HBZ impedes the Menin function and up-regulates the transcription of the hTERT gene in leukemic cells

International audiencen.

Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation

Embryonic stem (ES) cells hold great promise with respect to their potential to be differentiated into desired cell types. Of interest are organs derived from the definitive endoderm, such as the pancreas and liver, and animal studies have revealed an essential role for Nodal in development of the definitive endoderm. Activin A is a related TGFβ member that acts through many of the same downstream signaling effectors as Nodal and is thought to mimic Nodal activity. Detailed characterization of ES cell-derived endodermal cell types by gene expression analysis in vitro and functional analysis in vivo reveal that, despite their similarity in gene expression, Nodal and Activin-derived endodermal cells exhibit a distinct difference in functional competence following transplantation into the developing mouse embryo. Pdx1-expressing cells arising from the respective endoderm populations exhibit extended differences in their competence to mature into insulin/c-peptide-expressing cells in vivo. Our findings underscore the importance of functional cell-type evaluation during stepwise differentiation of stem cells.Stem Cell and Regenerative Biolog

c-Met is essential for wound healing in the skin

Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met–deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met–positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult

SARS-CoV-2 infects an <I>in vitro</I> model of the human developing pancreas through endocytosis

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic β-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences

The New Generation of Beta-Cells: Replication, Stem Cell Differentiation, and the Role of Small Molecules

Diabetic patients suffer from the loss of insulin-secreting β-cells, or from an improper working β-cell mass. Due to the increasing prevalence of diabetes across the world, there is a compelling need for a renewable source of cells that could replace pancreatic β-cells. In recent years, several promising approaches to the generation of new β-cells have been developed. These include directed differentiation of pluripotent cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, or reprogramming of mature tissue cells. High yield methods to differentiate cell populations into β-cells, definitive endoderm, and pancreatic progenitors, have been established using growth factors and small molecules. However, the final step of directed differentiation to generate functional, mature β-cells in sufficient quantities has yet to be achieved in vitro. Beside the needs of transplantation medicine, a renewable source of β-cells would also be important in terms of a platform to study the pathogenesis of diabetes, and to seek alternative treatments. Finally, by generating new β-cells, we could learn more details about pancreatic development and β-cell specification. This review gives an overview of pancreas ontogenesis in the perspective of stem cell differentiation, and highlights the critical aspects of small molecules in the generation of a renewable β-cell source. Also, it discusses longer term challenges and opportunities in moving towards a therapeutic goal for diabetes

