Leukemia 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.

The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivityconferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.

Continuous, non-cell cycle-dependent expression of cyclin E is a characteristic feature of mouse embryonic stem cells (mESCs). We studied the 5′ regulatory region of Cyclin E, also known as Ccne1, and identified binding sites for transcription factors of the naïve pluripotency network, including Esrrb, Klf4, and Tfcp2l1 within 1 kilobase upstream of the transcription start site. Luciferase assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChiP–qPCR) study highlighted one binding site for Esrrb that is essential to transcriptional activity of the promoter region, and three binding sites for Klf4 and Tfcp2l1. Knockdown of Esrrb, Klf4, and Tfcp2l1 reduced Cyclin E expression whereas overexpression of Esrrb and Klf4 increased it, indicating a strong correlation between the expression level of these factors and that of cyclin E. We observed that cyclin E overexpression delays differentiation induced by Esrrb depletion, suggesting that cyclin E is an important target of Esrrb for differentiation blockade. We observed that mESCs express a low level of miR-15a and that transfection of a miR-15a mimic decreases Cyclin E mRNA level. These results lead to the conclusion that the high expression level of Cyclin E in mESCs can be attributed to transcriptional activation by Esrrb as well as to the absence of its negative regulator, miR-15a.

Renal 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.

Continuous, non-cell cycle-dependent expression of cyclin E is a characteristic feature of mouse embryonic stem cells (ESCs). We studied the 5′ regulatory region of Cyclin E, also known as Ccne1, and identified binding sites for transcription factors of the naïve pluripotency network, including Esrrb, Klf4, and Tfcp2l1 within 1 kilobase upstream of the transcription start site. Luciferase assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChiP-qPCR) study highlighted one binding site for Esrrb that is essential to transcriptional activity of the promoter region, and three binding sites for Klf4 and Tfcp2l1. Knockdown of Esrrb, Klf4, and Tfcp2l1 reduced Cyclin E expression whereas overexpression of Esrrb and Klf4 increased it, indicating a strong correlation between the expression level of these factors and that of cyclin E. We observed that cyclin E overexpression delays differentiation induced by Esrrb depletion, suggesting that cyclin E is an important target of Esrrb for differentiation blockade. We observed that mESCs express a low level of miR-15a and that transfection of a miR-15a mimic decreases Cyclin E mRNA level. These results lead to the conclusion that the high expression level of Cyclin E in mESCs can be attributed to transcriptional activation by Esrrb as well as to the absence of its negative regulator, miR-15a. IntroductionCyclin E is a regulatory subunit of cyclin-dependent kinase (Cdk) 2 involved in many cellular processes including cell cycle progression, replication complex assembly, centrosome cycle, and epigenetic regulation. Its expression is regulated at both the transcriptional and protein level to achieve a timely control of cell division in connection with cell environment and fate decision. Deregulated expression of cyclin E has been shown to play a key role in tumorigenesis [1,2]. Transcription of the Cyclin E gene, also known as Ccne1, is activated during the G1 phase and depends on mitogenic input, which is integrated through E2F and Myc transcription factors [3,4]. E2F activity is regulated by phosphorylation of the retinoblastoma (Rb) protein in response to cyclin D/Cdk4 and cyclin D/Cdk6 kinase activity [5,6]. The miRNA miR-15a was shown to act as a negative regulator of Cyclin E in somatic cells [7,8]. Since, both Cyclin E and mir-15a are direct transcriptional targets of E2F, it raises the possibility that E2F, miR-15, and cyclin E constitute a feed-forward loop that modulates E2F activity and cell-cycle progression [8].There is a growing body of evidence showing that the cell cycle of mouse embryonic stem cells (ESCs) lacks some of the regulatory pathways that operate in somatic cells [9][10][11].These include extensive phosphorylation of the Rb family proteins despite little cyclin D/Cdk4 kinase activity [12], p16 ink4a -resistant residual cyclin D3/Cdk6 kinase activity [13], and lack of functional Chk/p53/p21 cip1 and Chk/Cdc25A pathways resulting in the absence of the DNA damage checkpoint in the G1 phase [14][15][16]. A key feature of the pluri...

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Introduction: Despite advances in cardioprotection, new therapeutic strategies capable of preventing acute myocardial ischemia-reperfusion injury and reducing secondary event of patients are still needed. Hypothesis: We hypothesized that limitation of SERCA2 phosphorylation on a serine/threonine residue at reperfusion could provide protection against reperfusion injury, via the activation of the Ca 2+ reuptake into SR/ER which confers detoxification of the cytosolic and mitochondrial Ca 2+ overload during reperfusion . Methods: Combining in silico analysis with in vitro and in vivo genetic approaches, we discovered that the phosphorylation of SERCA2 at serine 663 is a clinical and pathophysiological event of cardiac function. Results: We demonstrated that the phosphorylation level of SERCA2 at serine 663 is increased with heart damage in both patient and mouse ischemic hearts. Mechanistically, we demonstrated that preventing serine 663 phosphorylation significantly increased SERCA2 Ca 2+ pumping activity into the reticulum and protected against hypoxia/reoxygenation-induced cell death, by counteracting the cytosolic and mitochondrial Ca 2+ overload. To link this specific residue event to a physiological role of SERCA2 in heart, we demonstrated that gene therapy for the phosphoresistant form of SERCA2 at serine 663 improved the excitation/contraction coupling of cardiomyocytes and significantly reduced infarct size in an in vivo myocardial infarction model, whereas mice expressing a phosphomimetic form of SERCA2 developed a larger infarct size. Conclusions: Together, these findings establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 on its serine 663 residue.

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.