scholarly journals β-Catenin Accumulation is Associated with Increased Expression of Nanog Protein and Predicts Maintenance of MSC Self-Renewal

2017 ◽  
Vol 26 (2) ◽  
pp. 365-377 ◽  
Author(s):  
Sang-Jin Yu ◽  
Hyun-Je Kim ◽  
Eui Seok Lee ◽  
Chung-Gyu Park ◽  
Su Jin Cho ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Es Cells ◽  
Autologous Transplantation ◽  
Human Mesenchymal Stem Cells ◽  
Self Renewal ◽  
Ras Protein ◽  
Nanog Expression ◽  
Human Es Cells

Human mesenchymal stem cells (hMSCs) are self-renewing cells with the ability to differentiate into organized, functional network of cells. Recent studies have revealed that activation of the Wnt/β-catenin pathway by a glycogen synthase kinase (GSK)-3-specific pharmacological inhibitor, Bio, results in the maintenance of self-renewal in both mouse and human ES cells. The molecular mechanism behind the maintenance of hMSCs by these factors, however, is not fully understood. We found that rEGF enhances the level of β-catenin, a component of the Wnt/β-catenin signaling pathway. Furthermore, it was found that β-catenin upregulates Nanog. EGF activates the β-catenin pathway via the Ras protein and also increased the Nanog protein and gene expression levels 2 h after rEGF treatment. These results suggest that adding EGF can enhance β-catenin and Nanog expression in MSCs and facilitate EGF-mediated maintenance of MSC self-renewal. EGF was shown to augment MSC proliferation while preserving early progenitors within MSC population and thus did not induce differentiation. Thus, EGF not only can be used to expand MSC in vitro but also be utilized to autologous transplantation of MSCs in vivo.

scholarly journals Human embryonic stem cells for brain repair?

2007 ◽  
Vol 363 (1489) ◽  
pp. 87-99 ◽  
Author(s):  
Su-Chun Zhang ◽  
Xue-Jun Li ◽  
M Austin Johnson ◽  
Matthew T Pankratz
Keyword(s):  
Stem Cells ◽  
Motor Neurons ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dopamine Neurons ◽  
Neural Cells ◽  
Es Cell ◽  
Human Es Cells

Cell therapy has been perceived as the main or ultimate goal of human embryonic stem (ES) cell research. Where are we now and how are we going to get there? There has been rapid success in devising in vitro protocols for differentiating human ES cells to neuroepithelial cells. Progress has also been made to guide these neural precursors further to more specialized neural cells such as spinal motor neurons and dopamine-producing neurons. However, some of the in vitro produced neuronal types such as dopamine neurons do not possess all the phenotypes of their in vivo counterparts, which may contribute to the limited success of these cells in repairing injured or diseased brain and spinal cord in animal models. Hence, efficient generation of neural subtypes with correct phenotypes remains a challenge, although major hurdles still lie ahead in applying the human ES cell-derived neural cells clinically. We propose that careful studies on neural differentiation from human ES cells may provide more immediate answers to clinically relevant problems, such as drug discovery, mechanisms of disease and stimulation of endogenous stem cells.

4 GENE EXPRESSION IN CULTURES OF INNER CELL MASSES ISOLATED FROM IN VITRO-PRODUCED AND IN VIVO-DERIVED BOVINE BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 110 ◽  
Author(s):  
D. Pant ◽  
C. Keefer
Keyword(s):  
Es Cells ◽  
Primary Cultures ◽  
Embryonic Stem ◽  
Pcr Analysis ◽  
Beta Catenin ◽  
Rt Pcr ◽  
Self Renewal ◽  
Domestic Species

