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OTX2 is a transcription factor and known driver in medulloblastoma (MB), where it is amplified in a subset of tumours and overexpressed in most cases of group 3 and group 4 MB. Here we demonstrate a noncanonical role for OTX2 in group 3 MB alternative splicing. OTX2 associates with the large assembly of splicing regulators complex through protein-protein interactions and regulates a stem cell splicing program. OTX2 can directly or indirectly bind RNA and this may be partially independent of its DNA regulatory functions. OTX2 controls a pro-tumorigenic splicing program that is mirrored in human cerebellar rhombic lip origins. Among the OTX2-regulated differentially spliced genes, PPHLN1 is expressed in the most primitive rhombic lip stem cells, and targeting PPHLN1 splicing reduces tumour growth and enhances survival in vivo. These findings identify OTX2-mediated alternative splicing as a major determinant of cell fate decisions that drive group 3 MB progression., (© 2024. The Author(s).)

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses., (© 2024. The Author(s).)

Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase function. The OTUD7A protein-protein interaction network included synaptic, axonal, and cytoskeletal proteins and was enriched for ASD and epilepsy risk genes (Ank3, Ank2, SPTAN1, SPTBN1). The interactions between OTUD7A and Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7A L233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment, while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7A L233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Furthermore, our study highlights the utility of targeting CNV genes using cell type-specific proteomics to identify shared and unexplored disease mechanisms across NDDs., (© 2023. The Author(s).)

There are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID2) to identify protein-protein interaction (PPI) networks for 41 ASD risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout displays an association between mitochondrial activity and ASD risk genes. The PPI network shows an enrichment of 112 additional ASD risk genes and differentially expressed genes from postmortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data report that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and highlight biological insight into disease mechanisms and sub-cohorts of ASD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a β-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure., (© 2021 by The American Society of Hematology.)

Background: Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a homopolymeric, adhesive, platelet and endothelial protein that binds to von Willebrand factor and enhances platelet adhesion to fibrillar collagen ex vivo., Objectives: To examine the impact of Mmrn1 deficiency on platelet adhesive function, and the molecular motifs in fibrillar collagen that bind MMRN1 to enhance platelet adhesion., Methods: Mmrn1-deficient mice were generated and assessed for altered platelet adhesive function. Collagen Toolkit peptides, and other triple-helical collagen peptides, were used to identify multimerin 1 binding motifs and their contribution to platelet adhesion., Results: MMRN1 bound to conserved GPAGPOGPX sequences in collagens I, II, and III (including GPAGPOGPI, GPAGPOGPV, and GPAGPOGPQ) that enhanced activated human platelet adhesion to collagen synergistically with other triple-helical collagen peptides (P < .05). Mmrn1 -/- and Mmrn1 +/- mice were viable and fertile, with complete and partial platelet Mmrn1 deficiency, respectively. Relative to wild-type mice, Mmrn1 -/- and Mmrn1 +/- mice did not have overt bleeding, increased median bleeding times, or increased wound blood loss (P ≥ .07); however, they both showed significantly impaired platelet adhesion and thrombus formation in the ferric chloride injury model (P ≤ .0003). Mmrn1 -/- platelets had impaired adhesion to GPAGPOGPX peptides and fibrillar collagen (P ≤ .03) and formed smaller aggregates than wild-type platelets when captured onto collagen, triple-helical collagen mimetic peptides, von Willebrand factor, or fibrinogen (P ≤ .008), despite preserved, low shear, and high shear aggregation responses., Conclusions: Multimerin 1 supports platelet adhesion and thrombus formation and binds to highly conserved, GPAGPOGPX motifs in fibrillar collagens that synergistically enhance platelet adhesion., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/β-catenin signaling.

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133 high and CD133 low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

