Functional relationships between pluripotency regulators and cell cycle in the pluripotent embryonic stem cells

Les cellules souches embryonnaires de souris (mESC) présentent un cycle cellulaire atypique caractérisé par l'absence d'une voie Rb fonctionnelle et la forte expression de la cycline E pendant toutes les phases du cycle cellulaire. En conséquence, les mESC sont constitutivement amorcées pour la réplication de l'ADN. Pour comprendre comment la cycline E, un régulateur clé de la transition de la phase G1 à S, est régulée dans les mESC, nous avons analysé la régulation transcriptionnelle de son gène Ccne1 par des facteurs de transcription du réseau de pluripotence naïve. Nous avons observé que les facteurs Esrrb, Klf4 et Tfcp2l1 se lient à la région du promoteur de Ccne1 sur plusieurs sites situés entre 0 et 1kb en amont du site d'initiation de la transcription. Un test luciférase nous a permis de monter qu'une mutation de ces sites de liaison diminue ou abolie l'activité transcriptionnelle du promoteur. De plus, la surexpression inductible à la doxycycline des facteurs Esrrb, Klf4 et Tfcp2l1 augment le niveau d'expression d'ARNm de Ccne1. Ces résultats suggèrent que Esrrb, Klf4 et Tfcp2l1 contrôlent l'expression de la cycline E. Ils mettent en évidence un lien direct entre le réseau de pluripotence naïve et la régulation du cycle mitotique dans les mESC. Nous avons utilisé le système rapporteur FUCCI pour étudier en fonction du cycle cellulaire l'expression des facteurs de transcription qui forment le réseau de pluripotence naïve. Nous avons observé que l'expression de Esrrb, Klf4, Tfcp2l1 et Nanog oscille au cours du cycle cellulaire avec une diminution de l'expression entre la phase G1 précoce et le début de S, puis une ré-augmentation entre le début de S et la phase G2/M. Ces résultats suggèrent que le réseau de pluripotence naïve est déstabilisé transitoirement lors du passage de la phase G1 à la phase S du cycle cellulaireMouse embryonic stem cells (mESCs) display an unorthodox cell cycle characterised by the lack of a functional Rb pathway and robust expression of cyclin E during all cell cycle phases. Therefore, mESCs are constitutively primed for DNA replication. To understand how cyclin E, a key regulator of the G1-to-S phase transition, is regulated in mESCs, we analysed the transcriptional regulation of Ccne1 by transcription factors of the naive pluripotency network. We observed that Esrrb, Klf4 and Tfcp2l1 bound the Ccne1 promoter region on multiple sites between 0 and 1kb upstream transcription start site. Disrupting the binding sites reduced or abolished transcriptional activity in a luciferase assay. Moreover, the doxycyclin-inducible expression of Essrb, Klf4 and Tfcp2l1 up-regulated the Ccne1 mRNA level. Taken together, these results strongly suggest that Essrb, Klf4 and Tfcp2l1 control Cyclin E expression and highlight a direct connection between the naïve pluripotency network and regulation of the mitotic cycle in mESCs. We used the FUCCI reporter system to study cell-cycle dependent expression of the transcription factors that form the naïve pluripotency network. Esrrb, Klf4, Tfcp2l1 and Nanog expression oscillated during the cell cycle with a down-regulated expression between the early G1-phase and the beginning of S-phase, and then up-regulated expression between the beginning of S-phase and the G2/M-phase. These results suggest that the naive pluripotency network is destabilized transiently during the transition from the G1-phase to the S-phase of the cell cycl

Regulation of Cyclin E by transcription factors of the naïve pluripotency network in mouse embryonic stem cells

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Mild Therapeutic Hypothermia Protects from Acute and Chronic Renal Ischemia-Reperfusion Injury in Mice by Mitigated Mitochondrial Dysfunction and Modulation of Local and Systemic Inflammation

International audienceRenal ischemia-reperfusion (IR) injury can lead to acute kidney injury, increasing the risk of developing chronic kidney disease. We hypothesized that mild therapeutic hypothermia (mTH), 34 °C, applied during ischemia could protect the function and structure of kidneys against IR injuries in mice. In vivo bilateral renal IR led to an increase in plasma urea and acute tubular necrosis at 24 h prevented by mTH. One month after unilateral IR, kidney atrophy and fibrosis were reduced by mTH. Evaluation of mitochondrial function showed that mTH protected against IR-mediated mitochondrial dysfunction at 24 h, by preserving CRC and OX-PHOS. mTH completely abrogated the IR increase of plasmatic IL-6 and IL-10 at 24 h. Acute tissue inflammation was decreased by mTH (IL-6 and IL1-β) in as little as 2 h. Concomitantly, mTH increased TNF-α expression at 24 h. One month after IR, mTH increased TNF-α mRNA expression, and it decreased TGF-β mRNA expression. We showed that mTH alleviates renal dysfunction and damage through a preservation of mitochondrial function and a modulated systemic and local inflammatory response at the acute phase (2–24 h). The protective effect of mTH is maintained in the long term (1 month), as it diminished renal atrophy and fibrosis, and mitigated chronic renal inflammation

SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases

Abstract Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca2+ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca2+ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue

Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency

International audienceLeukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2-and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells