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-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

ObjectivesObjectives

- + + -- - + +

Effect of TNFEffect of TNF and cAMP on FOS and cAMP on FOS and cJUN protein expressionand cJUN protein expression

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cAMPTNF

68kDa

43kDa

FOS C-JUN

Component Two: BackgroundEffect of cAMP and TNFEffect of cAMP and TNF on 3TPluc activity on 3TPluc activity

Treatment (hour)0 2 4 6 8 10 12 14

3XTP

LUC

act

ivity

0

2

4

6

8

10

12

14

16cAMPTNFcAMP+TNF

cAMP 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

- + - - - - - - - -- - + - - - - - - -- - - + - - + + - +- - - - + - + - + +- - - - - + - + + +

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)

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 activation

TNFTNF 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 activation

c-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

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

SummarySummary

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

ConclusionsConclusions

MAP-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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