Copy-number variations (CNVs) are strong risk factors for neurodevelopmental and psychiatric disorders. The 15q13.3 microdeletion syndrome region contains up to ten genes and is associated with numerous conditions, including autism spectrum disorder (ASD), epilepsy, schizophrenia, and intellectual disability; however, the mechanisms underlying the pathogenesis of 15q13.3 microdeletion syndrome remain unknown. We combined whole-genome sequencing, human brain gene expression (proteome and transcriptome), and a mouse model with a syntenic heterozygous deletion (Df(h15q13)/+ mice) and determined that the microdeletion results in abnormal development of cortical dendritic spines and dendrite outgrowth. Analysis of large-scale genomic, transcriptomic, and proteomic data identified OTUD7A as a critical gene for brain function. OTUD7A was found to localize to dendritic and spine compartments in cortical neurons, and its reduced levels in Df(h15q13)/+ cortical neurons contributed to the dendritic spine and dendrite outgrowth deficits. Our results reveal OTUD7A as a major regulatory gene for 15q13.3 microdeletion syndrome phenotypes that contribute to the disease mechanism through abnormal cortical neuron morphological development., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Co-expression and cross-regulation of the four TCF/LEFs render their redundant and unique functions ambiguous. Here, we describe quadruple-knockout (QKO) mouse ESCs lacking all full-length TCF/LEFs and cell lines rescued with TCF7 or TCF7L1. QKO cells self-renew, despite gene expression patterns that differ significantly from WT, and display delayed, neurectoderm-biased, embryoid body (EB) differentiation. QKO EBs have no contracting cardiomyocytes and differentiate poorly into mesendoderm but readily generate neuronal cells. QKO cells and TCF7L1-rescued cells cannot efficiently activate TCF reporters, whereas TCF7-rescued cells exhibit significant reporter responsiveness. Surprisingly, despite dramatically different transactivation capacities, re-expression of TCF7L1 or TCF7 in QKO cells restores their tri-lineage differentiation ability, with similar lineage marker expression patterns and beating cardiomyocyte frequencies observed in EBs. Both factors also similarly affect the transcriptome of QKO cells. Our data reveal that a single TCF, regardless of its activation capacity, is sufficient for effective trilineage differentiation of ESCs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3α (GSK-3α) and GSK-3β dependency leads to aggressive AML. Although GSK-3α deletion alone has no effect, GSK-3β deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3β and GSK-3α uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3β provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3α- and GSK-3β-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133(+) treatment-refractory population., Experimental Design: Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved., Results: We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/β-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133(+) stem cells that drive glioblastoma relapse and mortality., Conclusions: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers., (©2015 American Association for Cancer Research.)

Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3β(-/-), and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α(-/-);Gsk-3β(-/-) ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α(-/-);Gsk-3β(-/-) ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3-dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3-mediated signal transduction in the epigenome., (© 2015 Meredith et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways. Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling. Genetic deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells (ESCs) has previously been shown to lead to the constitutive activation of the Wnt/β-catenin signaling pathway. However, in addition to Wnt signaling, all Gsk-3-regulated pathways, such as insulin signaling, are also affected simultaneously in Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-)ESCs. In an effort to better understand how specific signaling pathways contribute to the global pattern of gene expression in Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-)ESCs, we compared the gene expression profiles in Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-) ESCs to mouse ESCs in which either Wnt/β-catenin signaling or phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling are constitutively active. Our results show that Wnt signaling has a greater effect on up-regulated genes in the Gsk-3α(-) (/) (-); Gsk-3β(-) (/) (-)ESCs, whereas PI3K-dependent insulin signaling is more responsible for the down-regulation of genes in the same cells. These data show the importance of Gsk-3 activity on gene expression in mouse ESCs, and that these effects are due to the combined effects of multiple signaling pathways.

Brain tumors represent the leading cause of childhood cancer mortality, with medulloblastoma (MB) being the most frequent malignant tumor. In this review we discuss the morphological and molecular heterogeneity of this malignant childhood brain tumor and how this key feature has implicated the presence of a MB stem cell. We focus on evidence from cerebellar development, histopathological and molecular subtypes of MB, the recent identification of brain tumor-initiating cells (BTICs, also referred to as MB stem cells), and the current limitations in studying the interplay between MB stem cells and tumor heterogeneity., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Neural stem cells (NSCs) can be obtained from a variety of sources, but not all NSCs exhibit the same characteristics. We have examined how the level of glycogen synthase kinase-3 activity regulates NSCs obtained from different sources: the mouse embryonic striatum, embryonic hippocampus, and mouse ES cells. Growth of striatal NSCs is enhanced by mild inhibition of GSK-3 but not by strong inhibition that is accompanied by Wnt/TCF transcriptional activation. In contrast, the growth of hippocampal NSCs is enhanced by both mild inhibition of GSK-3 as well as stronger inhibition. Active Wnt/TCF signaling, which occurs normally in the embryonic hippocampus, is required for growth of neural stem and progenitor cells. In the embryonic striatal germinal zone, however, TCF signaling is normally absent and its activation inhibits growth of NSCs from this region. Using a genetic model for progressive loss of GSK-3, we find that primitive ES cell-derived NSCs resemble striatal NSCs. That is, partial loss of GSK-3 alleles leads to an increase in NSCs while complete ablation of GSK-3, and activation of TCF-signaling, leads to their decline. Furthermore, expression of dominant negative TCF-4 in the GSK-3-null background was effective in blocking expression of Wnt-response genes and was also able to rescue neuronal gene expression. These results reveal that GSK-3 regulates NSCs by divergent pathways depending on the tissue of origin. The responses of these neural precursor cells may be contingent on baseline Wnt/TCF signaling occurring in a particular tissue.

