Introduction Tumor necrosis factor- (TNF ) is a pro-inflammatory cytokine important in immune responses TNF inhibits cAMP-stimulated Cyp17 transcription.

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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