes car les souris déficientes pour Klf10 dans les hépatocytes présentent aussi une cytolyse hépatocellulaire (ALAT) accrue. De plus, la diminution de l’expression de Klf10 dans les hépatocytes primaires de souris diminue la viabilité cellulaire et augmente l’activation de la caspase 3 et de l’apoptose en réponse au TNFα. Enfin, l’expression hépatique de KLF10 corrèle avec les marqueurs circulants de la souffrance hépatique (ALAT) et de la mort hépatocytaire (kératine 18) chez les patients obèses. Nos données chez la souris, in vitro, et chez l’Homme indiquent que le développement des complications hépatiques pourrait suivre un rythme circadien et que KLF10 a des propriétés hépato-protectrices qui pourraient atténuer le développement des NAFLD
Non-alcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver diseases (NAFLDs), is a global public health problem without approved pharmacological therapy. NAFLD extend from non-pathogenic lipid accumulation, known as hepatic steatosis to hepatocellular carcinoma (HCC) through a wide spectrum of stages including NASH and fibrosis. NASH is featured by hepatic inflammation and hepatocyte cell death. Better understand NASH pathogenic cellular and molecular mechanisms is an important clinical requirement.Circadian timing system (CTS) is the main synchronizer of organismal physiology to environmental light/dark cycles. This CTS is comprised of a central pacemaker in the supra-chiasmatic nucleus of the hypothalamus and peripheral clocks localized in each single cell throughout the brain and body. Western society life style, including junk food consumption and erratic feeding, chronic jet lag, light exposure at night and shift-work, can disrupt the CTS. CTS disruption has been assessed as a risk factor for the development of chronic diseases including metabolic syndrome and cancer. The liver is the most rhythmic organ and evidence for an intricate link between CTS disruption and NAFLD development is most illustrated by (i) the genetic and environmental disruption of the CTS leads to dyslipidemia, hepatic steatosis as well as spontaneous NASH and HCC development (ii) the circadian hepatic transcriptome is rearranged in mice fed high fat diet and displaying hepatic steatosis, showing that metabolic disruption also impacts diurnal oscillation of transcripts. Krüppel-like factor 10 is a circadian transcription factor directly regulated by the circadian clock in the liver and help shaping the hepatic diurnal transcriptome and the control carbohydrate and lipid metabolism homeostasis. Beside from metabolism, this transcription factor has also been shown to regulate two NASH related processes, in very different contexts, namely inflammation and cell death. We thus aimed to evaluate the implication of circadian rhythms and the role of KLF10 during steatohepatitisHere, we show that hepatic steatosis and inflammation display diurnal rhythmicity in mice developing steatohepatitis upon feeding with a methionine and choline deficient diet (MCDD). Core clock gene oscillations remained mostly unaffected but rhythmic Klf10 expression was abolished in this model. Klf10 deficient mice (Klf10-/-) display enhanced liver injury despite the same level of hepatic steatosis and inflammation that control mice upon MCDD challenge. Specific genetic ablation of Klf10 only in hepatocytes phenocopied the phenotype of Klf10-/- mice upon MCDD. Silencing Klf10 in isolated primary hepatocytes also sensitized these cells to apoptosis along with increased caspase 3 activation in response to TNFα. We also show that the hepatic KLF10 expression correlates with liver injury (ALT activity) and the circulating keratin 18 hepatocyte death marker in a cohort of obese patients. Collectively our findings suggest that specific NASH features including steatosis and inflammation display diurnal oscillations and the associated altered circadian expression of Klf10 may aggravate liver injury through hepatocyte sensitization to cell death.Collectively, our results gathered from cellular and animal experiments as well as correlative study in Human indicate that hepatic steatosis and inflammation could be rhythmic during NASH and that KLF10 could be a hepatoprotective factor that could limit NAFLD progression

Neuroblastoma is a childhood solid tumor of a unique propensity to either regress spontaneously or grow relentlessly. Emerging evidence indicated that neuroblastoma contains heterogeneous populations of cells, and commitment of these cells to neuronal lineage may result in aggressive progression in patients, whereas to fibromuscular lineage may give a favorable outcome. However, mechanism(s) controlling the lineage commitment of neuroblastoma cells remains to be identified. Our preliminary data suggested that Kr?ppel-Like Factor 4 (KLF4) might promote neuroblastoma regression. KLF4 is a transcription factor regulating a variety of cellular functions, including proliferation and cell cycle progression. Recent studies have demonstrated that KLF4 may act as both tumor suppressor and oncogene in a cell-context dependent manner. Importantly, our preliminary data showed that low KLF4 expression is highly associated with poor clinical outcomes of the neuroblastoma patients. In addition, we found that overexpression of KLF4 suppresses neuroblastoma cell growth accompanied with loss of tumorigenicity. Morphologically, KLF4 overexpressing cells changed their morphologies to become epithelial-like, strongly substrate-adherent and expressing smooth muscle marker. Therefore, we hypothesized that KLF4 exerts its effects through two ways, it may (i) function to inhibit cell growth and reduce tumorigenicity; and (ii) promote differentiation of the neuroblastoma cells to the non-tumorigenic, fibromuscular-like cells. RT-PCR data revealed the differential expression of KLF4 in 11 neuroblastoma cell lines. In particular, a modest expression was found in Be(2)C, a cell line which was formerly demonstrated to differentiate and form tumor in mice xenograft assay. It was therefore chosen as the study model. To assess the effects of KLF4 knockdown on tumor growth, stable knockdown clones from Be(2)C cells were established by lentiviral transduction of KLF4-targeting shRNA. In parallel, clones that stably expressed non-target shRNA were used as controls. After the transduction, two stable knockdown clones showing significant KLF4 downregulation were isolated from single colony (monoclonal stable clones) and a pool of cells (polyclonal stable clones) respectively. The cell proliferation and growth rate of the stable clones were then measured by 5-bromo-2’-deoxyuridine (BrdU) proliferation assay and growth curve assay. The results have indicated that both monoclonal and polyclonal stable KLF4 knockdown clones grow faster than the control clones. In order to examine the tumorigenicity in vivo, the stable clones were xenotransplanted to severe combined immunodeficient mice. The stable KLF4 knockdown clones showed a significant higher growth rate and formed a larger tumor. The stable clones were also treated with BrdU for four weeks for differentiation towards fibromuscular lineage. As anticipated, the control clones showed fibromuscular features, like more flattened and epithelial-like morphology. In contrast, the stable KLF4 knockdown clones failed to present the fibromuscular features after treatment. In addition, immunocytochemistry staining of SMA and quantitative analysis of the immunocytochemistry further confirmed that only the control clones showed higher SMA expression after BrdU treatment, while there is no change in the SMA expression in the stable KLF4 knockdown clones. These results demonstrated that KLF4 functioned by inhibiting neuroblastoma cell proliferation and growth, reducing the tumorigenicity, and it was required for fibromuscular differentiation.
published_or_final_version
Surgery
Master
Master of Philosophy

