Cytokines and the regulation of steroidogenesis

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Cytokines and the regulation of steroidogenesis

• Buck Hales
• Department of Physiology Biophysics
• UIC

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Immune factors vs. Cyp17 mRNA
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Immune factors vs. Cyp17 mRNA
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Immune factors vs. testosterone
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IL-6 vs. testosterone production
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Calphostin C vs. AVP inhibition of P450c17 mRNA
18S
P450c17
- - - - - -
- - -
cAMP AVP Calphostin C
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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 32Pi

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

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Cyclic-AMP responsive regions of the Cyp17
promoter
CONTROL
100
cAMP
80
RELATIVE CAT ACTIVITY
60
40
20
-245
-2500
-1021
-346
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TNFa and PMA stimulate translocation of PKCa from
cytoplasm to membrane
control
No antibody
PMA
TNFa
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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.

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Percent of WT induction by cAMP
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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
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Putative regulatory motifs revealed by mutagenesis
?
-440
-250
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Effect of ATF2 on expression
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cAMP vs.ATF2 protein expression
99
80
73
66
68
43
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0
1h
3h
6h
9h
9h TNF
cAMP
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Summary and plans

• ATF2 may be the involved but is likely pairing
  with an as yet unidentified and cAMP-induced
  factor
• C/EBPb is a likely candidate-- cAMP induces its
  expression
• More detailed mutagenesis, expression cloning, in
  vivo foot printing

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