β-catenin, an adherens junction component and key Wnt pathway effector, regulates numerous developmental processes and supports embryonic stem cell (ESC) pluripotency in specific contexts. The β-catenin homologue γ-catenin (also known as Plakoglobin) is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. Here, we use β-catenin((+/+)) and β-catenin((-/-)) mouse embryonic stem cells (mESCs) to investigate the role of γ-catenin in Wnt signaling and mESC differentiation. Although γ-catenin protein is markedly stabilized upon inhibition or ablation of GSK-3 in wild-type (WT) mESCs, efficient silencing of its expression in these cells does not affect β-catenin/TCF target gene activation after Wnt pathway stimulation. Nonetheless, knocking down γ-catenin expression in WT mESCs appears to promote their exit from pluripotency in short-term differentiation assays. In β-catenin((-/-)) mESCs, GSK-3 inhibition does not detectably alter cytosolic γ-catenin levels and does not activate TCF target genes. Intriguingly, β-catenin/TCF target genes are induced in β-catenin((-/-)) mESCs overexpressing stabilized γ-catenin and the ability of these genes to be activated upon GSK-3 inhibition is partially restored when wild-type γ-catenin is overexpressed in these cells. This suggests that a critical threshold level of total catenin expression must be attained before there is sufficient signaling-competent γ-catenin available to respond to GSK-3 inhibition and to regulate target genes as a consequence. WT mESCs stably overexpressing γ-catenin exhibit robust Wnt pathway activation and display a block in tri-lineage differentiation that largely mimics that observed upon overexpression of β-catenin. However, β-catenin overexpression appears to be more effective than γ-catenin overexpression in sustaining the retention of markers of naïve pluripotency in cells that have been subjected to differentiation-inducing conditions. Collectively, our study reveals a function for γ-catenin in the regulation of mESC differentiation and has implications for human cancers in which γ-catenin is mutated and/or aberrantly expressed.

Brain tumors are the leading cause of childhood cancer mortality, with medulloblastoma (MB) representing the most frequent malignant tumor. The recent molecular classification of MB has reconceptualized the heterogeneity that exists within pathological subtypes by giving context to the role of key developmental signaling pathways in MB pathogenesis. The identification of cancer stem cell (CSC) populations, termed brain tumor-initiating cells (BTICs), in MB has provided novel cellular targets for the study of these aberrantly activated signaling pathways, namely, Sonic hedgehog (Shh) and Wingless (Wnt), along with the identification of novel BTIC self-renewal pathways. In this review, we discuss recent evidence for the presence of a MB stem cell that drives tumorigenesis in this malignant childhood tumor. We focus on evidence from cerebellar development, the recent identification of BTICs, the presence of activated developmental signaling pathways in MB, the role of epigenetic stem cell regulatory mechanisms, and how these developmental and epigenetic pathways may be targeted for novel therapeutic options.

Understanding the mechanisms regulating pluripotency in embryonic and induced pluripotent stem cells is required to ensure their safe use in clinical applications. Glycogen synthase kinase-3 (GSK-3) has emerged as an important regulator of pluripotency, based primarily on studies with small-molecule GSK-3 inhibitors. Here, we use mouse embryonic stem cells (ESCs) lacking GSK-3 to demonstrate that a single GSK-3 substrate, β-catenin, controls the ability of ESCs to exit the pluripotent state and to differentiate into neurectoderm. Unexpectedly, the effects of β-catenin on pluripotency do not appear to be dependent on TCF-mediated signaling, based on experiments utilizing a β-catenin C-terminal truncation mutant or highly efficient dominant-negative TCF strategies. Alternatively, we find that stabilized β-catenin forms a complex with and enhances the activity of Oct-4, a core component of the transcriptional network regulating pluripotency. Collectively, our data suggest previously underappreciated, divergent TCF-dependent and TCF-independent roles for β-catenin in ESCs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases consists of 2 highly related isoforms, alpha and beta. Although GSK-3beta has an important role in cardiac development, much remains unknown about the function of either GSK-3 isoform in the postnatal heart. Herein, we present what we believe to be the first studies defining the role of GSK-3alpha in the mouse heart using gene targeting. Gsk3a(-/-) mice over 2 months of age developed progressive cardiomyocyte and cardiac hypertrophy and contractile dysfunction. Following thoracic aortic constriction in young mice, we observed enhanced hypertrophy that rapidly transitioned to ventricular dilatation and contractile dysfunction. Surprisingly, markedly impaired beta-adrenergic responsiveness was found at both the organ and cellular level. This phenotype was reproduced by acute treatment of WT cardiomyocytes with a small molecule GSK-3 inhibitor, confirming that the response was not due to a chronic adaptation to LV dysfunction. Thus, GSK-3alpha appears to be the central regulator of a striking range of essential processes, including acute and direct positive regulation of beta-adrenergic responsiveness. In the absence of GSK-3alpha, the heart cannot respond effectively to hemodynamic stress and rapidly fails. Our findings identify what we believe to be a new paradigm of regulation of beta-adrenergic signaling and raise concerns given the rapid expansion of drug development targeting GSK-3.

