Regulation of peroxisome proliferator-activated receptor γ [gamma] in macrophages during inflammatory processes [Elektronische Ressource] / vorgelegt von Carla Jennewein

Jennec

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 21 Mo

Extrait

Aus dem Fachbereich Medizin
der Goethe-Universität
Frankfurt am Main

Institut für Biochemie I - Pathobiochemie


Regulation of peroxisome proliferator-activated erepctor γ in
macrophages during inflammatory processes


Dissertation

zur Erlangung des Doktorgrades der
theoretischen Medizin des Fachbereichs MedizGioenth ed-eUrn iversität
Frankfurt am Main

vorgelegt von
Carla Jennewein
Kaiserslautern




Frankfurt am Main 2009























Dekan: Prof. Dr. Josef M. Pfeilschifter
Referent: PD Dr. Andreas von Knethen
Koreferent: Prof. Dr. Dr. Kai Zacharowski

Tag der mündlichen Prüfung: 13. Januar 2010

















„Man merkt nie, was schon getan wurde;
man sieht immer nur das,
was noch zu tun bleibt.“

Marie Curie

Index I
Index
1 Summary .................................................... .1....................
2 Zusammenfassung ............................................. ..................... 3
3 Introduction ......................................................................... .5...................
3.1 Regulation of gene expression ................................... .................... 5
3.1.1 Transcription ............................................................... 5
3.1.2 Post-transcriptional regulation ........................................... .6. ........
3.2 Inflammation and macrophages ................................. ................ 10
3.2.1 Distinct macrophage phenotypes ..................................... .10. ...............
3.2.2 NFκB ....................................................................... 12
3.2.3 Resolution of inflammation ........................................................................ .13. .........
3.3 Peroxisome proliferator–activated receptor γ (PPARγ) ................. ........... 14
3.3.1 Structure .................................................................... 14
3.3.2 Transactivation and function........................................ .15. ..........
3.3.3 Anti-inflammatory properties .................................................................. .16. ...........
3.3.4 PPARγ in diseases ........................................................ .20. ...
3.4 The impact of apoptotic cells on macrophages..................... ............. 22
3.4.1 Apoptosis .................................................................... 22
3.4.2 Engulfment of apoptotic cells - effero c.y.t.os.i.s................................ .22. .................
3.4.3 Immunological consequences of phagocytosi sa pofoptotic cells ........................ 25
3.5 Aims of the study ............................................ .3.0 .....................
4 Material and Methods ......................................................... .................. 31
4.1 Material ....................................................... .3.1................
4.1.1 Cells ........................................................................ 31
4.1.2 Bacteria................................................................... 31
4.1.3 Chemicals and reagents .............................................................................. .3.2 ........
4.1.4 Antibodies ................................................................ .33
4.1.5 Plasmids ................................................................... 34
4.1.6 Oligonucleotides .......................................................... .3.5 ..
4.1.7 Instruments and Software .......................................................................... .3.6. ........Index II
4.2 Methods ...................................................... .3 .7..................
4.2.1 Cell biology ............................................................................................................ 37
4.2.2 Biochemistry ............................................................... .4.0
4.2.3 Molecular biology ......................................................... .4.1 ...
4.2.4 Microbiology............................................................... .4.6.
4.2.5 Statistical analysis ................................................................................................ .4.7
5 Results ...................................................... .4.8.................
5.1 PPARγ contributes to macrophage polarization towarsd an anti-
inflammatory phenotype in response to AC .................................... ................. 48
