Regulation of IL-6 signal transduction, contributions of the signal regulatory protein (SIRP) and cyclic AMP [Elektronische Ressource] / vorgelegt von Radoslaw, Mikolaj Sobota

Biochemie

Description

Regulation of IL-6 signal transduction, contributions of the Signal Regulatory Protein (SIRP) and cyclic AMP Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science, Biology Radoslaw, Mikolaj Sobota aus Czestochowa (Polen) Berichter: Univ.-Prof. Dr. rer. nat. Peter C. Heinrich Priv.-Doz. Dr. rer. nat. Christoph Peterhänsel Tag der mündlichen Prüfung: 5. Oktober 2007 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. follow your dreams... Content of this thesis is enclosed in following publications: • SIRP/SHPS contributes to interleukin-6 signalling. Sobota R.M., Müller J.P., Ullrich A., Noguchi T., Heinrich P.C., Schaper F. Manuscript to be submitted • Inhibition of interleukin-6-mediated signal transduction and gene expression through the adenylate cyclase pathway. Sobota R.M., Müller P.J., Heinrich P.C., Schaper F. Manuscript to be submitted Further publications: • Mechanisms of SOCS3 phosphorylation upon IL-6 stimulation: Contributions of Src- and receptor tyrosine kinases Sommer U.

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2007
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

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Regulation of IL-6 signal transduction, contributions of the Signal Regulatory Protein (SIRP) and cyclic AMP         Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch- Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation       vorgelegt von   Master of Science, Biology Radoslaw, Mikolaj Sobota  aus Czestochowa (Polen)  

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     Berichter: Univ.-Prof. Dr. rer. nat. Peter C. Heinrich  Priv.-Doz. Dr. rer. nat. Christoph Peterhänsel             Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Tag der mündlichen Prüfung: 5. Oktober 2007

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                                          

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follow your dreams...

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Content of this thesis is enclosed in following publications: •  SIRP/SHPS contributes to interleukin-6 signalling. Sobota R.M., Müller J.P., Ullrich A., Noguchi T., Heinrich P.C., Schaper F. Manuscript to be submitted  •  Inhibition of interleukin-6-mediated signal transduction and gene expression through the adenylate cyclase pathway. Sobota R.M., Müller P.J., Heinrich P.C., Schaper F. Manuscript to be submitted  Further publications: •  Mechanisms of SOCS3 phosphorylation upon IL-6 stimulation: Contributions of Src- and receptor tyrosine kinases  Sommer U., Schmid C.,Sobota R.M., Lehmann U., Stevenson N.J., Johnston J.A., Schaper F., Heinrich P.C., Haan S. The Journal of Biological Chemistry  2005, 280: 31478   •  Dual function of interleukin-1beta for the regulation of interleukin-6-induced suppressor of cytokine signalling 3 expression.  Yang X.P., Albrecht U., Zakowski V.,Sobota R.M.Haussinger D., Heinrich P.C., Ludwig S.,,  Bode J.G, Schaper F. The Journal of Biological Chemistry  2004, 279: 45279  •  The role of the inhibitors of interleukin-6 signal transduction SHP2 and SOCS3 for  desensitisation of interleukin-6 signalling Fischer P., Lehmann U.,Sobota R.M., Schmitz J.,Yoshimura A., Niemand C., Haan S., Johnston J.A., Behrmann I., Müller-Newen G., Heinrich P.C., Schaper F. Biochemical Journal2004, 378: 449  •  SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signalling through gp130. Lehmann U., Schmitz J., Weissenbach M.,Sobota R.M., Hortner M., Friederichs K., Behrmann I., Tsiaris W., Sasaki A., Schneider-Mergener J., Yoshimura A., Neel B.G., Heinrich P.C., Schaper F. The Journal of Biological Chemistry2003, 278: 661  •  Autocrine stimulation by oncostatin M but resistance to leukemia inhibitory factor in astrocytoma cells expressing OSM and LIF Kasza A., Rogowski K., Kilarski W.,Sobota R., Bernaś Dobrucki J., Travis J., Koj A., T., Bugno M. and Kordula T. Biochemical Journal2001, 355: 307  •  Signal transduction of IL-6, leukemia-inhibitory factor, and oncostatin M: Structural receptor requirements for signal attenuation Anhuf D., Weissenbach M., Schmitz J.,Sobota R., Hermanns H.M., Radtke S., Linnemann S., Behrman I., Heinrich P.C., Schaper F. The Journal of Immunology2000, 165: 2535  •  Parthenolide inhibits activation of signal transducers and activators transcription (STATs) induced by cytokines of IL-6 family Sobota. R., Szwed M., Kasza A., Bugno M., Kordula T. Biochem Biophys Res Commun2000, 267: 329