We have demonstrated that simvastatin and sphingosine 1-phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Kruppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, S1PR1, via a KLF2-dependent mechanism. S1PR1 luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 mu M), and S1PR1 and KLF2 protein expression detected by immunoblotting. In vivo, transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both S1PR1 and KLF2 gene expression (mRNA, protein) were significantly increased by simvastatin in vitro and in vivo. S1PR1 promoter activity was significantly increased by simvastatin (P < 0.05), which was significantly attenuated by KLF2 silencing (siRNA). Simvastatin induced KLF2 recruitment to the S1PR1 promoter, and consequently, significantly augmented the effects of the S1PR1 agonist on EC barrier enhancement (P < 0.05), which was significantly attenuated by KLF2 silencing (P < 0.05). These results suggest that simvastatin upregulates S1PR1 transcription and expression via the transcription factor KLF2, and consequently augments the effects of S1PR1 agonists on preserving vascular barrier integrity. These results may lead to novel combinatorial therapeutic strategies for lung inflammatory syndromes.

Le facteur de transcription Kruppel-like 6 (KLF6) est impliqué dans des rôles essentiels tels que la transduction du signal liée à la croissance, la prolifération et la différenciation cellulaire, le développement, l'apoptose et l'angiogenèse. En outre, KLF6 semble être un facteur clé lors du développement et la progression de cancers et son expression est finement régulée par plusieurs stimuli qui endommagent les cellules. Le but de cette thèse était de caractériser les signaux biochimiques régulant la fonction de KLF6 et d’analyser leurs effets sur la prolifération cellulaire et l'apoptose. Nous avons tout d’abord caractérisé le rôle des voies de transduction de signal JNK et p38 dans l'expression et la fonction KLF6. Puis nous avons étudié les effets de KLF6 en réponse au stress cellulaire généré par le rayonnement UV et analysé leurs conséquences sur l'onco-protéine c-Jun et l'apoptose. Enfin, nous avons déterminé des effets de KLF6 dans le contexte de stress cellulaire généré par l'infection à adénovirus qui est capable d'induire la prolifération cellulaire ou l'apoptose
The mammalian Krüppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Additionally, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several celldamaging stimuli. The aim of this thesis was to characterize the biochemical signals that regulate the function of KLF6 and analyze their effects on cell proliferation and apoptosis. We first characterized the role of JNK and p38 signal transduction pathways in the expression and function of KLF6. Then we studied the effects of KLF6 in response to cellular stress generated by UV radiation and analyzed their effects on the c-Jun onco-protein and on apoptosis. Finally, we determined the effects of KLF6 in the context of cellular stress generated by infection with adenovirus that is able to induce cell proliferation or apoptosis

El endotelio disfuncional presenta, entre otras caracteristicas, alteración en los mecanismos de vasodilatación, complicaciones trombóticas, disminución de la resistencia al estrés oxidativo, aumento de la expresión de moléculas de adhesión y de la secreción de moléculas proinflamatorias. El factor de transcripción endotelial KLF2 juega un importante papel en la regulación del fenotipo protector endotelial y su expresión depende de las fuerza hemodinámicas generadas por el flujo sanguíneo y de la administración exógena de estatinas. La hipertensión portal y el daño hepático por I/R son dos condiciones patológicas asociadas a disfunción endotelial. Los trastornos estructurales característicos de la cirrosis hepática, la mayor causa prevalente de hipertensión portal en nuestro entorno, se acompañan de variaciones en las fuerzas hemodinámicas que pueden modificar la expresión de KLF2 y su programa transcripcional vasoprotector. Asímismo, durante la isquemia asociada a la preservación de injertos hepáticos para transplante, la interrupción de las fuerzas hemodinámicas generadas por el flujo sanguíneo podría resultar en la reducción de los programas endoteliales vasoprotectores, que se debería en parte a la pérdida de expresión de KLF2. Los trabajos de investigación de la presente tesis doctoral amplian el conocimiento de los mecanismos moleculares responsables de la disfunción endotelial hepática, demostrando: 1. Que KLF2 está muy expresado en los hígados cirróticos y que su expresión se induce en las fases tempranas de la progresión de la enfermedad, representando un mecanismo compensador para mejorar los desórdenes vasculares característicos de los hígados cirróticos. 2. Que los hígados preservados en condiciones de transplante muestran un descenso tiempo-dependiente de KLF2, acompañado de daño hepático y aumentada resistencia vascular. Además, demostran que la modulación farmacologica de la expresión de KLF2 puede ser beneficiosa tanto en el tratamiento de la hipertensión portal como en la preservación de los injertos hepáticos para transplante.

Krüppel-like factor 6 (KLF6) a été identifié comme gène suppresseur de tumeur impliqué dans le contrôle de la croissance, de la différentiation cellulaire et dans la mort par apoptose. L'expression de KLF6 est altérée au cours de la carcinogenèse hépatique, et cette protéine joue un rôle prépondérant dans le contrôle de la prolifération des cellules tumorales hépatiques. Nous avons étudié le rôle de la région 3'UTR de l'ARNm de KLF6 dans la régulation de son expression. Nous avons établi que la demi-vie de l'ARNm de KLF6 est fortement diminuée dans les lignées cellules dérivées du foie. La région 3'UTR inhibe fortement l'expression et l'activité de gènes rapporteurs en corrélation avec la région entre les nucléotides 1835 et 2615
Krüppel-like factor 6 (KLF6) is a tumor suppressor gene that is involved in the regulation of cell cycle, cellular proliferation, differentiation and cell death by apoptosis. KLF6 expression is altered in liver carcinogenesis. However, little is known on the mechanisms governing KLF6 expression in HCC. In the current study, we asked whether 3’UTR may be responsible for down regulation of KLF6 mRNA in HCC. Our results demonstrated that KLF6 mRNA half-life was greatly decreased in cells derived from HCC. Moreover, 3'UTR strongly inhibited the activity and expression of the reporter gene. In addition, we found that most of KLF6 3’UTR destabilisation activity resides between nucleotides 1835 and 2615

