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Introduction

Introduction to AP-1. Introduction . Tumor necrosis factor- a (TNF a ) is a pro-inflammatory cytokine important in immune responses TNF a inhibits cAMP-stimulated Cyp17 transcription TNF a activates protein kinase C in Leydig cells

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Introduction

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  1. Introduction to AP-1 Introduction • 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. Objectives Component One: Status • 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. - + - - - - - + - - - - - + - - - - - + - + - - - - - + - - - - - + - - - - - + - + - - - - - + - - - - - + - - - - - + - + - - - - - + - - - - - + - - - - - + FOS c-jun junB junD cAMP and TNFa vs. DNA-protein interactions with canonical AP-1 ab control cAMP cA+TNF TNFa

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

  7. c-Jun Kinase:Stress Activated Protein Kinase/cJun Nuclear Kinase (SAPK/JNK) SAPK/JNKStress 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

  8. TNFa and cAMP vs. cJUN kinase activity 44 42 hrs 0 .25 .5 1 2 6 12 24 .25 .5 1 2 6 12 24 TNFa cAMP

  9. Time course of cJUN kinase activation

  10. TNFa and cAMP vs. MAPK activity ERK hrs 0 .25 .5 1 2 6 12 24 .25 .5 1 2 6 12 24 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. Summary 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

  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. Back to cytokines

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