1. 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-1

2. 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

3. - + + - - - + + Effect of TNF  and cAMP on FOS and cJUN protein expression - + + - - - + + cAMP TNF  68kDa 43kDa FOSC-JUN

4. Component Two: Background Effect of cAMP and TNF  on 3TPluc activity

5. cAMP and TNF  vs. DNA-protein interactions with canonical AP-1 - + - - - - - + - - - - - + - - - - - + - + - - - - - + - - - - - + - - - - - + - + - - - - - + - - - - - + - - - - - + - + - - - - - + - - - - - + - - - - - + FOS c-jun junB junD control ab cAMPcA+TNF TNF 

6. Super-shift with AP-1 antibodies vs. canonical AP-1 binding element - + - - - - - - - - - - + - - - - - - - - - - + - - + + - + - - - - + - + - + + - - - - - + - + + + AP-1 FOS c-jun junB junD

7. SAPK/JNK Stress 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 Kinase: Stress Activated Protein Kinase/cJun Nuclear Kinase ( SAPK/JNK)

8. TNF  and cAMP vs. cJUN kinase activity.25.5 1 2 6 12 240 hrs TNF  cAMP 44 42

9. Time course of cJUN kinase activation

10. TNF  and cAMP vs. MAPK activity.25.5 1 2 6 12 240 hrs TNF  cAMP ERK

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 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

15. 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

16. MAP-kinase signaling pathways RAS RAF MEK1,2 ERK1,2 MEKK1,+ SEK/JNKK SAPK/JNK1,2 MEKK4,+ MKK3 p38 ?Rac/Cdc42 Elk1, RskcJun, ATF2, Elk1 ATF2 mitogenStress/cytokine ? ?

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