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Publication
Journal: Nature
January/6/2014
Abstract
Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
Publication
Journal: Cancer Research
April/5/1998
Abstract
Mutation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of beta-catenin/Tcf-mediated transcription. To further explore the role of the APC/beta-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the gamma-catenin (CTNNG1), GSK-3alpha (GSK3A), or GSK-3beta (GSK3B) genes were detected. In contrast, mutations in the NH2-terminal regulatory domain of beta-catenin (CTNNB1) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the beta-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on beta-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that beta-catenin signaling plays a critical role in CR tumorigenesis.
Publication
Journal: Pharmacology and Therapeutics
November/29/2015
Abstract
Glycogen synthase kinase-3 (GSK3) may be the busiest kinase in most cells, with over 100 known substrates to deal with. How does GSK3 maintain control to selectively phosphorylate each substrate, and why was it evolutionarily favorable for GSK3 to assume such a large responsibility? GSK3 must be particularly adaptable for incorporating new substrates into its repertoire, and we discuss the distinct properties of GSK3 that may contribute to its capacity to fulfill its roles in multiple signaling pathways. The mechanisms regulating GSK3 (predominantly post-translational modifications, substrate priming, cellular trafficking, protein complexes) have been reviewed previously, so here we focus on newly identified complexities in these mechanisms, how each of these regulatory mechanism contributes to the ability of GSK3 to select which substrates to phosphorylate, and how these mechanisms may have contributed to its adaptability as new substrates evolved. The current understanding of the mechanisms regulating GSK3 is reviewed, as are emerging topics in the actions of GSK3, particularly its interactions with receptors and receptor-coupled signal transduction events, and differential actions and regulation of the two GSK3 isoforms, GSK3α and GSK3β. Another remarkable characteristic of GSK3 is its involvement in many prevalent disorders, including psychiatric and neurological diseases, inflammatory diseases, cancer, and others. We address the feasibility of targeting GSK3 therapeutically, and provide an update of its involvement in the etiology and treatment of several disorders.
Publication
Journal: Annual Review of Biochemistry
August/20/2012
Abstract
Excessive caloric intake without a rise in energy expenditure promotes adipocyte hyperplasia and adiposity. The rise in adipocyte number is triggered by signaling factors that induce conversion of mesenchymal stem cells (MSCs) to preadipocytes that differentiate into adipocytes. MSCs, which are recruited from the vascular stroma of adipose tissue, provide an unlimited supply of adipocyte precursors. Members of the BMP and Wnt families are key mediators of stem cell commitment to produce preadipocytes. Following commitment, exposure of growth-arrested preadipocytes to differentiation inducers [insulin-like growth factor 1 (IGF1), glucocorticoid, and cyclic AMP (cAMP)] triggers DNA replication and reentry into the cell cycle (mitotic clonal expansion). Mitotic clonal expansion involves a transcription factor cascade, followed by the expression of adipocyte genes. Critical to these events are phosphorylations of the transcription factor CCATT enhancer-binding protein β (C/EBPβ) by MAP kinase and GSK3β to produce a conformational change that gives rise to DNA-binding activity. "Activated" C/EBPβ then triggers transcription of peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which in turn coordinately activate genes whose expression produces the adipocyte phenotype.
Publication
Journal: Skeletal Muscle
October/19/2011
Abstract
A highly conserved signaling pathway involving insulin-like growth factor 1 (IGF1), and a cascade of intracellular components that mediate its effects, plays a major role in the regulation of skeletal muscle growth. A central component in this cascade is the kinase Akt, also called protein kinase B (PKB), which controls both protein synthesis, via the kinases mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3β (GSK3β), and protein degradation, via the transcription factors of the FoxO family. In this paper, we review the composition and function of this pathway in skeletal muscle fibers, focusing on evidence obtained in vivo by transgenic and knockout models and by muscle transient transfection experiments. Although this pathway is essential for muscle growth during development and regeneration, its role in adult muscle response to mechanical load is less clear. A full understanding of the operation of this pathway could help to design molecularly targeted therapeutics aimed at preventing muscle wasting, which occurs in a variety of pathologic contexts and in the course of aging.