Index

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INDEX INDEX..........................................................................................................................I  ABBREVIATIONS.....................................................................................................IV  1.  .............1................................................................................I....OI.NUDTCTNOR  1.1  Cytokines ................................................................................................................ 1  1.2  Interleukin 6-type cytokines.................................................................................. 1  1.3  Inkieurlte-n.6.............................................................................................2..............  1.4  IL-6 signal transduction ........................................................................................ 3  1.4.1   3 ...............................................................................................Jak/STAT pathway  1.4.2  pathways activated in response to IL-6.............................................. 4Alternative  1.5  Regulation of the IL-6 signalling pathway ........................................................... 5  1.5.1  SH2 domain containing tyrosine phosphatase 2 (SHP2) 5 .....................................  1.5.2   ................................................................................................... 6SOCS proteins  1.5.3  Signal regulatory protein (SIRP) .......................................................................... 6  1.6  Prostaglandins ....................................................................................................... 7  1.6.1  Prostaglandin E 1 - biological functions ................................................................. 8  1.6.2  Prostaglandin E-type receptors and signalling..................................................... 9  1.7  ..................................................................esinokemCh........................................9..  1.7.1  Monocyte chemotactic protein 1 (MCP-1/CCL2)................................................ 11  1.8  Aim of the study ................................................................................................... 11  2.  MATERIAL AND METHODS .............................................................................13  2.1   13Reagents ...............................................................................................................  2.1.1  Chemicals .......................................................................................................... 13  2.1.2  Cytokines, soluble receptors and inhibitors........................................................ 13  2.1.3  Tissue culture reagents...................................................................................... 13  2.1.4  Antibodies .......................................................................................................... 14  2.1.5  DNA Constructs ................................................................................................. 14  2.1.6  DNA oligonucleotides......................................................................................... 14  2.1.7  Cell lines............................................................................................................. 15  2.2  Methods ................................................................................................................ 15  2.2.1  Cell culture ......................................................................................................... 15  2.2.2  Prokaryotic cells ................................................................................................. 16  2.2.3   16 .....................................................................Preparation of competent bacteria  2.3  General protocols for molecular biology........................................................... 17  2.3.1   17 ...................................................DNA digestion with restriction endonucleases  2.3.2  Agarose gel electrophoresis............................................................................... 17  2.3.3  Isolation of DNA fragments from the agarose gel .............................................. 17  2.3.4  Dephosphorylation of DNA................................................................................. 17  2.3.5   18DNA-ligation .......................................................................................................  2.3.6   18 ...................................................................................Chemical Transformation  2.3.7   18Preparative DNA isolation ..................................................................................  2.3.8  Polymerase Chain Reaction (PCR).................................................................... 18  2.3.9  Isolation of total RNA ......................................................................................... 19  2.3.10  Reverse transcription reaction (RT) ................................................................... 19  2.3.11  Synthesis of DNA oligos..................................................................................... 20  2.4  Transient transfection ......................................................................................... 20 