Genetic modification of embryonic stem (ES) cells derived from domestic species could be exploited to produce transgenic animals; however, fully validated ES have not been obtained in domestic species. Recent findings regarding key transcription factors and regulation of pluripotency and self-renewal in murine ES cells may provide keys to enable the derivation of ES in domestic species. The aim of this study was to identify and monitor the expression of candidate genes, which are known to be involved in the maintenance of self-renewal and pluripotency in mouse and human ES cells, during the critical first steps in establishment of primary cultures. Inner cell masses (ICMs) were isolated via manual dissection of 25 to 30 commercial in vitro-produced (IVP) blastocysts (Bomed, Inc., Madison, WI, USA) in each of three separate replicates and from 10 in vivo-derived Day 7-8 bovine blastocysts. On the day of ICM isolation (Day 0), 4-5 ICM clumps were collected for RT-PCR analysis. The remaining isolated ICMs were cultured (4-5 ICM clumps per well) on mitomycin C (Sigma-Aldrich, St. Louis, MO, USA)-inactivated mouse embryonic fibroblasts (STO, ATCC, Manassas, VA, USA). The ICM clumps were cultured in 12-well tissue culture dishes in ES medium consisting of Knockout DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 15% FCS (Hyclone, Logan, UT, USA), 2 mM l-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (MP Biomedicals, Irvine, CA, USA), and non-essential amino acids (Sigma). Two to four cultured ICM clumps were collected for RT-PCR analysis on Days 1-4 from IVP embryos and on Days 2, 4, and 6 from in vivo-derived embryos. Total RNA was extracted from the collected samples using the Absolutely RNA Nanoprep Kit (Strategene, La Jolla, CA, USA). First-strand DNAs were synthesized using Superscript III (Invitrogen) and cDNAs were amplified with PfuUltra hotstart PCR mastermix (Stratagene). Primers were designed based on homology between human and mouse sequences and were validated using bovine tissues. In experiments spanning these critical first few days of culture, the pluripotency-related genes (Nanog, Oct-4, Sox-2) and components of the LIF (LIFR, Gp130), BMP (Bmpr1a, Id-1), and Wnt (Beta-catenin, Frizzled) pathways were expressed in the ICM cultures over the 4 days of IVP-ICM cultures and the 6 days of in vivo-derived ICM cultures. These results indicate that the markers of pluripotency and the components of signaling pathways implicated in the maintenance of murine embryonic stem cells are present in ICMs of Day 7-8 bovine blastocysts and continue to be expressed at least during the initial days of culture. Genes (NCAM, Lef1) associated with early differentiation, however, were also expressed. Whether their expression is an indicator of ICM differentiation or of residual contamination with trophectoderm remains to be determined. Further studies will determine whether stimulation of these pathways can facilitate efficient derivation and maintenance ruminant ES cells.

scholarly journals Impaired RAS Proteolysis Drives Clonal Hematopoietic Transformation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 356-356
Author(s):  
Sisi Chen ◽  
Rahul S. Vedula ◽  
Pau Castel ◽  
Antonio Cuevas Navarro ◽  
Simon J. Hogg ◽  
...  
Keyword(s):  
Stock Options ◽  
Fetal Liver ◽  
Advisory Committees ◽  
Self Renewal ◽  
Ras Protein ◽  
Myeloid Neoplasm ◽  
Ras Gtpases ◽  
Conditional Expression

Abstract Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells. Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D). In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients. Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L). While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. Figure 1 Figure 1. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Targeting Tau Hyperphosphorylation via Kinase Inhibition: Strategy to Address Alzheimer's Disease

2020 ◽  
Vol 20 (12) ◽  
pp. 1059-1073 ◽  
Author(s):  
Ahmad Abu Turab Naqvi ◽  
Gulam Mustafa Hasan ◽  
Md. Imtaiyaz Hassan
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Tau Protein ◽  
Glycogen Synthase ◽  
Paired Helical Filaments ◽  
Tau Hyperphosphorylation ◽  
Microtubule Stabilization ◽  
Extracellular Signal

Microtubule-associated protein tau is involved in the tubulin binding leading to microtubule stabilization in neuronal cells which is essential for stabilization of neuron cytoskeleton. The regulation of tau activity is accommodated by several kinases which phosphorylate tau protein on specific sites. In pathological conditions, abnormal activity of tau kinases such as glycogen synthase kinase-3 β (GSK3β), cyclin-dependent kinase 5 (CDK5), c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinase 1 and 2 (ERK1/2) and microtubule affinity regulating kinase (MARK) lead to tau hyperphosphorylation. Hyperphosphorylation of tau protein leads to aggregation of tau into paired helical filaments like structures which are major constituents of neurofibrillary tangles, a hallmark of Alzheimer’s disease. In this review, we discuss various tau protein kinases and their association with tau hyperphosphorylation. We also discuss various strategies and the advancements made in the area of Alzheimer's disease drug development by designing effective and specific inhibitors for such kinases using traditional in vitro/in vivo methods and state of the art in silico techniques.

scholarly journals GSK-3β in Pancreatic Cancer: Spotlight on 9-ING-41, Its Therapeutic Potential and Immune Modulatory Properties

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 610
Author(s):  
Robin Park ◽  
Andrew L. Coveler ◽  
Ludimila Cavalcante ◽  
Anwaar Saeed
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Potential ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
In Vivo Models ◽  
Gsk 3Β ◽  
Early Phase Clinical Trials