Mammalian central nervous system (CNS) neurons lose their ability to regenerate their axons after injury during development. For example, optic nerve injury studies in hamsters have shown that optic nerve axons injured around the time of birth retain the ability to regenerate to their target, but this ability is lost during development (So et al., 1981). The development of an inhibitory CNS environment has been implicated in the inability of the adult CNS to regenerate, however there is also support for this loss being a result of changes in developmental programs intrinsic to the neurons themselves (Goldberg et al., 2002a; Goldberg, 2004). While some molecules have been identified as being involved in intrinsic mechanisms controlling axon growth, there is still much to be discovered. Using genes shown to be regulated in retinal ganglion cells (RGCs) during development (Wang et al., 2007), I performed an overexpression screen in embryonic primary neurons measuring changes in neurite growth. Of these genes, the most significant effect in neurite growth was seen with overexpression of Krüppel-like factor 4 (KLF4), resulting in a greater than 50% decrease in growth. KLF4 is a member of the KLF family of transcription factors which all possess a DNA binding domain containing 3 zinc finger motifs. Outside of the nervous system, KLF4 has been implicated in cancer (Black et al., 2001; Rowland and Peeper, 2006), mitotic growth arrest (Shields et al., 1996) and most recently in the induction of pluripotency (Yamanaka, 2008; Zhao and Daley, 2008). In the CNS, KLF4 has recently been implicated in increasing the sensitivity of cortical neurons to NMDA insult (Zhu et al, 2009), though no effect of KLF4 on neurite growth or regeneration has yet been described. I found that KLF4 overexpression in RGCs results in decreased neurite growth and neurite initiation. KLF4 overexpression also leads to decreases in polarity acquisition in hippocampal neurons, though even when they acquire polarity, they still display decreased neurite growth. Additionally, KLF4 knockout targeted to RGCs leads to an increased neurite growth ability and increased neurite initiation in vitro. In vivo, KLF4 knockout increases RGC axon regeneration after optic nerve injury. Interestingly, KLF4 is one of 17 members of the KLF family, known for their ability to act redundantly and competitively amongst family members for their binding sites. Therefore, we looked to see if other KLFs could affect neurite growth ability. 15 of 17 KLF family members are expressed in RGCs, and their overexpression results in differential effects on neurite growth in both cortical neurons and RGCs. Additionally, many of the family members are developmentally regulated in a manner that typically correlates with their ability to affect neurite growth. For example, KLF6 and -7, whose expression decreases during development, when overexpressed, increase neurite growth, whereas KLF9, whose expression increases developmentally, when overexpressed, decreases neurite growth. Surprisingly, there are multiple KLFs expressed in RGCs that are neurite growth-suppressors, and further study has revealed that the combination of KLF growth enhancers with KLF growth suppressors results in a suppressive or neutral phenotype (Moore et al., 2009), suggesting that to further enhance regeneration after injury in vivo, we will need to additionally remove the growth suppression from other KLF family members. Taken together, these data suggest that KLFs may play an important role in the intrinsic loss of axon growth and regeneration seen during development. Further characterization of downstream targets of KLF4 and other KLF family members may reveal specific neuronal gene targets that could mediate the phenotypic effects of these transcription factors. It is my hope that by determining the developmental programs that underlie the loss of intrinsic axon growth ability of CNS neurons, we may ultimately determine how to revert adult CNS neurons to their embryonic axon growth ability.

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs.

Le diabète de type 2 (DT2) est une maladie multifactorielle dû à des facteurs environnementaux et génétiques. De nombreuses analyses génétiques ont identifié les régions du génome liées à cette maladie. Dans notre laboratoire, une liaison génétique avec le DT2 et l'obésité a été mise en évidence sur la région chromosomique 2p25-23 dans une population française (Vionnet et al. , 2000). Par une approche combinée de clonage positionnel et de gène candidat, nous avons récemment mis en évidence que le gène KLF11 (ou TIEG2) est impliqué dans la susceptibilité au DT2 à l'âge adulte au sein de familles comportant plusieurs sujets diabétiques (Neve et al. , 2005). Le gène KLF11 appartient à la famille Sp/XKLF (Krüppel like fingers) de facteurs transcriptionnels. La famille Sp/XKLF est constituée de 25 protéines qui ont la propriété de s'accrocher aux éléments situés dans le promoteur appelés éléments cis comme les boites GC riches (GGGGCGGGG) au GT/CACC. Plusieurs facteurs de cette famille ont été impliqués dans l'homéostasie du glucose, et sont de ce fait de bons gènes candidats pour l'étude des prédispositions génétique au DT2. Ce projet de thèse porte sur la compréhension des rôles potentiels des membres de la famille KLF dans la susceptibilité génétique au DT2. Premièrement, nous avons continué l'étude de KLF11 pour pouvoir comprendre son rôle dans le DT2. Nous avons précédemment montré que deux SNP en déséquilibre de liaison total, Q62R et AS1659 G>C (SNP1, Single Nucleotide Polymorphisms) du promoteur putatif de KLF11, sont associés au DT2. Dans cette étude, nous avons analysé dans une population danoise (INTER99 chez 4443 sujets) l'implication génétique de ces SNP dans la susceptibilité génétique au DT2 et dans la sensibilité à l'insuline. Chez les sujets tolérants au glucose l'allèle 62R a été associé à un niveau plus élevé d'insuline et de peptide-C à jeun, et d'indice HOMA de résistance à l'insuline (P= 0. 00004, P= 0. 006 et P=0. 00002, respectivement). Les SNP fonctionnels de KLF11 sont donc associés à la résistance à l'insuline dans les sujets tolérants au glucose dans une population danoise. Dans ce travail, nous avons également démontré que STAT3 est le facteur transcriptionnel qui s'accroche à la séquence présentant l'allèle sauvage du SNP1 et régule l'expression de KLF11. STAT3 a été décrit précédemment comme un facteur de transcription qui régule la voie de la néoglucogenèse. Nos résultats suggèrent que KLF11 pourrait être impliqué dans cette voie de signalisation métabolique. Deuxièmement, nous avons étudiés 9 gènes de la famille XKLF. Au total, 28 variants génétiques de type SNP ont été génotypé dans une première étude cas témoins formés par 700 individus. Seulement trois SNP des gènes KLF2, KLF15 et KLF16 ont montré une tendance d'association au DT2 (p<0. 10). Une étude de réplication sur 2000 individus ne nous a pas permis de confirmer cette association. Nous avons conclu que les variants analysés de ces 9 gènes de la famille KLF ne contribuent pas à la susceptibilité au DT2. Finalement, les gènes KLF10 et KLF11 ont une grande homologie et sont capables d'inhiber l'expression du gène SMAD7, impliqué dans la signalisation de TGFβ. Nous avons décidé de cribler les gènes KLF10 et SMAD7. Un variant dans le 3'-UTR du KLF10 a montré une faible association au DT2 dans un modèle récessif (P = 0. 009, Odds ratio = 0. 81, 95% intervalle de confiance= 0. 69-0. 95; P = 0. 042 en ajustant par l'âge, le sexe, l'IMC). En outre, le variant trouvé dans l'intron 2 de SMAD7 a été associé au DT2 dans un modèle récessif (P = 0. 007, Odds ratio = 0. 62, 95% intervalle de confiance= 0. 44-0. 88; P = 0. 034 en ajustant par l'âge, le sexe, l'IMC). Donc, KLF10 et SMAD7 montrent une association modeste au DT2 et ils pourraient contribuer à augmenter la susceptibilité à développer le DT2.