Signaltransduktion durch Met in der adulten Leber

1 INTRODUCTION 6 1.1 Met, the tyrosine kinase receptor 6 1.2 The structure of HGF/SF and its receptor, Met 6 1.3 Met signal transduction 7 1.4 Met signaling in development 9 1.5 Met function in the adult 10 1.6 Liver regeneration 11 1.6.1 Experimental models of liver regeneration 12 1.6.2 Hepatocyte proliferative capacity 13 1.6.3 Liver regeneration is a multi-step process 13 1.6.4 Factors that regulate hepatocyte growth 15 1.7 Cell cycle progression: entry into S-phase 16 1.7.1 Positive and negative regulation and degradation of cdks 18 1.8 The aim of this study 20 2 MATERIALS AND METHODS 21 2.1 Abbreviations 21 2.2 Materials 23 2.2.1 Bacterial strains 23 2.2.2 Vectors/plasmids 23 2.2.3 ES cell line 23 2.2.4 Antibodies 24 2.2.5 Mouse strains: 25 2.2.6 Cell Culture Media 25 2.3 Methods 27 2.3.1 Extraction and Purification of DNA 27 2.3.2 Polymerase chain reaction (PCR) 28 2.3.3 DNA sequencing 30 2.3.4 Southern blotting 31 2.3.5 Cell culture 32 2.3.6 Generation of conditional knockout mice 35 2.3.7 Partial hepatectomy 35 2.3.8 Histology and staining procedures 36 2.3.9 Protein biochemistry 39 3 RESULTS 45 3.1 Generation of conditional Met mutant mice 45 3.2 Elimination of Met function in an adult liver 48 3.3 Met function in the normal liver 49 3.4 Met is essential for liver regeneration 51 3.4.1 Exit from quiescence in the Met-deficient regenerating livers 58 3.4.2 Entry into S-phase during liver regeneration in conditional Met mutant mice 59 3.4.3 Negative regulators of cell cycle progression during liver regeneration in conditional Met mutant mice 61 3.4.4 Changes in the regulation of growth factors and cytokines in Met mutant mice after partial hepatectomy 62 3.4.5 Signaling pathways activated during liver regeneration in control and conditional Met mutant mice. 64 4 DISCUSSION 66 4.1 Analysis of Met function in the adult liver 67 4.2 Cell cycle progression in conditional Met mutant mice 69 4.3 Cytokines and growth factors are increased in the blood during liver regeneration 72 4.4 Signaling of cytokines and growth factor during liver regeneration 74 4.5 Outlook 76 5.0 Summary 71 Zusammenfassung 72 6.0 References 80The Met tyrosine kinase receptor and its ligand, HGF/SF, play important roles in embryonic development of the liver, placenta and muscle. The placenta defects are responsible for lethality of Met-/- and HGF/SF-/- mutants in uterus. This embryonic lethality had precluded a genetic analysis of Met functions in the adult. I used here conditional gene targeting in mice to examine the function of Met receptor in the adult liver. The Mx-cre transgenic strain was used to introduce a Met mutation in the adult liver, after growth of the organ had ceased. The ablation of the Met receptor had little effect on the adult liver and only the long-term loss of the receptor caused a stress and led to abnormal lipid accumulation in the liver. In contrast, Met signaling played a crucial role in the liver that was challenged by partial hepatectomy. Liver regeneration was severely impaired in mice carrying the Mx- induced Met mutation, and was accompanied by a decrease in hepatocyte proliferation. Analysis of cell cycle progression in conditional Met mutant mice revealed an impaired exit from quiescence, i.e. a reduced cyclin D expression. In addition, the expression or the activity of several S-phase molecules was reduced, indicating impaired entry into S-phase. Moreover, conditional Met mutant mice showed an abnormal up-regulation of cdk-inhibitor p21Cip1/Waf1 in the regenerating liver. Impaired liver regeneration was accompanied by compensatory physiological responses, for instance a prolonged up-regulation of HGF/SF and IL-6 in the blood. A biochemical analysis enabled me to determine the contribution of HGF/SF/Met to the activation of various of signaling pathways in the regenerating liver. Activation of Erk/MAP kinases depended exclusively on HGF/SF/Met signaling. In contrast, Met cooperated to activate other signaling pathways, for instance the Akt kinase. Thus, interplay between different signaling systems plays an important role during liver regeneration. The mutation of Met receptors in the adult liver allowed me to demonstrate that the HGF/SF/Met signaling system is not only important for liver development, but also for regeneration and homeostasis of this organ.Der Tyrosinkinase Rezeptor Met und sein Ligand Hepatocyte Growth Factor/Scatter Factor (HGF/SF) besitzen essentielle Funktionen in der Embryonalentwicklung der Leber, der Skelettmuskulatur, sowie der Plazentaentwicklung. Mäuse mit homozygoten Mutationen des Met- bzw. des HGF /SF-Gens sterben während der Embryogenese an den Folgen einer gestörten Plazentaentwicklung. Es war daher bisher nicht möglich, adulte Funktionen des HGF/SF/Met Signalsystems genetisch in Mäusen zu untersuchen. Ich habe Konditionelles Gene Targeting in Mäusen eingesetzt, um Funktionen des Met- Rezeptors in der adulten Leber zu untersuchen. Mit Hilfe eines induzierbaren Cre des transgenen Mausstamms Mx-Cre, wurde Met in der adulten Leber, d.h. nach Abschluß des Organwachstums mutiert. Nach Deletion von Met wurden keine unmittelbaren Organveränderungen in der adulten Leber beobachtet. Mutante Mäuse entwickelten jedoch während eines längeren Beobachtungszeitraums eine ausgeprägte Fetteinlagerung (Steatose) der Leber. Ich konnte außerdem zeigen, daß Met essentiell ist für die Leberregeneration. Nach partieller Hepatektomie war die Regeneration des Lebergewebes in Met mutanten Mäusen hochgradig gestört. Dies war begleitet von einer reduzierten Hepatozytenproliferation. Eine Zellzyklusanalyse ergab, daß die Cyclin D Expression reduziert, also der Wiedereintritt in den Zellzyklus in Met Mutanten gestört ist. Es konnte außerdem anhand der reduzierten Expression mehrerer S-Phase Marker eine gestörte Progression in die S-Phase beobachtet werden. Mäuse mit konditioneller Mutation von Met zeigten darüber hinaus eine abnorm gesteigerte Expression des Cdk-Inhibitors p21Cip1/Waf1 im regenerierenden Lebergewebe. Die gestörte Leberregeneration war ebenfalls begleitet von prolongiert erhöhten Blutkonzentrationen für HGF/SF und IL-6. Ich konnte biochemisch den relativen Beitrag des HGF/SF/Met Signalsystems zur Aktivierung unterschiedlicher, intrazellulärer Signalwege in der regenerierenden Leber bestimmen. So ist die Aktivierung von Erk/MAP Kinasen ausschließlich Met-abhängig. Demgegenüber kooperiert Met mit anderen Signalsystemen z. B. bei der Aktivierung von Akt Kinasen. Das Zusammenwirken mehrerer Signalwege spielt damit eine bedeutsame Rolle bei der Leberregeneration. Durch die konditionelle Mutation des Tyrosinkinase Rezeptors Met in der adulten Leber konnte ich zeigen, daß das HGF/SF/Met Signalsystem neben der Embryogenese der Leber auch für die adulte Biologie und Regeneration dieses Organs essentiell ist

