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Introduction
• Tumor necrosis factor- (TNF) is a pro-inflammatory cytokine important in immune responses
• TNF inhibits cAMP-stimulated Cyp17 transcription
• TNF activates protein kinase C in Leydig cells
• TNF action is known to involve AP-1 factors in other systems
Introduction to AP-1Introduction to AP-1Introduction to AP-1Introduction to AP-1
Component One: Status
• To determine if activation of AP-1 is involved in cAMP and/or TNF signal transduction in MA-10 cells
• To determine if cAMP and/or TNF activate c-Jun kinase in MA-10 cells
ObjectivesObjectivesObjectivesObjectives
- + + -- - + +
Effect of TNFEffect of TNF and cAMP on FOS and cAMP on FOS and cJUN protein expressionand cJUN protein expression
Effect of TNFEffect of TNF and cAMP on FOS and cAMP on FOS and cJUN protein expressionand cJUN protein expression
- + + -- - + +
cAMPTNF
68kDa
43kDa
FOS C-JUN
Component Two: BackgroundEffect of cAMP and TNFEffect of cAMP and TNF on 3TPluc activity on 3TPluc activityEffect of cAMP and TNFEffect of cAMP and TNF on 3TPluc activity on 3TPluc activity
Treatment (hour)
0 2 4 6 8 10 12 14
3XT
PL
UC
act
ivit
y
0
2
4
6
8
10
12
14
16
cAMPTNFcAMP+TNF
cAMP and TNFcAMP and TNF vs. DNA-protein vs. DNA-protein interactions with canonical AP-1interactions with canonical AP-1cAMP and TNFcAMP and TNF vs. DNA-protein vs. DNA-protein interactions with canonical AP-1interactions with canonical AP-1
- + - - - - - + - - - - - + - - - - - +
- + - - - - - + - - - - - + - - - - - +
- + - - - - - + - - - - - + - - - - - +
- + - - - - - + - - - - - + - - - - - +
FOSc-junjunBjunD
controlab cAMP cA+TNF TNF
Super-shift with AP-1 antibodies vs. Super-shift with AP-1 antibodies vs. canonical AP-1 binding elementcanonical AP-1 binding element
Super-shift with AP-1 antibodies vs. Super-shift with AP-1 antibodies vs. canonical AP-1 binding elementcanonical AP-1 binding element
- + - - - - - - - -- - + - - - - - - -- - - + - - + + - +- - - - + - + - + +- - - - - + - + + +
AP-1FOS
c-junjunBjunD
SAPK/JNKSAPK/JNKStress Activated Protein Kinase/c-Jun KinaseStress Activated Protein Kinase/c-Jun Kinase
• 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
c-Jun Kinasec-Jun Kinase::Stress Activated Protein Kinase/cJun Nuclear Stress Activated Protein Kinase/cJun Nuclear
Kinase (Kinase (SAPK/JNK)SAPK/JNK)
c-Jun Kinasec-Jun Kinase::Stress Activated Protein Kinase/cJun Nuclear Stress Activated Protein Kinase/cJun Nuclear
Kinase (Kinase (SAPK/JNK)SAPK/JNK)
TNFTNF and cAMP vs. cJUN kinase and cAMP vs. cJUN kinase activityactivity
TNFTNF and cAMP vs. cJUN kinase and cAMP vs. cJUN kinase activityactivity
.25 .5 1 2 6 12 24
0 .25 .5 1 2 6 12 24
hrs
TNF cAMP
4442
Time course of cJUN kinase activationTime course of cJUN kinase activationTime course of cJUN kinase activationTime course of cJUN kinase activation
TNFTNF and cAMP vs. MAPK activity and cAMP vs. MAPK activityTNFTNF and cAMP vs. MAPK activity and cAMP vs. MAPK activity
.25 .5 1 2 6 12 24
0 .25 .5 1 2 6 12 24
hrs
TNF cAMP
ERK
Time course of MAP kinase activationTime course of MAP kinase activationTime course of MAP kinase activationTime course of MAP kinase activation
c-Jun kinase transactivation assayc-Jun kinase transactivation assayc-Jun kinase transactivation assayc-Jun kinase transactivation assay
Effect of cAMP and TNF on c-Jun Effect of cAMP and TNF on c-Jun transactivation activitytransactivation activity
Effect of cAMP and TNF on c-Jun Effect of cAMP and TNF on c-Jun transactivation activitytransactivation activity
Overall Status
• 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
SummarySummarySummarySummary
Conclusions
• These studies suggest that TNF effects are promoter specific.
• TNF inhibits cAMP-induced Cyp17 transcription but stimulates cAMP-induced activation of canonical AP-1 target genes
ConclusionsConclusionsConclusionsConclusions
MAP-kinase signaling pathwaysMAP-kinase signaling pathwaysMAP-kinase signaling pathwaysMAP-kinase signaling pathways
RAS
RAF
MEK1,2
ERK1,2
MEKK1,+
SEK/JNKK
SAPK/JNK1,2
MEKK4,+
MKK3
p38
? Rac/Cdc42
Elk1, Rsk cJun, ATF2, Elk1
ATF2
mitogen Stress/cytokine
? ?
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