Tout au long de la vie, les spermatozoïdes sont produits dans le testicule à partir des cellules souches germinales (CSGs), au cours du processus de spermatogenèse. Les CSGs ont la capacité de s'auto-renouveler pour maintenir le stock de cellules souches et d'entrer en différenciation. L'infertilité masculine est responsable dans un cas sur deux des difficultés à enfanter au sein du couple. Chez les patients traités par radiothérapie et/ou chimiothérapie (plus particulièrement chez les enfants atteints de cancer), un des effets secondaires majeurs qui altèrent la qualité de vie après guérison est l'atteinte du stock de CSGs et les problèmes d'infertilité qui en découlent. Des solutions thérapeutiques telles que la transplantation de CSGs provenant de biopsies testiculaires réalisées avant les traitements anticancéreux sont actuellement étudiées. Des travaux récents de transplantation testiculaire de CSGs chez le primate non humain ont permis de restaurer, chez l'animal stérilisé, une spermatogénèse permettant la fécondation in vitro d'ovocytes à partir de spermatozoïdes issus des CSGs transplantées. Ces résultats très encourageants font entrer pour la première fois la transplantation des CSGs dans le champ de l'application préclinique. Cependant, l'identité du pool de CSGs humaines et les mécanismes moléculaires gouvernant leur auto-renouvellement restent peu connus. Mon travail de thèse a porté sur la caractérisation des CSGs chez l'Homme. Au cours de ce projet j'ai également utilisé le modèle murin afin de mieux appréhender certains mécanismes de régulation physiologique des CSGs. Dans un premier temps, ce travail de thèse a contribué à définir, à l'aide d'une combinaison de marqueurs cellulaires et d'un test fonctionnel, la transplantation testiculaire, une population de spermatogonies immatures enrichie en CSGs. Le profil d'expression de cette population a été réalisé par une analyse transcriptomique, et nous a permis de définir un réseau de régulateurs de la transcription préférentiellement exprimé dans cette population. Parmi ces régulateurs, nous nous sommes focalisés sur l'étude du répresseur de transcription bHLH Hes1 dans le modèle murin. Dans des conditions de culture de privation de sérum et de facteurs de croissance, induisant un arrêt prolifératif et la quiescence, notre étude tend à montrer un rôle protecteur de Hes1 contre la mort cellulaire. Nous avons également observé une diminution du nombre de cellules ayant le potentiel à régénérer une spermatogenèse dans les cultures de CSGs lorsque l'expression de Hes1 est diminuée à l'aide de siRNA. Dans un second temps, nous avons étudié l'effet de l'hypoxie sur l'auto-renouvellement et la différenciation des CSGs murines en culture. En effet, l'hypoxie est une composante importante de la niche des cellules souches qui régule leur destin cellulaire. Nous avons observé qu'une forte hypoxie (1% et 0,1% d'oxygène) a un effet délétère sur la capacité des CSGs murines à former des colonies, et qu'elle induit modérément la quiescence et un début de différenciation. Un effet positif sur la fonctionnalité des CSGs adultes murines a été observé à 3,5% d'oxygène, mais ces conditions n'ont pas d'effets bénéfiques sur la prolifération et le maintien des CSGs humaines à long terme. Enfin, mon travail de thèse a contribué à une meilleure compréhension des mécanismes responsables de la reprogrammation spontanée vers la pluripotence des CSGs in vitro. Si les facteurs de Yamanaka sont efficaces pour la reprogrammation des cellules somatiques testiculaires, ils ne permettent pas celle des CSGs adultes. Ces résultats suggèrent donc l'existence d'un mécanisme spécifique empêchant la reprogrammation par les facteurs de Yamanaka des cellules germinales adultes en un état pluripotent
Throughout life, sperm cells are produced from germinal stem cells (GSCs) during the process of spermatogenesis in the testis. GSCs have the ability to self-renew to maintain stem cell stock and to differentiate. Male infertility is responsible in one out of two cases of issues of procreation. In patients treated with radiotherapy and/or chemotherapy (particularly in children with cancer), one of the major side effects that affects the quality of life after healing is the attrition of the GSCs stock and the subsequent problems of infertility. The most severe infertility issue, the Sertoli-Cell-Only syndrome with a complete germ cell aplasia, results from the exhaustion of the GSCs population. Therapeutic solutions such as transplantation of GSCs obtained from testicular biopsies harvested prior to cancer treatments are being studied. Recent studies on testicular transplantation of GSCs in non-human primates have shown the restoration, in the sterilized animal, of spermatogenesis allowing in vitro fertilization of oocytes by intracytoplasmic microinjection of spermatozoa derived from transplanted GSCs. These very encouraging results bring for the first time the transplantation of GSCs into the field of preclinical application. However, the identity of the pool of human GSCs and the molecular mechanisms governing their self-renewal remain poorly known. My thesis work focused on the characterization of GSCs in humans. During this project I also used the mouse model of GSCs to better understand some mechanisms of the physiological regulation of GSCs. As a first step, this thesis work has contributed to define, using a combination of cell markers and the testicular transplantation, as functional test, a population of immature spermatogonia enriched in GSCs. The specific expression profile of this population was performed by transcriptomic analysis, and allowed us to define a transcription regulator network preferentially expressed in this population. Among the enriched transcriptional regulators in the human immature spermatogonia population, we focused on the study of the bHLH transcriptional repressor Hes1 in the murine model. Under serum and growth factors-deprived conditions of culture inducing proliferative arrest and quiescence, our study tends to show a protective role of HES1 against cell death. In addition, we also observed a decrease in the number of cells with the potential to regenerate spermatogenesis in GSCs cultures when Hes1 expression is decreased. Secondly, we studied the effect of hypoxia on the self-renewal and differentiation of murine GSCs in culture. Indeed, hypoxia is an important component of the stem cell niche that regulates their cell fate. We observed that high hypoxia (1% and 0.1% oxygen) has a deleterious effect on the ability of murine GSCs to form colonies, and moderately induce quiescence and the onset of differentiation of GSCs. A positive effect on the functionality of murine adult GSCs has been observed at 3.5% oxygen, but these conditions do not support the proliferation and maintenance at long-term of human GSCs. Finally, this work has contributed to a better understanding of the mechanisms responsible for the spontaneous reprogramming to pluripotency of GSCs in vitro. While Yamanaka factors are effective for reprogramming testicular somatic cells, they do not allow reprogrammation of adult GSCs and spermatogonial progenitors. These results suggest the existence of a specific mechanism preventing reprogramming by Yamanaka factors of adult germ cells into a pluripotent state