Background: Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by two genes that generate two related proteins: GSK-3alpha and GSK-3beta. Mice lacking a functional GSK-3alpha gene were engineered in our laboratory; they are viable and display insulin sensitivity. In this study, we have characterized brain functions of GSK-3alpha KO mice by using a well-established battery of behavioral tests together with neurochemical and neuroanatomical analysis., Results: Similar to the previously described behaviours of GSK-3beta(+/-) mice, GSK-3alpha mutants display decreased exploratory activity, decreased immobility time and reduced aggressive behavior. However, genetic inactivation of the GSK-3alpha gene was associated with: decreased locomotion and impaired motor coordination, increased grooming activity, loss of social motivation and novelty; enhanced sensorimotor gating and impaired associated memory and coordination. GSK-3alpha KO mice exhibited a deficit in fear conditioning, however memory formation as assessed by a passive avoidance test was normal, suggesting that the animals are sensitized for active avoidance of a highly aversive stimulus in the fear-conditioning paradigm. Changes in cerebellar structure and function were observed in mutant mice along with a significant decrease of the number and size of Purkinje cells., Conclusion: Taken together, these data support a role for the GSK-3alpha gene in CNS functioning and possible involvement in the development of psychiatric disorders.

The development of the brain requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. It has been suggested that glycogen synthase kinase 3 (GSK-3) acts as an integrating molecule for multiple proliferation and differentiation signals because of its essential role in the RTK, Wnt and Shh signaling pathways. We created conditional mutations that deleted both the alpha and beta forms of GSK-3 in mouse neural progenitors. GSK-3 deletion resulted in massive hyperproliferation of neural progenitors along the entire neuraxis. Generation of both intermediate neural progenitors and postmitotic neurons was markedly suppressed. These effects were associated with the dysregulation of beta-catenin, Sonic Hedgehog, Notch and fibroblast growth factor signaling. Our results indicate that GSK-3 signaling is an essential mediator of homeostatic controls that regulate neural progenitors during mammalian brain development.

In a recent issue of Chemistry & Biology, Bone et al. (2009) employed a chemical biology approach to probe the role of glycogen synthase kinase-3 (GSK-3), a key regulator of pluripotentiality, providing new insights and tools for modulating this process.

Based on extensive preclinical data, glycogen synthase kinase-3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b(-/-) embryoid bodies. While GSK-3alpha-deficient mice were born without a cardiac phenotype, no live-born Gsk3b(-/-) pups were recovered. The Gsk3b(-/-) embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation - GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3alpha and GSK-3beta in the heart using loss-of-function approaches, implicate GSK-3beta as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.

Dysregulation of the protein kinase glycogen synthase kinase 3 (GSK-3) has been implicated in the development of type 2 diabetes mellitus. GSK-3 protein expression and kinase activity are elevated in diabetes, while selective GSK-3 inhibitors have shown promise as modulators of glucose metabolism and insulin sensitivity. There are two GSK-3 isoforms in mammals, GSK-3alpha and GSK-3beta. Mice engineered to lack GSK-3beta die in late embryogenesis from liver apoptosis, whereas mice engineered to lack GSK-3alpha are viable and exhibit improved insulin sensitivity and hepatic glucose homeostasis. To assess the potential role of GSK-3beta in insulin function, a conditional gene-targeting approach whereby mice in which expression of GSK-3beta was specifically ablated within insulin-sensitive tissues were generated was undertaken. Liver-specific GSK-3beta knockout mice are viable and glucose and insulin tolerant and display "normal" metabolic characteristics and insulin signaling. Mice lacking expression of GSK-3beta in skeletal muscle are also viable but, in contrast to the liver-deleted animals, display improved glucose tolerance that is coupled with enhanced insulin-stimulated glycogen synthase regulation and glycogen deposition. These data indicate that there are not only distinct roles for GSK-3alpha and GSK-3beta within the adult but also tissue-specific phenotypes associated with each of these isoforms.