Publication
Journal: Free Radical Biology and Medicine
August/12/2014
Abstract
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes that encode detoxifying enzymes, drug transporters, antiapoptotic proteins, and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase-mediated degradation of Nrf2. Chemicals, including antioxidants, tocopherols including α-tocopherol (vitamin E), and phytochemicals, and radiation antagonize the Nrf2:INrf2 interaction and lead to the stabilization and activation of Nrf2. The signaling events involve preinduction, induction, and postinduction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinases in a preinduction response that phosphorylates specific residues on Nrf2 negative regulators, INrf2, Fyn, and Bach1, leading to their nuclear export, ubiquitination, and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2 cysteine 151 followed by PKC phosphorylation of Nrf2 serine 40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus, resulting in a coordinated activation of gene expression. This is followed by a postinduction response that controls the "switching off" of Nrf2-activated gene expression. GSK3β, under the control of AKT and PI3K, phosphorylates Fyn, leading to Fyn nuclear localization. Fyn phosphorylates Nrf2 Y568, resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provide protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
Publication
Journal: Journal of the National Cancer Institute
February/27/2014
Abstract
Wnt/β-catenin alterations are prominent in human malignancies. In non-small cell lung cancer (NSCLC), β-catenin and APC mutations are uncommon, but Wnt signaling is important in NSCLC cell lines, and Wnt inhibition reduces proliferation. Overexpression of Wnt-1, -2, -3, and -5a and of Wnt-pathway components Frizzled-8, Dishevelled, Porcupine, and TCF-4 is common in resected NSCLC and is associated with poor prognosis. Conversely, noncanonical Wnt-7a suppresses NSCLC development and is often downregulated. Although β-catenin is often expressed in NSCLCs, it was paradoxically associated with improved prognosis in some series, possibly because of E-cadherin interactions. Downregulation of Wnt inhibitors (eg, by hypermethylation) is common in NSCLC tumor cell lines and resected samples; may be associated with high stage, dedifferentiation, and poor prognosis; and has been reported for AXIN, sFRPs 1-5, WIF-1, Dkk-1, Dkk-3, HDPR1, RUNX3, APC, CDX2, DACT2, TMEM88, Chibby, NKD1, EMX2, ING4, and miR-487b. AXIN is also destabilized by tankyrases, and GSK3β may be inactivated through phosphorylation by EGFR. Preclinically, restoration of Wnt inhibitor function is associated with reduced Wnt signaling, decreased cell proliferation, and increased apoptosis. Wnt signaling may also augment resistance to cisplatin, docetaxel, and radiotherapy, and Wnt inhibitors may restore sensitivity. Overall, available data indicate that Wnt signaling substantially impacts NSCLC tumorigenesis, prognosis, and resistance to therapy, with loss of Wnt signaling inhibitors by promoter hypermethylation or other mechanisms appearing to be particularly important. Wnt pathway antagonists warrant exploration clinically in NSCLC. Agents blocking selected specific β-catenin interactions and approaches to increase expression of downregulated Wnt inhibitors may be of particular interest.
Publication
Journal: Nature Structural and Molecular Biology
May/2/2013
Abstract
Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
Publication
Journal: Nature
January/31/2011
Abstract
We report that, in the rat, administering insulin-like growth factor II (IGF-II, also known as IGF2) significantly enhances memory retention and prevents forgetting. Inhibitory avoidance learning leads to an increase in hippocampal expression of IGF-II, which requires the transcription factor CCAAT enhancer binding protein β and is essential for memory consolidation. Furthermore, injections of recombinant IGF-II into the hippocampus after either training or memory retrieval significantly enhance memory retention and prevent forgetting. To be effective, IGF-II needs to be administered within a sensitive period of memory consolidation. IGF-II-dependent memory enhancement requires IGF-II receptors, new protein synthesis, the function of activity-regulated cytoskeletal-associated protein and glycogen-synthase kinase 3 (GSK3). Moreover, it correlates with a significant activation of synaptic GSK3β and increased expression of GluR1 (also known as GRIA1) α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid receptor subunits. In hippocampal slices, IGF-II promotes IGF-II receptor-dependent, persistent long-term potentiation after weak synaptic stimulation. Thus, IGF-II may represent a novel target for cognitive enhancement therapies.
Publication
Journal: Cell Stem Cell
April/7/2014
Abstract
Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.
Publication
Journal: Nature Cell Biology
July/8/2015
Abstract
The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3β/β-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.
Publication
Journal: Cell
January/17/2011
Abstract
The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3β signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.
Publication