Krüppel-like factor 5 (KLF5) encodes a zinc finger transcription factor involved in growth, differentiation and apoptosis of a number of different tissues, and it has previously been implicated as either a tumour suppressor or oncogene in various solid tumours. Expression of Klf5 is up-regulated during granulocyte-macrophage differentiation of the murine myeloid cell line FDB1 in response to expression of a constitutively active mutant form of the human GM-CSF/IL-3/IL-5 receptor common β subunit (FIΔ mutant)¹. The aim of this work was thus to characterise the role of KLF5 in the myeloid lineage and to determine whether deregulation of this gene may contribute to Acute Myeloid Leukaemia (AML). We show that KLF5 mRNA expression is increased during granulocyte-macrophage differentiation of both cell lines and primary haemopoietic cells. KLF5 expression levels increase to the greatest extent during granulocyte differentiation, and accordingly we demonstrate that knock-down of Klf5 in the 32D cell line model attenuates granulocyte differentiation. Enforced expression of KLF5 in the FDB1 cell line model and in primary murine bone marrow progenitors functions to induce terminal myeloid differentiation, coupled with cell cycle arrest and cell death. Consistent with a potential role as a promoter of differentiation and tumour suppressor, we show that mean KLF5 mRNA expression is significantly reduced in AML samples compared to normal controls (mononuclear cells and bone marrow CD34⁺ samples). KLF5 expression is selectively decreased in French-American-British (FAB) subtypes M1, M3 and M4, and in samples positive for the FLT3-ITD mutation, relative to primitive bone marrow CD34⁺ controls. We identify methylation of the KLF5 5’ proximal promoter and intron 1 as potential mechanisms for down-regulation in AML, and accordingly demonstrate that treatment of leukaemia cell lines with demethylating agents re-activates KLF5 expression in cells displaying hypermethylation of KLF5. Aberrant methylation of the KLF5 proximal promoter is associated with samples displaying monosomy 7 or deletions in chromosome 7q, and methylation of KLF5 intron 1 is significantly higher in samples with normal karyotype than in those displaying abnormal cytogenetics. We demonstrate expression and splicing of an alternative exon within KLF5 intron 1, which is encompassed by the region shown to be hypermethylated in a subset of AML samples. It is likely that this exon represents an alternative transcription start site which is regulated by an alternative promoter associated with a high level of interspecies conservation. Translation of this alternative KLF5 isoform is predicted to code for an N-terminal-truncated KLF5 protein, and further research is now required to determine the regulation and functional significance of this isoform in normal haemopoiesis and in AML. Finally, we demonstrate that enforced expression of KLF5 in the leukaemia cell line U937 induces changes associated with myeloid differentiation, and enhances differentiation in response to differentiation-inducing reagents. Collectively, these data are consistent with KLF5 acting as a tumour suppressor in the myeloid lineage.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science and School of Paediatrics and Reproductive Health, 2011

博士
國立臺灣大學
微生物學研究所
98
The zinc finger Kruppel-like transcription factor 4 (KLF4) has been implicated in cancer formation and stem cell regulation. However, the function of KLF4 in tumorigenesis and stem cell regulation are poorly understood due to limited knowledge of its targets in these cells. In this study, we have revealed a surprising link between KLF4 and regulation of telomerase, which offers important insight into how KLF4 contributes to cancer formation and stem cell regulation. KLF4 sufficiently activated expression of the human telomerase catalytic subunit, hTERT, in telomerase-low ALT and fibroblast cells, while down-regulation of KLF4 reduced its expression in cancerous and stem cells which normally exhibits high expression. Furthermore, KLF4-dependent induction of hTERT was mediated by a KLF4 binding site in the proximal promoter region of hTERT. In human embryonic stem cells, expression of hTERT replaced KLF4 function to maintain their self-renewal. Therefore, our findings demonstrate that hTERT is one of the major targets of KLF4 in cancer and stem cells to maintain long-term proliferation potential.

