Publications by authors named "Christian Schmedt"

26 Publications

  • Page 1 of 1

STIM1 thermosensitivity defines the optimal preference temperature for warm sensation in mice.

Cell Res 2019 02 3;29(2):95-109. Epub 2019 Jan 3.

State Key Laboratory of Membrane Biology; Tsinghua-Peking Joint Center for Life Sciences; IDG/McGovern Institute for Brain Research; School of Pharmaceutical Sciences, Tsinghua University, 100084, Beijing, China.

Mammals possess a remarkable ability to sense subtle temperature deviations from the thermoneutral skin temperature of ~33 °C, which ensures precise warm sensation. However, the underlying mechanisms remain unclear. Here we show that STIM1, an endoplasmic reticulum (ER) resident transmembrane protein that responds to both ER Ca depletion and heat, mediates temperature-induced Ca influx in skin keratinocytes via coupling to Orai Ca channels in plasma membrane. Behaviorally, the keratinocyte-specific knockout of STIM1 shifts the optimal preference temperature (OPT) of mice from ~32 °C to ~34 °C, resulting in a strikingly reversed preference between 32 °C and 34 °C. Importantly, the thermally inactive STIM1-ΔK knock-in mice show altered OPT and warm preference behaviors as well, demonstrating the requirement of STIM1 thermosensitivity for warm sensation. Furthermore, the wild-type and mutant mice prefer temperatures closer to their respective OPTs, but poorly distinguish temperatures that are equally but oppositely deviated from their OPTs. Mechanistically, keratinocyte STIM1 affects the in vivo warm responses of sensory neurons by likely involving TRPA1 as a downstream transduction channel. Collectively, our data suggest that STIM1 serves as a novel in vivo thermosensor in keratinocytes to define the OPT, which might be utilized as a peripheral reference temperature for precise warm sensation.
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http://dx.doi.org/10.1038/s41422-018-0129-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355819PMC
February 2019

Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.

Nat Methods 2018 11 8;15(11):941-946. Epub 2018 Oct 8.

Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4 T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34 hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.
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http://dx.doi.org/10.1038/s41592-018-0149-1DOI Listing
November 2018

Induction of muscle stem cell quiescence by the secreted niche factor Oncostatin M.

Nat Commun 2018 04 18;9(1):1531. Epub 2018 Apr 18.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA.

The balance between stem cell quiescence and proliferation in skeletal muscle is tightly controlled, but perturbed in a variety of disease states. Despite progress in identifying activators of stem cell proliferation, the niche factor(s) responsible for quiescence induction remain unclear. Here we report an in vivo imaging-based screen which identifies Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, as a potent inducer of muscle stem cell (MuSC, satellite cell) quiescence. OSM is produced by muscle fibers, induces reversible MuSC cell cycle exit, and maintains stem cell regenerative capacity as judged by serial transplantation. Conditional OSM receptor deletion in satellite cells leads to stem cell depletion and impaired regeneration following injury. These results identify Oncostatin M as a secreted niche factor responsible for quiescence induction, and for the first time establish a direct connection between induction of quiescence, stemness, and transplantation potential in solid organ stem cells.
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http://dx.doi.org/10.1038/s41467-018-03876-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906564PMC
April 2018

Common PIEZO1 Allele in African Populations Causes RBC Dehydration and Attenuates Plasmodium Infection.

Cell 2018 04 22;173(2):443-455.e12. Epub 2018 Mar 22.

Howard Hughes Medical Institute, Department of Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.
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http://dx.doi.org/10.1016/j.cell.2018.02.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889333PMC
April 2018

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

Cell 2018 01 21;172(3):534-548.e19. Epub 2017 Dec 21.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.
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http://dx.doi.org/10.1016/j.cell.2017.11.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684025PMC
January 2018

The microprotein Minion controls cell fusion and muscle formation.

Nat Commun 2017 06 1;8:15664. Epub 2017 Jun 1.

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA.

Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, their function remains to be determined. Skeletal muscle development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process. Here we report the identification of Minion (microprotein inducer of fusion), a smORF encoding an essential skeletal muscle specific microprotein. Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally and demonstrate a marked reduction in fused muscle fibres. The fusogenic activity of Minion is conserved in the human orthologue, and co-expression of Minion and the transmembrane protein Myomaker is sufficient to induce cellular fusion accompanied by rapid cytoskeletal rearrangement, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component programme for the induction of mammalian cell fusion. Moreover, these data also significantly expand the known functions of smORF-encoded microproteins.
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http://dx.doi.org/10.1038/ncomms15664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461507PMC
June 2017

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease.

PLoS One 2015 29;10(6):e0131071. Epub 2015 Jun 29.

The Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California, United States of America.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131071PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488288PMC
April 2016

Inhibition of c-Kit is not required for reversal of hyperglycemia by imatinib in NOD mice.

PLoS One 2014 15;9(1):e84900. Epub 2014 Jan 15.

Genetics and Neglected Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.

Aim/hypothesis: Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice.

Methods: The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit(T670I) mice and NOD.c-Kit(wt) littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit(T670I) allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored.

Results: In vitro expansion of HSCs from NOD.c-Kit(wt) mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit(T670I) mice was insensitive to imatinib. However, in vivo treatment with imatinib lowered blood glucose levels in both strains of mice.

Conclusions/interpretation: The HSC experiment confirmed that, in NOD.c-Kit(T670I) mice, c-Kit is resistant to imatinib. As both NOD.c-Kit(T670I) and NOD.c-Kit(wt) mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084900PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893161PMC
December 2014

Identification of the Plasmodium berghei resistance locus 9 linked to survival on chromosome 9.

Malar J 2013 Sep 11;12:316. Epub 2013 Sep 11.

Department of Pediatrics, University of California, San Diego, School of Medicine, La Jolla, CA, USA.

Background: One of the main causes of mortality from severe malaria in Plasmodium falciparum infections is cerebral malaria (CM). An important host genetic component determines the susceptibility of an individual to develop CM or to clear the infection and become semi-immune. As such, the identification of genetic loci associated with susceptibility or resistance may serve to modulate disease severity.

Methodology: The Plasmodium berghei mouse model for experimental cerebral malaria (ECM) reproduces several disease symptoms seen in human CM, and two different phenotypes, a susceptible (FVB/NJ) and a resistant mouse strain (DBA/2J), were examined.

Results: FVB/NJ mice died from infection within ten days, whereas DBA/2J mice showed a gender bias: males survived on average nineteen days and females either died early with signs of ECM or survived for up to three weeks. A comparison of brain pathology between FVB/NJ and DBA/2J showed no major differences with regard to brain haemorrhages or the number of parasites and CD3+ cells in the microvasculature. However, significant differences were found in the peripheral blood of infected mice: For example resistant DBA/2J mice had significantly higher numbers of circulating basophils than did FVB/NJ mice on day seven. Analysis of the F2 offspring from a cross of DBA/2J and FVB/NJ mice mapped the genetic locus of the underlying survival trait to chromosome 9 with a Lod score of 4.9. This locus overlaps with two previously identified resistance loci (char1 and pymr) from a blood stage malaria model.

Conclusions: Survival best distinguishes malaria infections between FVB/NJ and DBA/2J mice. The importance of char1 and pymr on chromosome 9 in malaria resistance to P. berghei was confirmed. In addition there was an association of basophil numbers with survival.
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http://dx.doi.org/10.1186/1475-2875-12-316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848760PMC
September 2013

Immortalized clones of fibroblastic reticular cells activate virus-specific T cells during virus infection.

Proc Natl Acad Sci U S A 2012 May 1;109(20):7823-8. Epub 2012 May 1.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.

Fibroblastic reticular cells (FRCs) are lymphoid stromal cells essential to T-cell migration and survival. Although FRCs are targets of multiple viral infections, little is known about their role during infection due to the cells' scarcity and difficulty in isolating in vivo. To initiate studies of interactions among FRCs, viruses, and immune cells, we isolated and immortalized CD45(-)gp38(+)CD35(-)CD31(-)CD44(+)VCAM1(+) cell lines from C57BL/6 mice designated as immortalized FRC. Using these cloned cell lines, we have established that FRCs express the major histocompatibility complex (MHC) II molecule, a factor necessary for stimulation of CD4(+) T cells thought to be expressed primarily by antigen-presenting cells, along with other T-cell stimulatory ligands in an IFN-γ-dependent manner. In this environment, lymphocytic choriomeningitis virus (LCMV)-infected iFRCs activated naive LCMV-specific CD4(+) and CD8(+) T cells while limiting expansion of effector LCMV-specific T cells. Thus, FRCs effectively presented antigen along with activating signals during viral infection using both MHC I and MHC II molecules, illustrating a previously undescribed interaction with CD4(+) T cells and indicating a unique role for FRCs.
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http://dx.doi.org/10.1073/pnas.1205850109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356638PMC
May 2012

