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
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 TNF and cAMP on FOS and cJUN protein expression cAMP TNF 68kDa 43kDa FOSC-JUN
Component Two: Background Effect of cAMP and TNF on 3TPluc activity
cAMP and TNF vs. DNA-protein interactions with canonical AP FOS c-jun junB junD control ab cAMPcA+TNF TNF
Super-shift with AP-1 antibodies vs. canonical AP-1 binding element AP-1 FOS c-jun junB junD
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)
TNF and cAMP vs. cJUN kinase activity hrs TNF cAMP 44 42
Time course of cJUN kinase activation
TNF and cAMP vs. MAPK activity hrs TNF cAMP ERK
Time course of MAP kinase activation
c-Jun kinase transactivation assay
Effect of cAMP and TNF on c-Jun transactivation 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 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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