Tumor necrosis factor-a (TNFa) is a pro-inflammatory cytokine important in immune responses

1
Introduction
Introduction to AP-1

• Tumor necrosis factor-a (TNFa) is a
  pro-inflammatory cytokine important in immune
  responses
• TNFa inhibits cAMP-stimulated Cyp17 transcription
• TNFa activates protein kinase C in Leydig cells
• TNFa action is known to involve AP-1 factors in
  other systems

2
Component One Status
Objectives

• To determine if activation of AP-1 is involved in
  cAMP and/or TNFa signal transduction in MA-10
  cells
• To determine if cAMP and/or TNFa activate c-Jun
  kinase in MA-10 cells

3
Effect of TNFa and cAMP on FOS and cJUN protein
expression
FOS
C-JUN
68kDa
43kDa
cAMP TNFa
- - - -
- - - -
4
Effect of cAMP and TNFa on 3TPluc activity
Component Two Background
5
cAMP and TNFa vs. DNA-protein interactions with
canonical AP-1
control
ab
cAMP
cATNF
TNFa
6
Super-shift with AP-1 antibodies vs. canonical
AP-1 binding element
AP-1 FOS c-junjunB junD
- - - - - - - - - - - - -
- - - - - - - - - - - -
- - - - - - - - - -
-
7
SAPK/JNKStress Activated Protein Kinase/c-Jun
Kinase
c-Jun KinaseStress Activated Protein
Kinase/cJun Nuclear Kinase (SAPK/JNK)

• A serine/threonine kinase
• Phosphorylates c-Jun in the amino-terminus
• Modulates transactivational activity
• MAP-kinase, distinct from Erk and P38
• Activity depends on dual phosphorylation by
  serine/threonine and tyrosine kinases

8
TNFa and cAMP vs. cJUN kinase activity
44 42
.25 .5 1 2 6 12 24
0
.25 .5 1 2 6 12 24
hrs
TNFa
cAMP
9
Time course of cJUN kinase activation
10
TNFa and cAMP vs. MAPK activity
ERK
.25 .5 1 2 6 12 24
0
.25 .5 1 2 6 12 24
hrs
TNFa
cAMP
11
Time course of MAP kinase activation
12
c-Jun kinase transactivation assay
13
Effect of cAMP and TNF on c-Jun transactivation
activity
14
Overall Status
Summary

• Cyclic-AMP alone, but not TNF alone, stimulates
  3TPluc activity
• TNF enhances cAMP-stimulated 3TPluc activity
• TNF stimulates c-Jun kinase (JNK) activity
• TNF increases transactivational activity of c-jun
• Cyclic-AMP induces Fos, c-Jun, junB and junD
  nuclear binding activity

15
Conclusions
Conclusions

• These studies suggest that TNFa effects are
  promoter specific.
• TNF inhibits cAMP-induced Cyp17 transcription but
  stimulates cAMP-induced activation of canonical
  AP-1 target genes

16
MAP-kinase signaling pathways
mitogen
Stress/cytokine
RAS
?
Rac/Cdc42
?
?
RAF
MEKK1,
MEKK4,
MEK1,2
SEK/JNKK
MKK3
ERK1,2
SAPK/JNK1,2
p38
Elk1, Rsk
cJun, ATF2, Elk1
ATF2
17
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