Despite treatment with agents that enhance beta-cell function and insulin action, reduction in beta-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3beta (Gsk-3beta). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3beta to regulation of beta-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/-) exhibit insulin resistance and a doubling of beta-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3beta (Gsk-3beta+/-) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced beta-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2-/-), like the Ir+/- mice, are insulin resistant, but develop profound beta-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3beta activity associated with a marked reduction of beta-cell proliferation and increased apoptosis. Irs2-/- mice crossed with Gsk-3beta+/- mice preserved beta-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2-/- mice had increased cyclin-dependent kinase inhibitor p27(kip1) that was limiting for beta-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of beta-cell mass in Gsk-3beta+/- Irs2-/- mice was accompanied by suppressed p27(kip1) levels and increased Pdx1 levels. To separate peripheral versus beta-cell-specific effects of reduction of Gsk3beta activity on preservation of beta-cell mass, mice homozygous for a floxed Gsk-3beta allele (Gsk-3(F/F)) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce beta-cell-specific knockout of Gsk-3beta (betaGsk-3beta-/-). Like Gsk-3beta+/- mice, betaGsk-3beta-/- mice also prevented the diabetes of the Irs2-/- mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within beta-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of beta-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve beta-cells and prevent diabetes onset.

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.

In mammalian cells, glycogen synthase kinase-3 (GSK-3) exists as two homologs, GSK-3alpha and GSK-3beta, encoded by independent genes, which share similar kinase domains but differ substantially in their termini. Here, we describe the generation of an allelic series of mouse embryonic stem cell (ESC) lines with 0-4 functional GSK-3 alleles and examine GSK-3-isoform function in Wnt/beta-catenin signaling. No compensatory upregulation in GSK-3 protein levels or activity was detected in cells lacking either GSK-3alpha or GSK-3beta, and Wnt/beta-catenin signaling was normal. Only in cells lacking three or all four of the alleles was a gene-dosage effect on beta-catenin/TCF-mediated transcription observed. Indeed, GSK-3alpha/beta double-knockout ESCs displayed hyperactivated Wnt/beta-catenin signaling and were severely compromised in their ability to differentiate, but could be rescued to normality by re-expression of functional GSK-3. The rheostatic regulation of GSK-3 highlights the importance of considering the contributions of both homologs when studying GSK-3 functions in mammalian systems.

Epithelial cells usually exist as sheets of immotile, tightly packed, well-coupled, polarized cells with distinct apical, basal and lateral surfaces. Remarkably, these cells can dramatically alter their morphology to become motile, fibroblast-like mesenchymal cells in a process of epithelial-mesenchymal transition (EMT). This process and the reverse, mesenchymal-epithelial transition, occur repeatedly during normal embryonic development. A phenomenon similar to physiological EMT occurs during the pathophysiological progression of some cancers. Tumours of epithelial origin, as they transform to malignancy, appear to exploit the innate plasticity of epithelial cells, with EMT conferring increased invasiveness and metastatic potential. Key to the maintenance of epithelial cell identity is the expression of E-cadherin, a protein that is required for tight intercellular adhesion along the lateral surfaces of adjacent epithelial cells. Loss of functional E-cadherin is a critical event in EMT. An important regulator of E-cadherin expression is the protein Snail, a zinc-finger transcriptional repressor. Snail contains several consensus sites for the kinase, glycogen synthase kinase-3 (GSK-3), and accumulating evidence indicates that it is a GSK-3 substrate. Phosphorylation of Snail by GSK-3 facilitates its proteasomal degradation. Conversely, inhibition of GSK-3 leads to Snail accumulation, E-cadherin downregulation, and development of EMT in cultured epithelial cells. Several signalling pathways implicated in the progression of EMT, including the Wnt and phosphoinositide 3-kinase pathways, use GSK-3 to mediate their responses. In these pathways, GSK-3's regulation of other transcriptional effectors like beta-catenin works in concert with changes in Snail to orchestrate the EMT process. This review focuses on the emerging role of GSK-3 as a modulator of cell fate and EMT in the contexts of development, in vitro cell culture and cancer., (2007 S. Karger AG, Basel)

Objective: Added transforming growth factor beta (TGFbeta) inhibits the proliferation of immature cardiomyocytes. We have now examined the hypothesis that suppression of endogenous TGFbeta signaling will boost the proliferative response (DNA synthesis) of cardiac myocytes to serum and/or to the mitogenic factor fibroblast growth factor-2 (FGF-2)., Methods and Results: Overexpression of a kinase-deficient TGFbeta type II receptor (TGFbetaRIIDeltaKD) resulted in a 2.8-fold increase in cardiomyocyte DNA synthesis in serum-rich cultures, an effect requiring active FGFR-1 since it was not observed in the presence of excess kinase-deficient FGFR-1. This finding suggested that endogenous TGFbeta-TGFbetaRII suppressed endogenous FGFR-1-mediated signals that stimulate or are permissive for DNA synthesis. TGFbeta had no effect, however, on the FGF-2-induced acute stimulation of extracellular signal regulated kinase1/2. FGF-2, added in the absence or presence of TGFbeta inhibition, elicited a 3- or a 13-fold stimulation of DNA synthesis, respectively, pointing to a synergistic effect., Conclusion: Inhibition of TGFbetaRII-transduced signaling upregulates the proliferative response of cardiomyocytes to serum, and greatly potentiates the stimulatory effect of FGF-2. A combinatorial strategy including activation of FGF-2 and inhibition of TGFbeta-triggered signal transduction may be required for maximal stimulation of immature cardiomyocyte DNA synthesis.