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2021
RESUMEN La placenta es un órgano temporal clave durante el embarazo ya que ejerce la conexión entre la madre y el feto por lo que su correcta formación y funcionamiento es fundamental para el bienestar fetal y el progreso del embarazo. El desarrollo temprano de la placenta humana se considera, en algunos aspectos, similar a la carcinogénesis. Ambos se desarrollan en un ambiente con bajos niveles de oxígeno, su desarrollo es modulado por el sistema inmunológico, la expresión de protooncogenes, microARNs y genes supresores tumorales. Además, las células placentales y tumorales tienen una alta capacidad para proliferar, fusionarse, migrar e invadir. La principal diferencia entre la progresión del cáncer y el desarrollo de la placenta es que, en esta última, estos procesos están estrictamente regulados. La desregulación de algunos de estos procesos se asocia con complicaciones del embarazo como preeclampsia, retardo del crecimiento intrauterino, placentas cretas, entre otras. Durante el desarrollo placentario las células progenitoras de la placenta, los citotrofoblastos (CTB), proliferan rápidamente y comienzan a diferenciarse, algunas para dar origen por fusión celular al sinciciotrofoblasto (STB) multinucleado y algunas hacia el trofoblasto extravelloso migratorio y altamente invasivo que permite anclar la placenta al útero materno. KLF6 es un factor de transcripción ubicuo con un dominio de transactivación ácido N-terminal y un dominio de unión al ADN tipo dedos de zinc C-terminal. KLF6 es altamente expresado en la placenta. Los ratones Klf6-/- mueren en el día embrionario 12,5 mostrando deterioro del desarrollo de la placenta. Previamente, se demostró que KLF6 es necesario para la fusión célula-célula en cultivos primarios de citotrofoblastos vellosos (CTBv), así como en la línea celular BeWo derivada de trofoblasto y modula la expresión de la proteína inhibidora del ciclo celular p21, de sincitina-1 (Syn-1) y de la subunidad β de la gonadotrofina coriónica humana (β-hCG). Además, la inmunoreactividad de KLF6 es mayor en el lecho placentario de las placentas provenientes de embarazos con preeclampsia que en las de los embarazos sin complicaciones. Sin embargo, se desconoce el mecanismo por el cual KLF6 regula la diferenciación del CTBv y no existen reportes sobre el rol de KLF6 en el proceso de migración e invasión de los citotrofoblastos extravellosos (CTBev). En contextos tumorales, KLF6 actúa como un gen supresor de tumores. De hecho, la disminución en los niveles de KLF6 aumenta la tumorigenicidad y la metástasis en numerosos tipos de cáncer. No obstante, también se ha informado el efecto contrario en células normales y cancerosas. En esta tesis se estudió el mecanismo por el cual KLF6 participa en la diferenciación de los CTBv los cuales fusionan para formar el STB. Se demostró que KLF6 es suficiente para desencadenar la fusión de células BeWo. Además, KLF6 incrementó la expresión de moléculas importantes involucradas en este proceso, entre ellas β-hCG, Syn-1 y p21, y disminuyó la proliferación celular. Mientras que, una mutante de KLF6 que carece del dominio ácido, implicado en la actividad transcripcional (KLF6Δac), disminuyó la sincicialización in vitro de cultivos primarios de CTBv, así como, la expresión de Syn-1 y β-hCG. Además, la fusión celular desencadenada por KLF6 se redujo en células silenciadas para p21. Estos resultados sugieren que KLF6 induce la sincicialización de CTBv a través de un mecanismo que involucra su dominio regulador de la transcripción de una manera dependiente de p21. Por otro lado, se demostró que KLF6 está involucrado en la diferenciación del CTBev. La disminución en la expresión de KLF6 o la sobreexpresión de la mutante carente del dominio acídico (KLF6Δac) incrementó la migración, la actividad de la metaloproteinasa 9 y la expresión de moléculas importantes involucradas en la migración y la transición epitelio-mesenquimal (EMT), como β-catenina, conexina-43, integrinas α5 y β1. Además, provocó cambios morfológicos compatibles con la EMT confirmados por estudios de microscopía de fuerza atómica y Raman. En línea con los datos obtenidos in vitro, se demostró que placentas cretas, caracterizadas por ser anormalmente invasivas, poseen niveles de inmunomarcación para KLF6 más bajos con mayor localización citoplasmática en comparación con placentas normales. Los resultados obtenidos en esta tesis sugieren que KLF6 es un regulador maestro de la diferenciación celular en la vía sincicial y que su dominio de transactivación N-terminal es necesario para esta función. Mientras que, en la diferenciación de los CTBev, KLF6 actúa regulando negativamente la migración a través de la modulación de la expresión de moléculas importantes involucradas en este proceso. En resumen, los resultados actuales permiten proponer a KLF6 como un regulador clave de la diferenciación del trofoblasto a través de la vía vellosa y extravellosa, y sugieren que la desregulación en su nivel de expresión o la pérdida de su actividad transcripcional contribuiría al desarrollo de patologías como preeclampsia y placentas cretas.
ABSTRACT The placenta is a key temporary organ during pregnancy. It establishes critical relationships between the mother and the fetus, so its correct formation and functioning is essential for fetal well-being and the outcome of the pregnancy. Early human placental development resembles carcinogenesis in otherwise healthy tissues. The placental and tumor development share numerous features. Both take place in an environment with low levels of oxygen, their development is modulated by the immune system and the expression of protooncogenes, microRNAs and tumor suppressor genes. Furthermore, placental and tumor cells have a high capacity to proliferate, fuse, migrate and invade. The main difference between cancer progression and placenta development is the tight regulation of these processes in the placenta. However, the dysregulation of these processes is associated with pregnancy complications such as preeclampsia, intrauterine growth restriction and placentas cretas, among others. During development, placental progenitor cells, the cytotrophoblasts (CTB), rapidly proliferate and begin to differentiate, some towards the villous pathway to form the multinucleated syncytiotrophoblast (STB) by cell-cell fusion and some towards the highly invasive and migratory extravillous trophoblasts (EVT) that anchor the placenta to the uterus. KLF6 is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C‐terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta. Klf6-/- mice die at day E12.5 showing impaired placenta development. Previously, it was demonstrated that KLF6 is required for cell–cell fusion in primary culture of human villous cytotrophoblasts (vCTB) isolated from term placenta, as well as in the BeWo trophoblast-derived cell line and it modulates the expression of the cell cycle inhibitory protein p21, syncytin-1 (Syn-1), and the β subunit of the human chorionic gonadotropin (β-hCG). Additionally, KLF6 immunoreactivity is higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. However, the mechanism by which KLF6 regulates CTBv differentiation is unknown and there are no reports about the role of KLF6 in the process of migration and invasion of the EVT. In cancer, KLF6 acts mainly as a tumor suppressor gene. Indeed, KLF6 knockdown increases the tumorigenicity and metastasis in numerous types of cancer. Nevertheless, the opposite effect has been also reported in normal and cancer cells. In this thesis, the mechanism by which KLF6 participates in CTB differentiation through the villous and extravillous pathways was explored. It was demonstrated that KLF6 is sufficient to trigger cell fusion and to increase the expression of important molecules involved in this process, such as β-hCG, Syn-1 and p21, in addition, it downregulates cell proliferation. Furthermore, a KLF6 mutant lacking the acidic domain involved in its transcriptional activity (KLF6Δac) impaired the in vitro syncytialization of villous trophoblast cells isolated from human term placentas and decreased Syn-1 and β-hCG expression. Moreover, cell fusion triggered by KLF6 was reduced in cells silenced for p21. These results suggest that KLF6 induces vCTB syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner. Additionally, the KLF6 involvement in EVT differentiation was established. The decrease in KLF6 expression or the overexpression of the transcriptionally inactive mutant (KLF6Δac) increase cell migration, metalloproteinase 9 activity, and the expression of molecules involved in migration and epithelial mesenchymal transition (EMT) such as β-catenin, connexin-43, integrin α5 and β1. In addition, it causes morphological changes consistent with EMT, confirmed by atomic force microscopy and Raman studies. In line with the data obtained in vitro, it was shown that placentas cretas, characterized by being abnormally invasive, have lower KLF6 immunostaining levels and more cytoplasmic localization compared to normal placentas. The results obtained in this thesis suggest that KLF6 is a master regulator of cell differentiation into the syncytial pathway and that its N-terminal transactivation domain is required for this function. While in EVT differentiation, KLF6 negatively regulates migration by modulating the expression of important molecules involved in this process. In summary, current results allow proposing KLF6 as a key regulator of trophoblast differentiation through the villous and extravillous pathways and suggest that dysregulation in its expression level or the loss of its transcriptional activity may contribute to the development of pregnancy pathologies like preeclampsia and placentas cretas.
2023-07-31
Fil: Panzetta de Dutari, Graciela María Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.
Fil: Panzetta de Dutari, Graciela María del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.
Fil: Miranda, Andrea Lis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.
Fil: Caputto, Beatriz Leonor. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.
Fil: Caputto, Beatriz Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.
Fil: Genti de Raimondi, Susana Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.
Fil: Genti de Raimondi, Susana Del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.
Fil: Vivas, Laura Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina.
Fil: Varone, Cecilia L. Universidad Nacional de Buenos Aires; Argentina.
Fil: Varone, Cecilia L. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina.