Oxysterols direct immune cell migration via EBI2.

Nature 2011 Jul 27;475(7357):524-7. Epub 2011 Jul 27.

Euroscreen S.A., 6041 Gosselies, Belgium.

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.
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http://dx.doi.org/10.1038/nature10280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297623PMC
July 2011

Loss of CD4 T-cell-dependent tolerance to proteins with modified amino acids.

Proc Natl Acad Sci U S A 2011 Aug 18;108(31):12821-6. Epub 2011 Jul 18.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.
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http://dx.doi.org/10.1073/pnas.1110042108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150954PMC
August 2011

TLE3 is a dual-function transcriptional coregulator of adipogenesis.

Cell Metab 2011 Apr;13(4):413-427

Howard Hughes Medical Institute and Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program.
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http://dx.doi.org/10.1016/j.cmet.2011.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089971PMC
April 2011

Deletion of the potassium channel Kv12.2 causes hippocampal hyperexcitability and epilepsy.

Nat Neurosci 2010 Sep 1;13(9):1056-8. Epub 2010 Aug 1.

Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.

We found the voltage-gated K+ channel Kv12.2 to be a potent regulator of excitability in hippocampal pyramidal neurons. Genetic deletion and pharmacologic block of Kv12.2 substantially reduced the firing threshold of these neurons. Kv12.2-/- (also known as Kcnh3-/-) mice showed signs of persistent neuronal hyperexcitability including frequent interictal spiking, spontaneous seizures and increased sensitivity to the chemoconvulsant pentylenetetrazol.
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http://dx.doi.org/10.1038/nn.2610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928878PMC
September 2010

Autoimmune-associated PTPN22 R620W variation reduces phosphorylation of lymphoid phosphatase on an inhibitory tyrosine residue.

J Biol Chem 2010 Aug 9;285(34):26506-18. Epub 2010 Jun 9.

Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.

A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.
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http://dx.doi.org/10.1074/jbc.M110.111104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924087PMC
August 2010

HTS-Compatible Patient-Derived Cell-Based Assay to Identify Small Molecule Modulators of Aberrant Splicing in Myotonic Dystrophy Type 1.

Curr Chem Genomics 2010 Mar 19;4:9-18. Epub 2010 Mar 19.

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3'UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG(5)) and DM1 patient (DMPK CTG(1000)) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.
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http://dx.doi.org/10.2174/1875397301004010009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874217PMC
March 2010

Efficient transposition of Tol2 in the mouse germline.

Genetics 2009 Dec 5;183(4):1565-73. Epub 2009 Oct 5.

Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA

Insertional mutagenesis screens play an integral part in the annotating of functional data for all sequenced genes in the postgenomic era. Chemical mutagenesis screens are highly efficient but identifying the causative gene can be a laborious task. Other mutagenesis platforms, such as transposable elements, have been successfully applied for insertional mutagenesis screens in both the mouse and rat. However, relatively low transposition efficiency has hampered their use as a high-throughput forward genetic mutagenesis screen. Here we report the first evidence of germline activity in the mouse using a naturally active DNA transposon derived from the medaka fish called Tol2, as an alternative system for high-throughput forward genetic mutagenesis screening tool.
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http://dx.doi.org/10.1534/genetics.109.100768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787440PMC
December 2009

Inducible ablation of melanopsin-expressing retinal ganglion cells reveals their central role in non-image forming visual responses.

PLoS One 2008 Jun 11;3(6):e2451. Epub 2008 Jun 11.

The Salk Institute for Biological Studies, La Jolla, California, United States of America.

Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002451PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396502PMC
June 2008

Ablation of Csk in neural crest lineages causes corneal anomaly by deregulating collagen fibril organization and cell motility.

Dev Biol 2008 Mar 16;315(2):474-88. Epub 2008 Jan 16.

Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Src family kinases (SFKs) have been implicated in the regulation of cell motility. To verify their in vivo roles during development, we generated mutant mice in which Csk, a negative regulator of SFKs, was inactivated in neural crest lineages using the Protein zero promoter in a Cre-loxP system. Inactivation of Csk caused deformities in various tissues of neural crest origins, including facial dysplasia and corneal opacity. In the cornea, the stromal collagen fibril was disorganized and there was an overproduction of collagen 1a1 and several metalloproteases. The corneal endothelium failed to overlie the central region of the eye and the peripheral endothelium displayed a disorganized cytoskeleton. Corneal mesenchymal cells cultured from mutant mice showed attenuated cell motility. In these cells, p130 Crk-associated substrate (Cas) was hyperphosphorylated and markedly downregulated. The expression of a dominant negative Cas (Cas Delta SD) could suppress the cell motility defects. Fluorescence resonance energy transfer analysis revealed that activation of Rac1 and Cdc42 was depolarized in Csk-inactivated cells, which was restored by the expression of either Csk or Cas Delta SD. These results demonstrate that the SFKs/Csk circuit plays crucial roles in corneal development by controlling stromal organization and by ensuring cell motility via the Cas-Rac/Cdc42 pathways.
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http://dx.doi.org/10.1016/j.ydbio.2008.01.004DOI Listing
March 2008

Epidermal hyperplasia and papillomatosis in mice with a keratinocyte-restricted deletion of csk.

Carcinogenesis 2007 Oct 10;28(10):2074-81. Epub 2007 May 10.

Department of Biomedical Engineering and Orthopaedic Research Center/ND20, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

The Src family kinases (SFKs) are believed to play critical roles in malignant transformation, as well as in growth, invasion and dissemination of neoplastic tissue. Inhibition of SFK-mediated signal transduction and activation of downstream targets inhibits tumor progression. To determine whether constitutive activity of SFK per se is sufficient to induce tumorigenesis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of the SFK-negative regulator csk (Csk-K5 mice). Even though expression levels of SFKs were lower in C-terminal Src kinase (Csk)-null keratinocytes, activity levels were higher than in control keratinocytes. At the age of 3 months, all Csk-K5 mice displayed signs of chronic inflammation in dermis and epidermal hyperplasia. About 19% of Csk-K5 mice (7 out of 36) developed papillomatous lesions. However, these lesions did not show any signs of neoplastic transformation over the next 8 months. Epidermal hyperplasia and hyperkeratosis in Csk-K5 mice were associated with an increased number of stem cells in the interfollicular epidermis, an increased proliferation of basal keratinocytes and a delayed terminal differentiation of the suprabasal keratinocytes. Our results clearly demonstrate that even though SFK-mediated signaling promotes tumor progression, elevated activity of SFKs in vivo alone is not sufficient to induce neoplastic transformation.
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http://dx.doi.org/10.1093/carcin/bgm112DOI Listing
October 2007

C-terminal Src kinase controls development and maintenance of mouse squamous epithelia.

EMBO J 2007 Mar 15;26(5):1234-44. Epub 2007 Feb 15.

Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.
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http://dx.doi.org/10.1038/sj.emboj.7601595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1817640PMC
March 2007

The ubiquitously expressed Csk adaptor protein Cbp is dispensable for embryogenesis and T-cell development and function.

Mol Cell Biol 2005 Dec;25(23):10533-42

Laboratory of Lymphocyte Signaling, The Rockefeller University, 1230 York Avenue, Box 301, New York, NY 10021, USA.

Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of "lipid rafts" is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.
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http://dx.doi.org/10.1128/MCB.25.23.10533-10542.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1291250PMC
December 2005

C-terminal SRC kinase controls acute inflammation and granulocyte adhesion.

Immunity 2004 Feb;20(2):181-91

University College London, Department of Immunology and Molecular Pathology, The Windeyer Institute of Medical Sciences, 46 Cleveland Street, London W1T 4JF, United Kingdom.