Index

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2.5  Retroviral infection .............................................................................................. 20  2.6  Cell lysis ............................................................................................................... 20  2.6.1  Measurements of the protein concentration....................................................... 21  2.6.2   21Immunoprecipitation (IP) ....................................................................................  2.7  SDS-polyacrylamide gel electrophoresis .......................................................... 21  2.7.1  Semi-dry protein transfer.................................................................................... 22  2.7.2   ................................................................................................. 23Western blotting  2.8   23 .......................................................Fluorescence-assisted cell sorting (FACS)  2.9  Determination of MCP-1 expression .................................................................. 24  2.10  Measurement of cAMP ........................................................................................ 24  2.11  Luciferase reporter gene assay 24 ..........................................................................  3.  RESULTS ..........................................................................................................26  3.1  SHP2 associates with SIRPα 26 .........................................upon IL-6 stimulation   3.2  Characterisation of SIRPαdeficient cells .......................................................... 27   3.2.1  Genotyping of knockout cells ............................................................................. 27  3.2.2  SIRPαexpression on the surface of MEF cells.................................................. 28  3.2.3  IL-6 receptor cell surface expression ................................................................. 29  3.3  Delayed STAT3 activation in cells lacking SIRPα............................................. 29   3.3.1  SIRPαenhances activation of SOCS3 gene expression ................................... 30  3.3.2  SIRPαenhances IL-6-dependent c-fos induction .............................................. 30  3.3.3  Absence of SIRPαenhances tyrosine phosphorylation of SHP2....................... 31  3.3.4  The SHP2 binding site within gp130 is crucial for enhanced SHP2 phosphorylation in SIRPαdeficient cells............................................................ 32  3.3.5  SIRPαinfluences IL-6 mediated ERK activation................................................ 33  3.4   ........................................................................cAMP modulates IL-6 signalling. 34  3.4.1  Pertussis toxin (PTX) downregulates IL-6 dependent ERK activity ................... 34  3.4.2  Forskolin counteracts IL-6 induced ERK activation............................................ 35  3.5  Prostaglandin E 1 (PGE 1 ) counteracts IL-6-induced ERK activation ................ 36  3.5.1  Three out of four E-series prostaglandin receptors (EP) are expressed in primary dermal fibroblasts............................................................................................... 38  3.5.2  PGE 1 as well as the EP2 receptor agonist R - butaprost stimulate cAMP production .......................................................................................................... 39  3.5.3  R butaprost, a specific prostaglandin EP2 receptor agonist, mimics PGE 1  effects on IL-6-induced ERK activation .............................................................. 39  3.5.4  PKA but not Epac mediates cAMP effects on IL-6-induced ERK activation ...... 40  3.5.5  The PKA inhibitor H89 restores the PGE 1 -mediated inhibitory effect on ERK phosphorylation..................................................................................................41  3.5.6  PKA phosphorylates c-Raf-1 kinase in response to PGE 1 ................................. 42  3.5.7  c-Src is not directly involved in PGE 1 mediated inhibition of ERK 1/2 phosphorylation. 3  ................................................................................................. 4 3.5.8  cAMP inhibits IL-6-induced gene expression of chemokine MCP-1 .................. 44  4.  DISCUSSION .....................................................................................................46  4.1  Regulation of signalling ...................................................................................... 46  4.1.1  SIRPα- SHP2 interaction and its possible consequences in IL-6 signalling ..... 46  4.1.2   46 ..............................................Receptor surface expression and gene induction  4.1.3  Hyper-activation of SHP2 as a consequence of missing substrate?.................. 47  4.1.4   ....... 48Transient ERK induction as a possible effect of uncontrolled SHP2 action  4.2   49 ...........................................................Cross-talk between signalling pathways 

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Index

4.2.1  4.2.2  PGE 1 through EP2-receptor acts on IL-6-induced ERK activation..................... 51  4.2.3  Protein kinase A mediates cAMP action ............................................................ 51  4.2.4  Raf-1 kinase as a target of PKA......................................................................... 52  4.2.5  Are c-Src and PKA acting antagonistically in regard to ERK regulation? .......... 52  4.2.6  Anti-inflammatory properties of Prostaglandin E 1 ............................................... 53  5.  OUTLOOK .........................................................................................................54  5.1  Looking for new proteins in familiar pathways................................................. 54  5.2  Signalling networks- illustration of cross-talks ................................................ 54  6.  US...YRAMM................................................................................................56......  7.  .................57........FERE..SECNER........................................................................  CURRICULUM VITAE ..............................................................................................69  ACKNOWLEDGEMENTS .........................................................................................70  

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ctivatioedERKa-6emidtaibstI-LPAMhiinc0...5...................................................n

Abbreviations

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ABBREVIATIONS  Akt AKR thymoma oncogene homologue AMPadenosine monophosphate AP phase acute  APP phase protein acute APS ammonium peroxodisulfate ATP adenosine triphosphate BSA bovine serum albumin BSF stimulatory factor B-cell cAMP3'-5'-cyclic AMP  CD of differentiation cluster CIS SH2 containing protein cytokine-inducible CMV Cytomegalie-Virus  CNTF neurotrophic factor ciliary CLC cytokine cardiotrophin-like CT cardiotrophin C3GCrk SH3 guanine nucleotide exchange factor  ddH 2 Odouble distilled water DMEMDulbecco s Modified Eagle Medium ´ DMSOdimethylsulfoxid DNA deoxyribonucleic acid DOS of sevenless daughter DTT 1,4-Dithio-DL-threitol E. coli coli Escherichia ECL enhanced chemoluminescence EDTA ethylenediamine-tetraacetic acid EGF epidermal growth factor ELISAenzyme linked immunosorbent assay EPE-series prostaglandin receptor  Epacexchange protein activated by cAMP  Epo erythropoietin Erk extracellular regulated kinase ESS SH2 subdomain extended EtOHethanol  Ex Exon FACS activated cell sorting fluorescence FAK focal adhesion kinase FCS fetal calf serum FERM  Four-point-one ezrin radixin moesin Gab Grb2-associated binder GDPguanosine diphosphate  GTP guanosine triphosphate gp glycoprotein GPCR  G-protein coupled receptor Grb growth factor receptor bound protein h hour HEPESyh)lyxtedyor2(H-N-haetulnsnsforeäupip-zareN-ni-2-´ HIV immunodeficiency virus human HRP horseradish peroxidase

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