碩士
國立臺灣大學
化學工程學研究所
94
During atherogenesis, inflammation plays an important role. Thrombomodulin (TM), in vascular endothelial cells, has anti-coagulation and anti-inflammation properties. Transcription factor, Kruppel-like factor 2 (KLF2) has been shown to participate in the regulation of the expression of TM and endothelial nitric oxide synthase and thus is important for regulating thrombotic function. Endothelial cells are constantly under the influence of flow-induced hear stress and the atherosclerotic lesions are closely related to this hemodynamic effect. In the present study, we focused on the study of gene expression of TM and KLF2 in endothelial cells exposed to shear stress. Human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were used to probe into the signaling mechanisms involved in this shear-induced TM and KLF2 expression. ECs exposed to shear stress of 25 dyn/cm2 transiently induced the TM mRNA expression with peak induction about 1.6-folds after exposure to shear stress for 4 to 6 hrs before it returned to basal level. However, KLF2 mRNA expression in those ECs was found to be a sustained one with peak induction more than 85-folds. While mRNA expression of TM and KLF2 were significantly induced in shear-induced ECs, protein expression of TM and KLF2 had no obvious increase. Furthermore, TM mRNA and KLF2 mRNA were significantly increased in ECs exposed to higher shear stress (25 dyn/cm2) in contrast to those ECs exposed to low shear stress (4.5 dyn/cm2) indicating that TM mRNA induction is sensitive to shear force. However, the protein expression of TM and KLF2 from shear-treated ECs show no significant difference as compared to those control ECs. This indicates that protein stability may be involved in those shear-treated ECs. ECs under shear flow condition constantly release nitric oxide (NO). The role of NO in this shear-induced TM and KLF2 mRNA expression was examined. ECs exposed to a NO donor (NOC18) significantly suppressed the TM and KLF2 mRNA level with a dose-dependent manner. Consistently, shear stress to ECs increased the TM promoter activity. Interestingly, ECs pretreated with an eNOS inhibitor (L-NAME) did not enhance the shear-induced TM promoter activity. Furthermore, the protection effect of shear flow on cytokine-treated ECs was examined. ECs treated with tumor necrosis factor (TNF) greatly reduced the TM and KLF2 expression. ECs pretreated with TNF followed with shear flow, however, significantly attenuated the TNF-induced suppression of TM mRNA levels. In contrast, the basal KLF2 and the shear-induced KLF2 mRNA levels were not affected by TNF pretreatment. In summary, shear flow to ECs increases the TM and KLF2 mRNA expression in a dose-dependent manner. However, this induction can not be reflected by the TM protein levels indicating that the stability of TM mRNA and protein may be involved. Furthermore, shear flow exerts its protective effect by attenuating the TNF-induced suppression of TM mRNA expression. ECs treated with NO suppress the TM and KLF2 mRNA expression. Although the detailed up-regulation mechanism of TM remains unclear, this study suggests that shear flow plays an important role in regulating TM expression and consequently affects endothelial integrity.

La génération des cellules hématopoïétiques, aussi connue sous le nom d'hématopoïèse, est contrôlée par l’activité conjuguée de facteurs de transcription lignée-spécifiques permettant l’expression, en temps et lieu, de gènes spécifiques nécessaires pour le développement cellulaire. Dans le cadre de notre étude, nous avons étudié les facteurs de transcription KLF2 et KLF4 qui jouent des rôles cruciaux dans la formation des lymphocytes B et T. KLF2 et KLF4 activent la transcription de gènes spécifiques via leur interaction avec le co-activateur (CBP). Leurs interactions avec CBP requièrent le domaine de transactivation (TAD) qui est localisé dans la région N-terminal des facteurs KLF2 et KLF4. Des études préalables ont montré que des domaines TAD sont aussi présents chez la protéine suppresseur de tumeur p53 et que ces domaines sont requis pour les interactions entre la protéine p53 et le co-activateur CBP. Récemment, plusieurs structures des TADs de p53 en complexe avec les domaines TAZ2 et KIX de CBP ont permis de démontrer que ces TADs sont de nature acide et contiennent un motif ΦΧΧΦΦ crucial pour la formation des interactions. De plus, il s’avère que ces TADs sont similaires aux TADs de KLF2 et KLF4. L’étude présentée dans ce mémoire relate la caractérisation structurelle et fonctionnelle des interactions formées par les facteurs de transcription KLF2 et KLF4 avec leur partenaire d'interaction, CBP, pour activer la transcription de gènes spécifiques. Nos analyses ont été faites en utilisant différentes techniques telles que le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire (RMN) ainsi que des expériences de transactivation chez la levure. Notre étude permet une meilleure compréhension des rôles opposés mais complémentaires qu'ont les protéines KLF2 et KLF4 au cours du développement et de la différentiation des lymphocytes B et T en plus de fournir les détails mécanistiques à la base de leurs interactions. Ces informations seront potentiellement utiles pour le développement d'outils à des fins thérapeutiques dans le cadre des leucémies, notamment.
Hematopoietic development is regulated through a combinatorial interplay between lineage-specific activators and the general transcription factors that enables cell-specific patterns of gene expression. In this study, the transcription factors KLF2 and KLF4 play crucial roles in lymphocytes B and T development by activating transcription of specific genes through interactions with the co-activator (CBP). These interactions involve the transactivation domains (TAD) localized in the N-terminal region of KLF2 and KLF4 factors. Previous studies have shown that TADs are also found in the tumor suppressor protein p53 and these TADs are responsible for the interactions between the p53 protein and the coactivator CBP. Recently, several structures of p53TADs in complex with the TAZ2 and KIX domains of CBP have shown that these TADs are acidic and possess a ΦΧΧΦΦ motif crucial for the formation of the interaction. Interestingly, these TADs are similar to the ones found on KLF2 and KLF4. This thesis provides a structural and functional characterization of the interactions formed by the transcription factors KLF2 and KLF4, which have opposing roles, and competes for the same interacting partner CBP to activate transcription. The analysis is done using isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy and a yeast activation assay. This study brings a greater understanding on the opposing roles yet complementary of KLF2 and KLF4 proteins involved in B and T lymphocytes specific lineages selection and also provides information for potential therapeutic research regarding disease such as leukemia.