5.1.1 Activation of PPARγ in response to AC .................................. .4.8. ..............
5.1.2 PPARγ attenuates NFκB transactivation agnetd gteanre expression ................... 50
5.1.3 Identification of PPARγ domains requiNrFedκ Bf oir nhibition ............................. 54
5.1.4 SUMOylation of PPARγ prevents co-repressor removal ....................................... 55
5.2 Regulation of PPARγ expression during the inflammoarty response ....... .. 60
5.2.1 PPARγ1 expression during monocyte differenttioan and upon LPS exposure ..... 61
5.2.2 Post-transcriptional regulation of PPARγ1 Am .R.N...................... .6.5. ......................
5.2.3 miR-27b destabilizes PPARγ1 mRNA .................................................. .6.7. ................
6 Discussion ................................................... .7 .2...................
6.1 PPARγ contributes to macrophage polarization in rpeosnse to AC ........ .... 72
6.2 Regulation of PPARγ during the inflammatory respoen s................ .......... 77
6.3 Concluding remarks ...........................................2. .................... 8
7 References .................................................. .8. 4....................
8 Appendix ......................................................................... .9. .6...................
Buffers and solutions ................................................. ... .9.6...........
9 Publications ............................................... .1.0.3. ....................
10 Danksagung ....................................................................0.4. ................... 1
11 Curriculum vitae ............................................5. ................... 10
12 Erklärung ................................................. .1.0.6 ....................List of figures III
List of figures
Figure 3.1 miRNA processing and mRNA degrada..ti..on... ................................ .8. ..........
Figure 3.2 NFκB signaling pathway upon TLRi4on a. c.t.i.v.a.t............................ .13. ...........
Figure 3.3 PPARγ domain structure. ...................................................... .15
Figure 3.4 Transrepression mechanisms of PPAR.γ.. ..................................... .17. .........
Figure 3.5 Immunological consequences of AC on opmhaacgres. ........................... .26. ................
Figure 5.1 Transactivation of PPARγ in respAonC.se .t.o ................................... .4.8. .......
Figure 5.2 Unaltered PPARγ expression in respono sAe C.t................................. .4.9. ..........
Figure 5.3 Time-dependent inhibition of NtyF κiB na cretsipovnise to AC. ................ .50. ...........
Figure 5.4 NFκB reporter activity is restoredW 2i64n. 7R Ad/n PPARγ macrophages. .............. 51
Figure 5.5 PPARγ-dependent reduction of cytokinpree sesixon............................ .5.2 .............
Figure 5.6 PPARγ-deficient macrophages impaeinrueda taedt tTNFα mRNA expression. ......... 53
Figure 5.7 Domain analysis of PPARγ. .................................................. .54
Figure 5.8 Trichostatin A reverses AC-provokedb iintihion of NFκB transactivation. ...6. ............. 5
Figure 5.9 Interfering with SUMOylation of PPsAtoRreγd rNeFκB inhibition. ......... .5.6. ...............
Figure 5.10 Impact of PIAS1 on TNFα expression.. ........................................ .5.7. ......
Figure 5.11 Impact of NCoR on TNFα expression. ........................................ .5.8. .......
Figure 5.12 PPARγ antagonizes the removal of NC.oR.... .............................................. .5.9 ...........
Figure 5.13 p38-dependent NFκB inhibition isen t or eAspC.on ............................ .6.0. ..........
Figure 5.14 Differential expression of PPARγ mdounriocnytge/macrophage differentiation. .. 61
Figure 5.15 Time dependent reduction of PPARγ1 m RiNAn macrophages. .............................. 62
Figure 5.16 NFκB-dependent PPARγ1 mRNA decrease.. ........................................... .6.3. ..............
Figure 5.17 Time-dependent decrease of PPARγ prnote in response to LPS ..............4. ............. 6
Figure 5.18 Impact of LPS on PPARγ promoter actiyv.i .t................................... .6.5. .........
Figure 5.19 Altered mRNA half-life upon LPS ex.p o.su.r.................................. .6.5. .........
Figure 5.20 Sequence of the AU-rich PPARγ-3’UT.R... ..................................................... .6.6. ........
Figure 5.21 3’UTR-dependent reduction of luci fexraprsession. ........................ .6.7. ...........
Figure 5.22 Impact of the miR-27 binding nsi the e w3i’UtTh