To establish whether the widely expressed regulator of Src family kinases Csk contributes to the control of acute inflammation in vivo, we inactivated csk in granulocytes by conditional mutagenesis (Cre/loxP). Mutant mice (Csk-GEcre) developed acute multifocal inflammation in skin and lung. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation with hyperinduction of integrins. This hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Hyperphosphorylation of key signaling proteins such as Syk and Paxillin in mutant granulocytes further supported breakdown of the activation threshold set by Csk. By enforcing the need for ligand engagement Csk thus prevents premature granulocyte recruitment while supporting the motility of stimulated cells through negative regulation of cell adhesion.
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http://dx.doi.org/10.1016/s1074-7613(04)00023-8DOI Listing
February 2004

B cell immunity regulated by the protein kinase C family.

Ann N Y Acad Sci 2003 Apr;987:125-34

Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, New York 10021, USA.

Protein kinase C (PKC) is a family of serine/threonine kinases which mediate essential cellular signals required for activation, proliferation, differentiation, and survival. Several PKC members are expressed in B lineage cells and activated by stimulation of the B cell receptor (BCR), thus suggesting a contribution of PKCs to the B cell-mediated immune response. To understand the individual roles of PKCs for B cell immunity, mice deficient for PKCbetaI/II (PKCbeta) or PKCdelta were analyzed. PKCbeta and PKCdelta play essential but distinctive roles in B cell immunity. In addition to its role in B cell activation and humoral immunity, PKCbeta was recently shown to control NF-kappaB activation and survival of mature B cells. PKCdelta on the other hand specifically regulates the induction of tolerance in self-reactive B cells. Thus, individual PCKs regulate B cell immunity specifically.
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http://dx.doi.org/10.1111/j.1749-6632.2003.tb06040.xDOI Listing
April 2003

Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell development.

Nat Immunol 2003 Mar 3;4(3):274-9. Epub 2003 Feb 3.

Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, NY 10021, USA.

The nature of signals that govern the development of immunoglobulin heavy chain-dependent B cells is largely unknown. Using mice deficient for the B cell-expressed Src-family protein tyrosine kinases (SFKs) Blk, Fyn and Lyn, we show an essential role of these kinases in pre-B cell receptor (pre-BCR)- mediated NF-kappaB activation and B cell development. This signaling defect is SFK specific, as a deficiency in Syk, which controls pre-B cell development, does not affect NF-kappaB induction. Impaired NF-kappaB induction was overcome by the activation of protein kinase C (PKC)-lambda, thus suggesting the involvement of PKC-lambda in pre-BCR-mediated SFK-dependent activation of NF-kappaB. Our data show the existence of a functionally distinct SFK signaling module responsible for pre-BCR-mediated NF-kappaB activation and B cell development.
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http://dx.doi.org/10.1038/ni893DOI Listing
March 2003

Protein kinase C beta controls nuclear factor kappaB activation in B cells through selective regulation of the IkappaB kinase alpha.

J Exp Med 2002 Jun;195(12):1647-52

Laboratory of Lymphocyte Signaling, Rockefeller University, New York, NY 10021, USA.

Activation of the nuclear factor (NF)-kappaB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-kappaB is critically dependent on serine phosphorylation of the IkappaB protein by the multi-component IkappaB kinase (IKK) containing two catalytic subunits (IKKalpha and IKKbeta) and one regulatory subunit (IKKgamma). Using mice deficient for protein kinase C beta (PKCbeta) we show an essential role of PKCbeta in the phosphorylation of IKKalpha and the subsequent activation of NF-kappaB in B cells. Defective IKKalpha phosphorylation correlates with impaired B cell antigen receptor-mediated induction of the pro-survival protein Bcl-xL. Lack of IKKalpha phosphorylation and defective NF-kappaB induction in the absence of PKCbeta explains the similarity in immunodeficiencies caused by PKCbeta or IKKalpha ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Bruton's tyrosine kinase (Btk), which regulates the activity of NF-kappaB and PKCbeta, suggests PKCbeta as a likely serine/threonine kinase component of the Btk-dependent NF-kappaB activating signal transduction chain downstream of the BCR.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2193563PMC
http://dx.doi.org/10.1084/jem.20020408DOI Listing
June 2002