碩士
臺北醫學大學
臨床醫學研究所
97
Breast cancer is the fourth cause of female cancer deaths in Taiwan with increased incidence and young age tendency (age?T40 years old, 29.3%). In recent years, the distinct age distribution and more aggressive clinical behavior in the young patient are noted in Taiwanese women and this phenomenon is different from that in the Western countries. Besides, due to the heterogeneity of breast cancer, designation of an ideal treatment protocol for breast cancer could not only be based on the traditionally histological, clinical, and biological markers (such as ER, PR and HER-2/neu) but also some new prognostic factors. Therefore, the specific study of breast cancer in Taiwan women becomes an important issue. Kruppel-like factors (KLF) belong to a group of zinc finger like transcription factors and are involved in regulating cell proliferation. KLFs have more than twenty subtypes. The studies of Kruppel-like factors in breast cancer are increased recently and are mainly focused on their roles in tumorigenesis. The KLFs are considered as new prognostic factors in breast cancers in some studies. Among them, KLF4 and KLF5 are most important and are broadly studied, but most studies are mainly in Western countries. In order to provide better treatment strategies for native breast cancers, the aim of this study is to evaluate the correlation of KLF4 and KLF5 expression with pathologic changes and clinical behaviors of breast cancers in Taiwanese women. In the literatures, KLF4 has both tumor suppressor gene and oncogene functions. KLF4 can promote the proliferation of cancer cells and also can regulate production of extracellular matrix. More aggressive clinical manifestations may be associated with the cellular location of KLF4 in cancer cells. The patients have poor prognosis when nuclear localization of KLF4 in cancer cells. KLF5 also has both tumor suppressor gene and oncogene functions. KLF5 can facilitate the proliferation and transformation of cancer cells. Increased expression of KLF5 is a poor prognostic factor and is positively correlated with the expression of HER-2/neu and Ki-67 in breast cancer. KLF5 also has increased expression in breast cancer patients younger than 50 years old. In this study, we used immunohistochemistry method to evaluate both staining intensity and staining pattern of expression of KLF4 and KLF5 in non-tumor and tumor parts (including invasive and in situ cancers) of breast tissues. We also analyzed the associations of expression status of KLF4 and KLF5 with histological features, clinical presentation and other prognostic factors of breast cancer. We enrolled 60 breast cancer patients with the mean age 47 years old and the mean tumor size was 2.7 cm. The clinical presentation was stage I: 30.0%; stage II: 43.3%; stage III: 21.7%; and stage IV: 5.0%. The follow-up period of these patients ranged from 8 to 59 months (mean 27 months) and only one patient died of disease. Pathologically, most of them were invasive ductal carcinoma (IDC) (90.0%) and showed moderately differentiation (66.7%). The accompanied ductal carcinoma in situ (DCIS), if present, was predominantly highest grade (60.0%). The immunohistochemical study of KLF4 in cancer cells showed cytoplasmic and nuclear expression. The intensity of tumor part was stronger than non-tumor part in 43.3% patients. We evaluated the association of the immunohistochemical results of KLF4 and KLF5 and clinical manifestations of these patients. We found that more KLF4 nuclear expression in tumor cells positively correlated with more advanced stage (p=0.006) and larger size of the tumor (size more than 2 cm in maximal diameter, p=0.035). KLF4 expression was also age-related. KLF4 intensity was stronger in tumor part than non-tumor part in patients older than 50 years old (p=0.007) and, in this setting, the invasive cancer tended to be poorly differentiated (p=0.033). Besides, consistent expression of KLF4 between DCIS and invasive cancers was also found: stronger intensity in DCIS accompanied with stronger intensity in invasive cancers (p=0.002), more predominant nuclear expression in DCIS with more predominant nuclear expression in invasive cancers (p<0.001). The expression of KLF5 in cancer cells was mainly cytoplasmic. The intensity of tumor part was stronger than non- tumor part in 58.3% patients. For KLF5, invasive breast cancers with negative or weak cytoplasmic expression showed better differentiation compared with strong cytoplasmic expression (p=0.035). Consistent expression of KLF5 between DCIS and invasive cancers was also found: stronger intensity in DCIS with stronger intensity in invasive cancers (p<0.001) and more predominant cytoplasmic expression in DCIS with more predominant cytoplasmic expression in invasive cancers (p<0.001). Moreover, there was no association between the following factors and the KLF4 expression intensity and pattern, respectively: ER (p=0.271 and p=0.925), PR (p=0.191 and p=0.448), HER-2/neu (p=0.136 and p=0.454), p53 (p=1.000 and p=0.925), and p21 (p=0.572 and p=0.367). There was also no correlation between the following factors and the KLF5 expression intensity and staining pattern, respectively: ER (p=1.000 and p=0.512), PR (p=1.000 and p=1.000), and HER-2/neu (p=0.520 and p=0.443). Our study found that KLF4 expression is positive association with tumor stage, tumor size, and age but could not conduct the conclusion that nuclear KLF4 expression was an adverse prognostic factor proposed in the literatures. In the other hand, KLF5 expression was associated with the differentiation of invasive cancers. We also found that KLF5 nuclear localization was mainly restrictedly in non-tumor breast ducts and lobules (16.7%) and loss of nuclear expression in DCIS and invasive cancers, the finding not mentioned in literatures before. Although we didn’t study the biologic function of KLF5, it maybe presented a possible tumor suppressor gene-like function of KLF5. We found that there were associations of KLF4 and KLF5 expressions and clinical manifestations in breast cancers but the expressions of KLF4 and KLF5 were not enough to predict the prognosis and survival rate. The major cause was due to too short follow up period of our patients to exactly evaluate the association of survival rate and expressions of KLF4 and KLF5. Therefore, well-designed retrospective studies with adequate follow up period for studying correlation of expressions of KLF4 and KLF5 and prognosis and survival rate of breast cancers are necessary.