1. Cytokines and the regulation of steroidogenesis Buck Hales Department of Physiology & Biophysics UIC

2. Immune factors vs. Cyp17 mRNA

4. Immune factors vs. testosterone

5. IL-6 vs. testosterone production

8. Calphostin C vs. AVP inhibition of P450c17 mRNA - + - + + + - - + - + + - - - + - + cAMP AVP Calphostin C P450c17 18S

9. MARKS Phosphorylation Myristoylated alanine-rich C kinase substrate 88 kDa heat stable substrate for PKC Phosphorylation of MARKS is measure of PKC activation Analyze by immunoprecipitation post metabolic labeling with 32 Pi

10. Effect of AVP on MARKS phosphorylation con cAMP AVP 4PDD IL-1 PMA MARKS 94 66 45 31

11. PKC inhibits cAMP-induced P450c17 PMA and AVP both inhibit P450c17 and testosterone production in Leydig cells Both PMA and AVP stimulate MARKS phosphorylation Calphostin C blocks the inhibitory effects of AVP on P450c17 expression Activation of PKC inhibits P450c17

12. Cyclic-AMP responsive regions of the Cyp17 promoter RELATIVE CAT ACTIVITY 20 40 60 80 100 CONTROL cAMP -2500-1021-346 -245

19. TNF  and PMA stimulate translocation of PKC  from cytoplasm to membrane control PMA TNF  No antibody

21. Site-directed mutagenesis of Cyp17 CRR (-346 to –245) Oligos were designed to place an XhoI once every ten base pairs within the 100 base pair CRR. This resulted in changing as few as three (mutant 6) to as many as six (mutant 1 and 7) of every ten nucleotides. Mutagenesis was performed with Altered Sites (Promega) and all mutants were verified by sequencing.

22. Percent of WT induction by cAMP

23. Putative sites revealed by mutants gcaacctgat gacattaatt attaactgtg cagcactttt gacattacag CTCGAGtgat CTcGAGaatt CtCGaGtgtg cTCGaGtttt CTcGAGacag mut 1 mut 2 mut 3 mut 4 mut 5 cacgcactct gaaaccttga tcttaatctg atagcatttg cctctgggag cTcgAGctct CTCGAGttga CTCGaGtctg CtCgAGtttg cACGAgggag mut 6 mut 7 mut 8 mut 9 mut 10 ATF2/cjun AhR/Arnt (core sequence) SF-1

24. -440 -250 ATF2/c-jun mutants 2,5,9 C/EBP  AhR/ARNT mutant 6 SF-1 mutant 7 ARE Putative regulatory motifs revealed by mutagenesis ?

25. Effect of ATF2 on expression

26. cAMP vs.ATF2 protein expression 80 73 66 99 68 43 29 01h3h6h9h +TNF + cAMP

27. Summary and plans ATF2 may be the involved but is likely pairing with an as yet unidentified and cAMP-induced factor C/EBP  is a likely candidate-- cAMP induces its expression More detailed mutagenesis, expression cloning, in vivo foot printing

28. Immune-Endocrine control of Leydig cell function Return…. Overview and significance of Immune-endocrine interactions in the regulation of Leydig cell function Cytokine signaling