Mitogenic stimulation of cardiomyocytes is associated with decreased gap junction coupling and protein kinase C (PKC)-mediated phosphorylation of the gap junction protein connexin43 (Cx43). Identification of and interference with the amino acid(s) that becomes phosphorylated in response to stimulation are important steps towards defining the relationship between Cx43 phosphorylation and cell cycle. Using immunoblotting and phosphospecific antibodies we were able to show that serine-262 (S262) on Cx43 becomes phosphorylated in response to growth factor or PKC stimulation of cardiomyocytes. To examine the effect of Cx43, S262 phosphorylation and cell-cell contact (and/or coupling) on DNA synthesis, we overexpressed wild-type (wt) or mutant Cx43, carrying a S262-to-alanine (S262A, simulating the unphosphorylated state) or a S262-to-aspartate (S262D, simulating constitutive phosphorylation) substitutions in cultures of cell-cell contact forming or isolated cardiomyocytes. Overexpression of wt-Cx43 caused a significant decrease in DNA synthesis irrespective of the presence of cell-cell contact. In cell-cell contact forming cultures, the S262D mutation reversed while the S262A mutation increased the inhibitory effect of Cx43. In the absence of cell-cell contact, the S262-Cx43 mutations had no significant effect on Cx43 inhibition of DNA synthesis. Dye-coupling, evaluated by scrape-loading, indicated increased gap junction permeability in S262A (compared to wt or S262D) overexpressing myocytes. We conclude that Cx43 inhibits cardiomyocyte DNA synthesis irrespectively of cell-cell contact or coupling. Cell-cell contact, and possibly gap junction-mediated communication is required, however, in order to reverse Cx43 inhibition of DNA synthesis by S262 phosphorylation.

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCepsilon). Here we report that activation of PKCepsilon is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.

Glycogen synthase kinase 3 (GSK-3) is a multifunctional serine/threonine kinase found in all eukaryotes. The enzyme is a key regulator of numerous signalling pathways, including cellular responses to Wnt, receptor tyrosine kinases and G-protein-coupled receptors and is involved in a wide range of cellular processes, ranging from glycogen metabolism to cell cycle regulation and proliferation. GSK-3 is unusual in that it is normally active in cells and is primarily regulated through inhibition of its activity. Another peculiarity compared with other protein kinases is its preference for primed substrates, that is, substrates previously phosphorylated by another kinase. Several recent advances have improved our understanding of GSK-3 regulation in multiple pathways. These include the solution of the crystal structure of GSK-3, which has provided insight into GSK-3's penchant for primed substrates and the regulation of GSK-3 by serine phosphorylation, and findings related to the involvement of GSK-3 in the Wnt/beta-catenin and Hedgehog pathways. Finally, since increased GSK-3 activity may be linked to pathology in diseases such as Alzheimer's disease and non-insulin-dependent diabetes mellitus, several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture experiments, these new inhibitors hold promise as therapeutic agents.

Gap junctions are plasma membrane intercellular communication channels that in addition to ensuring electrical coupling and coordinated mechanical activity, can act as growth suppressors. To define the role of a non-channel forming domain of connexin-43 (Cx43), the main constituent of cardiomyocyte gap junctions, on growth regulation, we expressed its C-terminal portion (CT-Cx43) in cardiomyocytes and HeLa cells. In addition to broad cytoplasmic localization, CT-Cx43 was also localized to the nucleus of both cell types, detected by immunofluorescence as well as immunoblotting of subcellular fractions. Furthermore, stable expression of CT-Cx43 in HeLa cells induced a significant decrease in proliferation. It is therefore suggested that plasma membrane localization and formation of channels are not required for growth inhibition by Cx43, and that nuclear localization of CT-Cx43 may exert effects on gene expression and growth.