Glycogen synthase kinase-3 beta is a ubiquitously and constitutively expressed molecule with pleiotropic function. It acts as a protooncogene in the development of several solid tumors including pancreatic cancer through its involvement in various cellular processes including cell proliferation, survival, invasion and metastasis, as well as autophagy. Furthermore, the level of aberrant glycogen synthase kinase-3 beta expression in the nucleus is inversely correlated with tumor differentiation and survival in both in vitro and in vivo models of pancreatic cancer. Small molecule inhibitors of glycogen synthase kinase-3 beta have demonstrated therapeutic potential in pre-clinical models and are currently being evaluated in early phase clinical trials involving pancreatic cancer patients with interim results showing favorable results. Moreover, recent studies support a rationale for the combination of glycogen synthase kinase-3 beta inhibitors with chemotherapy and immunotherapy, warranting the evaluation of novel combination regimens in the future.

scholarly journals Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Jian-Ping Zhang ◽  
Wei-Jing Zhang ◽  
Miao Yang ◽  
Hua Fang
Keyword(s):  
Cell Apoptosis ◽  
Ischemia Reperfusion Injury ◽  
Glycogen Synthase ◽  
Ischemia Reperfusion ◽  
Inflammatory Factors ◽  
Therapeutic Effects ◽  
Protective Effects ◽  
Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

scholarly journals STEM-29. THE HISTONE VARIANT macroH2A2 ORCHESTRATES AN ACTIONABLE CHROMATIN PROGRAM OF STEMNESS IN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii202-ii202
Author(s):  
Ana Nikolic ◽  
Anna Bobyn ◽  
Katrina Ellestad ◽  
Xueqing Lun ◽  
Michael Johnston ◽  
...  
Keyword(s):  
Chromatin Accessibility ◽  
Histone Variant ◽  
Clinical Settings ◽  
Glioblastoma Cells ◽  
Self Renewal ◽  
Viral Mimicry ◽  
Experimental Findings ◽  
Preclinical Work

Abstract Glioblastoma cells with the crucial stemness property of self-renewal constitute therapy-resistant reservoirs that seed tumor relapse. Effective targeting of these cells in clinical settings has been hampered by their relative quiescence, which invalidates the cell replication bias of most current treatments. Furthermore, although their dependence on specific chromatin and transcriptional states for the maintenance of stemness programs has been proposed as a vulnerability, these nuclear programs have been challenging to target pharmaceutically. Therefore the identification of targetable chromatin paradigms regulating self-renewal would represent a significant advancement for this incurable malignancy. Here we report a new role for the histone variant macroH2A2 in modulating a targetable epigenetic network of stemness in glioblastoma. By integrating transcriptomic, bulk and single-cell epigenomic datasets we generated from patient-derived models and surgical specimens, we show that macroH2A2 represses a transcriptional network of stemness through direct regulation of chromatin accessibility at enhancer elements. Functional assays in vitro and in vivo further showcase that macroH2A2 antagonizes self-renewal and stemness in glioblastoma preclinical models. In agreement with our experimental findings, high expression of macroH2A2 is a positive prognostic factor in clinical glioblastoma cohorts. Reasoning that increasing macroH2A2 levels could be an effective strategy to repress stemness programs and ameliorate patient outcome, we embarked on a screen to identify compounds that could elevate macroH2A2 levels. We report that an inhibitor of the chromatin remodeler Menin increases macroH2A2 levels, which in turn repress self-renewal. Additionally, we provide evidence that Menin inhibition induces viral mimicry programs and the demise of glioblastoma cells. Menin inhibition is being tested in clinical trials for blood malignancies (NCT04067336). Our preclinical work therefore reveals a novel and central role for macroH2A2 in an epigenetic network of stemness and suggests new clinical approaches for glioblastoma.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

scholarly journals Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

2010 ◽  
Vol 108 (1) ◽  
pp. 379-384 ◽  
Author(s):  
Shiva K. Tyagarajan ◽  
Himanish Ghosh ◽  
Gonzalo E. Yévenes ◽  
Irina Nikonenko ◽  
Claire Ebeling ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Gabaergic Synapse ◽  
Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

scholarly journals Generation and Characterization of Smac/DIABLO-Deficient Mice

2002 ◽  
Vol 22 (10) ◽  
pp. 3509-3517 ◽  
Author(s):  
Hitoshi Okada ◽  
Woong-Kyung Suh ◽  
Jianping Jin ◽  
Minna Woo ◽  
Chunying Du ◽  
...  
Keyword(s):  
Physiological Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Caspase Activation ◽  
Proapoptotic Protein ◽  
Apoptosis Proteins ◽  
Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

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