Dérégulation de la ménine par HBZ - un nouveau regard sur le mécanisme d'activation de la télomérase pendant la leucémogénèse induite par HTLV-1

Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Reactivation of telomerase, a critical event in tumor progression observed in late phases of ATL development, has been shown to be caused by HBZ (HTLV-1 bZIP factor), a regulatory protein encoded by the negative strand of the HTLV-1 genome. The HBZ-mediated up-regulation of the telomerase catalytic subunit is dependent on JunD, which in the cellular context occurs in the complex with menin, the product of the MEN-1 tumor suppressor gene. Interaction with menin represses JunD-dependent transcription and converts JunD into a growth suppressor, whereas it acts as a growth promoter in the absence of menin. My results demonstrate that the viral protein HBZ abrogates tumor suppressor function of menin, resulting in the activation of JunD transcriptional activity and finally in the up-regulation of its target gene, the human telomerase reverse transcriptase (hTERT). I showed that HBZ, JunD and menin can coexist in the same protein complex and that HBZ and menin exert opposite effects on JunD transcriptional activity. Moreover menin inhibits the JunD-mediated activation of the hTERT proximal promoter and HBZ is able to counteract this effect. Finally, I proposed that HBZ, by recruiting p300 histone acetyltransferase, reverses the histone deacetylation conducted by menin-recruited HDACs and therefore up-regulates the expression of the hTERT gene.Altogether, my work led to the identification of the molecular mechanism leading to the functional impairment of the menin tumor suppressor, which results in the deregulation of AP-1 signaling in HTLV-1 infected cells. Finally this work gave new insights into the mechanism of the transcriptional up-regulation of the hTERT gene upon HTLV-1 infection, being a key event during the development of Adult T-cell leukemia and a necessary step towards the progression into more aggressive courses.La leucémie T de l’adulte (ATL) est une pathologie lympho-proliférative aiguë associée à l’infection par le virus HTLV-1 (human T-cell leukemia virus type 1). La réactivation de la télomérase observée lors de la phase tardive du développement de l’ATL est un évènement crucial dans la progression tumorale. Elle est induite au niveau transcriptionnel par la protéine HBZ (HTLV-1 bZIP factor) et est dépendante du facteur de transcription JunD. Ce dernier est normalement associé en complexe avec le produit du gène suppresseur de tumeur MEN-1, la ménine, dont l’interaction avec JunD réprime la transcription JunD-dépendante et convertit JunD en inhibiteur de croissance.Mes résultats démontrent que la protéine virale HBZ inhibe la fonction suppresseur de tumeur de la ménine, induisant l’activité transcriptionnelle de JunD et donc l’activation de la transcription de son gène cible : la transcriptase inverse télomérase humaine (hTERT). J’ai démontré que HBZ, JunD et la ménine peuvent coexister dans un même complexe protéique et que HBZ et la ménine ont des effets opposés sur l’activité transcriptionnelle de JunD. En effet la ménine inhibe l’activation du promoteur proximal d’hTERT par JunD, alors que HBZ est capable de contre balancer cet effet. Finalement, je propose qu’en recrutant l’histone acétyltransférase p300, HBZ réverse la déacétylation des histones induite par le recrutement des HDACs par la ménine et par conséquent active le promoteur d’hTERT.L’ensemble de ces résultats a permis d’identifier les mécanismes moléculaires aboutissant à l’inhibition fonctionnelle de la protéine suppresseur de tumeur ménine, résultant en la dérégulation de la voie AP-1 dans les cellules infectées par HTLV-1. Finalement, ce travail apporte de nouvelles précisions sur le mécanisme de la surexpression transcriptionnelle de la télomérase lors de l’infection par HTLV-1, une étape importante de la mise en place et du développement de la leucémie T de l’adulte vers des stades plus agressifs

