Investigating the role of the JAK/STAT and MAPK ... - UCL Discovery

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“And the newness that was in every stale thing when we looked at it as children” 2 Patrick Kavanagh “You got to know when to hold em, know when to fold em, know when to walk away and know when to run” Kenny Rogers
Abstract The signal transducer and activator of transcription (STAT) proteins are a family of transcription factors which transduce extracellular signals from cytokines, growth factors and G-proteins to the nucleus. STATs become activated by phosphorylation and translocate to the the nucleus where they bind to specific target promoters. STAT1 has previously been shown to have a role in inducing apoptosis in the myocardium following ischaemia/reperfusion injury (I/R), however the role of STAT3 in myocardial apoptosis is less clear. Here it is shown that STAT3 is phosphorylated in cardiac cells both in vito and in vivo in response to I/R injury and plays a protective role by reducing the levels of apoptosis. Several modulators of STAT3 activity were found to be upregulated following I/R, including JAK2, SOCS3 and GRIM-19. STAT3 was also found to be important in regulating DNA damage and repair through altered activity of DNA damage response proteins. Administration of the antioxidant tempol in vivo, reduced infarct size in a rat model of I/R injury and this was accompanied by a reduction in STAT1 and STAT3 phosphorylation. Increasing STAT1 phosphorylation with IFN- treatment abolished the protective effect of tempol, suggesting that inhibition of STAT1 phosphorylation may be a key protective effect of tempol infusion. Affymetrix microarray analysis of hearts from the in vivo I/R model identified several novel gene expression changes and uncovered transcriptional reduction in large numbers of genes involved in mitochondrial respiration and transport. In addition, this approach identified several possible new regulators of cardiac protection mediated by tempol and the urocortin hormones. The mitogen activated protein kinase (MAPK) family is involved in sensing cellular stress and play key roles in I/R injury and inflammation. MAPK activity is balanced by MAPK phosphatases (MKPs) such as MKP-1 and the role of MKP-1 in modulating the immune response was investigated. Mice deficient in MKP-1 were more susceptible to endotoxic shock and had elevated levels of serum cytokines. MKP-1 was found to be upregulated following toll-like recptor (TLR) stimulation and this was dependent on the signaling adaptors MyD88 and Trif. Macrophages deficient in MKP-1 had increased phosphorylation of p38 MAPK and JNK following TLR stimulation and secreted elevated amounts of the proinflammatory cytokines TNF- , IL-12 and the anti-inflammatory cytokine IL-10. The temporal control and regulation of cytokine production in response to TLR stimulation was dissected using pharmacological inhibition of MAPKs. MKP-1 was not found to contribute to T cell differentiation but did have a role to play in the adaptive immune response as MKP-1deficient mice failed to recover from an experimental model of multiple sclerosis. 3
Page 1: Investigating the role of the JAK/S
Page 5 and 6: Acknowledgements First and foremost
Page 7 and 8: Table of Contents Abstract 3 Dedica
Page 9 and 10: 2.4.3 Hypoxia/Reoxygenation of Neon
Page 11 and 12: Chapter 6: Regulation of the MAPK P
Page 13 and 14: Figure 3.11 - STAT3 mRNA expression
Page 15 and 16: Figure 6.6 - Prolonged MAPK activit
Page 17 and 18: Abbreviations 7-AAD 7-amino-actinom
Page 19 and 20: SOCS Supressor of cytokine signalli
Page 21 and 22: 1.1 Myocardial Infarction Coronary
Page 23 and 24: Reperfusion induced arrhythmias are
Page 25 and 26: eceptors such as Fas or TNFR1; thes
Page 27 and 28: 1.2.3 Bcl-2 Proteins The intrinsic
Page 29 and 30: addition to caspase inhibition. XIA
Page 31 and 32: staurosporine induced cell death. L
Page 33 and 34: stress thus occurs when excess ROS
Page 35 and 36: 1.3 The JAK/STAT Pathway 1.3.1 JAK/
Page 37 and 38: Receptor SOCS3 Cytokines/Growth Fac
Page 39 and 40: Dimerization of STATs appears to be
Page 41 and 42: Fig 1.7. STAT import/export cycle.
Page 43 and 44: Kinase Stimulus Cell Type Reference
Page 45 and 46: domain determines other target sele
Page 47 and 48: 1.3.8 Inhibition of STAT DNA Bindin
Page 49 and 50: (BRM/SWI2-related gene 1) the ATPas
Page 51 and 52: deficient mammary glands (Abell et
Page 53 and 54:
and COX-2 induction. Adenoviral med
Page 55 and 56:
compensatory hypertrophy, mediated
Page 57 and 58:
1.5.3 Urocortins and Ischaemia As w
Page 59 and 60:
Ott et al. recently showed that the
Page 61 and 62:
1.6.4 The SAM Complex The outer mem
Page 63 and 64:
etain H2AX activity for longer than
Page 65 and 66:
esulting in an ATM-MDC1-H2AX positi
Page 67 and 68:
1.8.3 TLR Adaptors TLR signalling i
Page 69 and 70:
1.9 The Adaptive Immune System 1.9.
Page 71 and 72:
Fig 1.14. Outline of T-helper cell
Page 73 and 74:
1.10.3 IL-12 IL-12 is a potent pro-
Page 75 and 76:
1.11.2 p38 MAPK p38 is induced by a
Page 77 and 78:
negative JNK overcomes LPS induced
Page 79 and 80:
1.12 Inflammatory Diseases 1.12.1 M
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10 producing Th2 response appears t
Page 83 and 84:
2.1 Reagents The following TLR liga
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2.3.2 Endotoxic shock Toxic shock o
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2.4 Cell Culture 2.4.1 Freezing and
Page 89 and 90:
emove red blood cells. CD4 and CD8
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2.5.2 ELISA ELISA was used as a met
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mM NaCl, 1 mM EDTA, 1 mM EGTA and p
Page 95 and 96:
To measure gene promoter regulation
Page 97 and 98:
uffer (200 mM MES buffer, 2 M NaCl,
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was added (10 mM RbCl, 50 mM , 100
Page 101 and 102:
2.9 Cell Death Measurements 2.9.1 T
Page 103 and 104:
Chapter 3: Investigating the Role o
Page 105 and 106:
3.2 Overexpression of STAT3 Protect
Page 107 and 108:
A B % Cell Death 50 40 30 20 10 0 G
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cell death were examined. Using thi
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A B 7AAD GFP %Cell Death 100 75 50
Page 113 and 114:
3.4 Deletion of STAT3 Sensitises Ce
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Next, wild type and STAT3 knockout
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3.5 STAT3 becomes Phosphorylated an
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While STAT3 is phosphorylated at bo
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Two STAT3 target genes, SOCS3 and c
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A STAT3 B C Fold Change Fold Change
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3.6 Oxidative stress induces STAT3
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3. 7 Activation of STAT1 and STAT3
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Next, the extent of DNA damage and
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A B C pSTAT3 Y705 pSTAT3 S727 Total
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3.9 I/R injury in the brain induces
Page 135 and 136:
3.10 Reperfusion Induced Myocardial
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Tempol infusion before the onset of
Page 139 and 140:
In order to examine the tissue dist
Page 141 and 142:
Fig 3.22. Effect of I/R and drug in
Page 143 and 144:
3.12 Discussion STAT transcription
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may be involved. JAK2 activity has
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studies have begun to address the r
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4.1 Aims In the previous chapter, t
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all 15 arrays to be included in dow
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C D A Log intensity Raw Intensity B
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A B C I/R Ucn1 Probe Sets Annotated
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Table 4.2. The 20 genes with the hi
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A B 209 245 150 111 133 30 44 41 30
Page 161 and 162:
A B 161
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E Activation Inhibition Translocati
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Number of Genes 20 15 10 5 0 No Ide
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4.6 Differential Expression mediate
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Number of Genes Number of Genes 20
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Ucn1 Ucn2 Fig 4.10 Network analysis
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A B D qPCR Expression (log2) qPCR E
Page 175 and 176:
Table 4.5. Genes differentially reg
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A C Fold Change Fold Change 17.5 15
Page 179 and 180:
A C Fold Change Fold Change 30 20 1
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order to examine if neonatal cardia
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4.10 Differential Regulation of MAP