We examined the effect of fibroblast growth factor (FGF)-2 on myocardial resistance to injury when administered after the onset of ischemia, in vivo and ex vivo, and the role of FGF-2 receptors and protein kinase C (PKC). FGF-2 was injected into the left ventricle of rats undergoing permanent surgical coronary occlusion leading to myocardial infarction (MI). After 24 h, FGF-2-treated hearts displayed significantly reduced injury, determined by histological staining and troponin T release, and improved developed pressure compared with untreated controls. An FGF-2 mutant with diminished affinity for the tyrosine kinase FGF-2 receptor 1 (FGFR1) was not cardioprotective. FGF-2-treated hearts retained improved function and decreased damage at 6 wk after MI. In the ex vivo heart, FGF-2 administration during reperfusion after 30-min ischemia improved functional recovery and increased relative levels of PKC subtypes alpha, epsilon, and zeta in the particulate fraction, in a chelerythrine-preventable mode; it also decreased loss of energy metabolites. We conclude that intramyocardial FGF-2 administration shortly after the onset of ischemia confers protection from acute and chronic cardiac dysfunction and damage; FGF-2 delivered during reperfusion protects from ischemia-reperfusion injury; and protection by FGF-2 requires intact binding to FGFR1 and is likely mediated by PKC.

Fibroblast growth factor-2 (FGF-2) is a mitogen found in CUG-initiated 21-25 kDa ("hi") or AUG-initiated 16-18 kDa ("lo") forms. Previously we demonstrated that "hi"-but not "lo"-FGF-2 caused a distinct nuclear phenotype characterized by apparently condensed chromatin present as separate clumps in the nucleus of cardiac myocytes. In this manuscript we investigated whether these effects were related to apoptosis or mitosis and whether they reflected a direct effect of "hi" FGF-2 on chromatin. Myocytes overexpressing "hi" FGF-2 and presenting the clumped chromatin phenotype: (i) were not labeled above background with antibodies to phosphorylated histones H1 and H3 used as indicators of mitotic chromatin condensation; (ii) did not stain positive for TUNEL; (iii) their nuclear lamina, visualized by anti-laminB immunofluorescence, appeared intact; (iv) neither caspase inhibitors, nor Bcl-2 or "lo" FGF-2 overexpression prevented the manifestation of the compacted nuclear phenotype. Purified recombinant "hi" FGF-2 was more potent than "lo" FGF-2 in promoting the condensation/aggregation of chick erythrocyte chromatin partially reconstituted with histone H1 in vitro. We conclude that the DNA phenotype induced by "hi" FGF-2 in cardiac myocytes likely reflects a direct effect on chromatin structure that does not require the engagement of mitosis or apoptosis. By affecting chromatin compaction "hi" FGF-2 may contribute to the regulation of gene expression., (Copyright 2001 Wiley-Liss, Inc.)

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKCepsilon) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (> or = 45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKK1 did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKCE significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of > or = 45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKCepsilon.

Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Gap junctions (GJ), composed of connexins, are membrane channels that connect and enable communication between neighboring cells and which, in addition to being essential for the coordinated electrical and contractile activity of the heart, may regulate intercellular transmission of signals affecting proliferative growth. Alterations in GJ permeability that have been associated with the regulation of growth can occur acutely through phosphorylation of connexins: fibroblast growth factor-2 (FGF-2) causes decreased coupling and increased phosphorylation of a major cardiomyocyte connexin, connexin43 (Cx43), while stimulating proliferation of cardiomyocytes. On the other hand, transforming growth factor-β (TGFβ) prevents the effects of FGF-2 on Cx43 phosphorylation, as well as canceling the FGF-2-induced proliferation. Parallel to its link with growth regulation, Cx43 phosphorylation emerges as a functionally important end point for delineating cardiac signal transduction pathways elicited by diverse physiologic or pathologic stimuli., (Copyright © 1998 Elsevier Science Inc. All rights reserved.)

A sequence-specific monoclonal antibody directed against the gap junction protein connexin43 (Cx43) is shown here to be specific for the unphosphorylated form of this protein. In tissues and cultured cells containing different phosphorylated and unphosphorylated forms of Cx43, the antibody detected only the latter as shown by Western blotting of native and alkaline phosphatase-treated samples. Immunohistochemically, this monoclonal antibody did not recognize gap junctions in the vast majority of cultured cardiac myocytes, where nearly all detectable Cx43 is phosphorylated. In contrast, it was able to detect some intracellular Cx43 in tracheal smooth muscle cells and an epithelial cell line (Cl-9 cells), producing patterns of labeling consistent with those seen using a polyclonal antibody that recognizes both phosphorylated and unphosphorylated forms of Cx43. Immunostaining of gap junctions in the cultured cells indicates that both phosphorylated and unphosphorylated Cx43 are present in some assembled gap junctions, suggesting that assembled junctions do not contain exclusively the phosphorylated form of the protein. Annular gap junctions, believed to form as part of the pathway for internalization and degradation of gap junctions, were only occasionally and sparsely labeled by the monoclonal antibody, indicating that complete protein dephosphorylation is not required for uptake and degradation of gap junctions. Furthermore, the ability of this antibody to recognize only unphosphorylated Cx43, and not any of the phosphorylated forms present in the tissues and cell types examined, suggests that a unique phosphorylation site, perhaps present in the epitope recognized by this antibody, must be phosphorylated prior to phosphorylation of Cx43 at other sites.

Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ-mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti-phosphotyrosine-specific antibodies in FGF-2-treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2-triggered and tyrosine phosphorylation-dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.

Gap junctions (GJ) are membrane specializations responsible for intercellular communication and for ensuring electrical and/or metabolic coupling between cells. They are composed of connexins, a family of related proteins. Connexin-43 (Cx43) is a major connexin of the rat heart, expressed by myocytes as well as non-muscle cells. In this communication we have examined expression of Cx43 by cardiac fibroblasts and regulation of its expression by an endogenous mitogen, basic fibroblast growth factor (bFGF). Recombinant human bFGF, administered to cultured cells which had been maintained in 0.5% serum for 48 h, induced dose-dependent and statistically significant increases in Cx43 mRNA as well as protein accumulation, at 6 h after addition. Intercellular communication was also increased at 6 h but not 30 min after bFGF treatment, as assessed using a scrape-loading protocol. It is concluded that the bFGF-induced stimulation of Cx43 expression caused increased coupling between cardiac fibroblasts. This would be of importance in injured myocardium, the increased bFGF content of which might stimulate electrical coupling involving fibroblasts of the scar tissue.

Initiation of translation from alternate codons in the same mRNA results in multiple forms of basic fibroblast growth factor (bFGF). High molecular weight species of bFGF make use of leucine translation initiation sites located upstream of the methionine residue used to produce the 18 kiloDalton (kDa) form. Although the addition of exogenous 18 kDa bFGF is known to stimulate DNA synthesis and proliferation of several cell types including embryonic chicken cardiac myocytes, little is known about the role of high molecular weight forms of bFGF. We modified the rat bFGF cDNA to yield high (22/21.5 kDa) or low (18 kDa) molecular weight species of bFGF. Expression of 22/21.5 kDa or 18 kDa bFGF in transfected embryonic chicken ventricular myocyte cultures was confirmed by protein blotting. Expression of both high and low molecular weight species of bFGF was associated with (i) a three-fold increase in overall thymidine incorporation as well as cardiomyocyte labelling index (fraction of cardiomyocyte nuclei incorporating tritiated thymidine); (ii) a two- to three-fold increase in cell number; (iii) an eight-fold increase in protein synthesis; and (iv) a three-fold decrease in myosin accumulation. Subcellular localization of bFGF in the transfected myocyte cultures was also assessed by immunofluorescence microscopy. Over-expression of cDNAs yielding high molecular weight bFGF resulted in predominantly nuclear bFGF staining. By contrast, both cytoplasmic and nuclear staining were observed following over-expression of 18 kDa bFGF. Over-expression of 22/21.5 kDa bFGF was associated with the formation of multiple DNA-containing "clumps" resembling condensed chromatin in cardiac myocyte nuclei. These DNA "clumps" were not observed in cardiac myocyte cultures over-expressing 18 kDa bFGF. These data indicate that over-expression of high as well as low molecular weight forms of bFGF can stimulate cardiac myocyte proliferative potential and decrease myosin accumulation. However, these forms possess distinct subcellular localizations and can have different biological functions in the nucleus.

Control of cardiomyocyte growth and differentiation may be exercised, in part, at the level of expression of endogenous growth factors such as bFGF (basic fibroblast growth factor), believed to act locally, in an autocrine or paracrine fashion. Examination of bFGF accumulation by extract fractionation and immunoblotting indicated a 4-fold increase of an 18-kDa bFGF in adult compared to newborn rat heart ventricles. In contrast, a 22-kDa bFGF species, found to be the predominant form in newborn ventricles, displayed a 2.5-fold decrease in extracts from adult (compared to neonatal) ventricles. Since newborn rats are physiologically hypothyroid, the effect of thyroid status on the accumulation of 22-kDa bFGF in the heart was examined. A 21.5- to 22-kDa bFGF showed a 5-fold increase in extracts from hyperthyroid rat heart ventricles, compared to those from euthyroid controls. The 21.5- to 22-kDa bFGF was essentially unaffected by thyroid status in extracts from brain. Our data point to a correlation between immature or differentiated cardiac phenotype and the predominance of 21- to 22.5-kDa or 18-kDa bFGF species, respectively. Elevated levels of 21.5- to 22-kDa bFGF detected in cardiac ventricles in either physiological or experimentally induced hypothyroidism indicates that thyroid hormone may, directly or indirectly, down-regulate accumulation of the higher molecular mass forms of bFGF in the heart.