Dérégulation de la ménine par HBZ - un nouveau regard sur le mécanisme d'activation de la télomérase pendant la leucémogénèse induite par HTLV-1

La leucémie T de l adulte (ATL) est une pathologie lympho-proliférative aiguë associée à l infection par le virus HTLV-1 (human T-cell leukemia virus type 1). La réactivation de la télomérase observée lors de la phase tardive du développement de l ATL est un évènement crucial dans la progression tumorale. Elle est induite au niveau transcriptionnel par la protéine HBZ (HTLV-1 bZIP factor) et est dépendante du facteur de transcription JunD. Ce dernier est normalement associé en complexe avec le produit du gène suppresseur de tumeur MEN-1, la ménine, dont l interaction avec JunD réprime la transcription JunD-dépendante et convertit JunD en inhibiteur de croissance.Mes résultats démontrent que la protéine virale HBZ inhibe la fonction suppresseur de tumeur de la ménine, induisant l activité transcriptionnelle de JunD et donc l activation de la transcription de son gène cible : la transcriptase inverse télomérase humaine (hTERT). J ai démontré que HBZ, JunD et la ménine peuvent coexister dans un même complexe protéique et que HBZ et la ménine ont des effets opposés sur l activité transcriptionnelle de JunD. En effet la ménine inhibe l activation du promoteur proximal d hTERT par JunD, alors que HBZ est capable de contre balancer cet effet. Finalement, je propose qu en recrutant l histone acétyltransférase p300, HBZ réverse la déacétylation des histones induite par le recrutement des HDACs par la ménine et par conséquent active le promoteur d hTERT. L ensemble de ces résultats a permis d identifier les mécanismes moléculaires aboutissant à l inhibition fonctionnelle de la protéine suppresseur de tumeur ménine, résultant en la dérégulation de la voie AP-1 dans les cellules infectées par HTLV-1. Finalement, ce travail apporte de nouvelles précisions sur le mécanisme de la surexpression transcriptionnelle de la télomérase lors de l infection par HTLV-1, une étape importante de la mise en place et du développement de la leucémie T de l adulte vers des stades plus agressifs.Adult T-cell leukemia (ATL) is an aggressive lymphoproliferative disorder associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Reactivation of telomerase, a critical event in tumor progression observed in late phases of ATL development, has been shown to be caused by HBZ (HTLV-1 bZIP factor), a regulatory protein encoded by the negative strand of the HTLV-1 genome. The HBZ-mediated up-regulation of the telomerase catalytic subunit is dependent on JunD, which in the cellular context occurs in the complex with menin, the product of the MEN-1 tumor suppressor gene. Interaction with menin represses JunD-dependent transcription and converts JunD into a growth suppressor, whereas it acts as a growth promoter in the absence of menin. My results demonstrate that the viral protein HBZ abrogates tumor suppressor function of menin, resulting in the activation of JunD transcriptional activity and finally in the up-regulation of its target gene, the human telomerase reverse transcriptase (hTERT). I showed that HBZ, JunD and menin can coexist in the same protein complex and that HBZ and menin exert opposite effects on JunD transcriptional activity. Moreover menin inhibits the JunD-mediated activation of the hTERT proximal promoter and HBZ is able to counteract this effect. Finally, I proposed that HBZ, by recruiting p300 histone acetyltransferase, reverses the histone deacetylation conducted by menin-recruited HDACs and therefore up-regulates the expression of the hTERT gene. Altogether, my work led to the identification of the molecular mechanism leading to the functional impairment of the menin tumor suppressor, which results in the deregulation of AP-1 signaling in HTLV-1 infected cells. Finally this work gave new insights into the mechanism of the transcriptional up-regulation of the hTERT gene upon HTLV-1 infection, being a key event during the development of Adult T-cell leukemia and a necessary step towards the progression into more aggressive courses.LYON-ENS Sciences (693872304) / SudocSudocFranceF

How to make beta cells?

Insulin-producing beta cells are lost or insufficient in diabetic patients, presenting the medical challenge for new beta cells. Currently, there are three strategies that offer promise. One involves the generation of beta cells de novo by directing the differentiation of either embryonic stem cells or induced pluripotent cells to the beta cell lineage. The second is based on the conversion of another terminally differentiated cell to beta cells in a process called reprogramming. The third approach is to promote the replication of existing beta cells either in vivo or in vitro. Significant progress is evident for each strategy, but it remains unclear which approach will ultimately prove successful.Stem Cell and Regenerative Biolog