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4.11 Uracil Metabolism is Altered b
Page 187 and 188:
4.12 Reduced Expression of Mitochon
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Gene Gene Title FC p value Nqo2 NAD
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ATP thus allowing protons to be pum
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A Fold Change 1.00 0.75 0.50 0.25 0
Page 195 and 196:
Fig 4.19. Tempol and Ucn1 upregulat
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A B Fold Change [MDA] µmol/g prote
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exogenously added IL-17 could induc
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The subunits that comprise the mito
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Taken together, these observations
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mediator of Ucn1 and Ucn2 cardiopro
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5.1 Aims In chapter 3 it was shown
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To show that reduced DNA repair in
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A pATM S1981 GAPDH C H2AX GAPDH STA
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5.4. STAT3 Facilitates DNA Damage M
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The STAT3 dependent regulation of M
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5.5 Discussion Efficient repair of
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(Cimprich and Cortez, 2008). Chk1 c
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Chapter 6: Regulation of the MAPK P
Page 223 and 224:
700 bp 450 bp +/+ -/- +/- Fig 6.1 M
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The pathological consequences of en
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6.3 MKP-1 Negatively Regulates p38
Page 229 and 230:
A B AP-1 AP-1 Fold Change 1500 1000
Page 231 and 232:
A B C MKP-1 Actin D MKP-1 Actin Act
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A B MKP-1 Actin Fig 6.9. MKP-1 upre
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A B IL-10 (pg/ml) 700 600 500 400 3
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B A Fold Change 1000 750 500 250 0
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6.6 Dynamic Regulation of TNF- is M
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levels seen by 5 hr LPS challenge i
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Previous studies have shown that IL
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6.7 MKP-1 Activity Promotes IL-12 E
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B A Fold Change 6000 4000 2000 0 +/
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A B IL-12p70 pg/ml IL-12p70 500 +/+
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% Weight 105 100 95 90 DSS 0 5 10 1
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6.10 Loss of MKP-1 does not Effect
Page 255 and 256:
Taken together, these results demon
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Since loss of MKP-1 does not appear
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mice leads increased NO levels (Zha
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Fig 6.26. Model of MKP-1 mediated t
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of DSS induced colitis. Other possi
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wild type mice but failed to do so
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STAT3 was found to be active during
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Both Ucn1 and Ucn2 were found to lo
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cytokine production (IL-2, IL-4, IF
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(A) Table of Antibodies Appendix 1
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IL-6_F ACTGCCTTCCCTACTTCACA IL-6_R
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Alexander WS, Starr R, Fenner JE, S
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Bevan MJ: Helping the CD8(+) T-cell
Page 281 and 282:
Chen P, Li J, Barnes J, Kokkonen GC
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Deveraux QL, Takahashi R, Salvesen
Page 285 and 286:
Garcia R, Bowman TL, Niu G, Yu H, M
Page 287 and 288:
Hirota H, Izumi M, Hamaguchi T, Sug
Page 289 and 290:
Kassel O, Sancono A, Kratzschmar J,
Page 291 and 292:
Kuwata H, Watanabe Y, Miyoshi H, Ya
Page 293 and 294:
Lufei C, Ma J, Huang G, Zhang T, No
Page 295 and 296:
Minners J, McLeod CJ, Sack MN: Mito
Page 297 and 298:
KL, JA, TL, R, TE: Activation of ST
Page 299 and 300:
F, Y, D, C, SB, PT, RJ, Monte F. Pr
Page 301 and 302:
Roucou X, Montessuit S, Antonsson B
Page 303 and 304:
Shen Y, Schlessinger K, Zhu X, Meff
Page 305 and 306:
Szabo SJ, Sullivan BM, Peng SL, Gli
Page 307 and 308:
Valledor AF, Xaus J, Comalada M, So
Page 309 and 310:
Yamamoto M, Sato S, Hemmi H, Hoshin
Page 311 and 312:
Zhang J, Yang J, Roy SK, Tininini S
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