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Cytokines and the regulation of steroidogenesis
Buck Hales
Department of Physiology & Biophysics
UIC
Immune factors vs. Cyp17 mRNA
Control
cAM
P
cAM
P + IL-6
cAM
P + IL-1
cAM
P + IL-1
cAM
P + TN
F-cA
MP
+ C8
Inte
gra
ted
Op
tica
l Den
sity
(Rat
io o
f P
450c
17 t
o c
yclo
ph
ilin
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Immune factors vs. Cyp17 mRNA
Control
cAM
P
cAM
P+IL-6
cAM
P+IL-1
cAM
P + IL-1
cAM
P + TN
F-T
esto
ster
on
e(n
g/1
06 Ley
dig
cel
ls/2
4h)
0
50
100
150
200
250
300
350
400
Immune factors vs. testosterone
IL-6(ng/ml)C
ontrol
cAM
P
cAM
P+1
cAM
P+10
cAM
P+100
cAM
P+1000
Tes
tost
ero
ne
(ng
/106 L
eyd
ig c
ells
/24h
)
0
200
400
600
800
1000
1200
1400
1600
1800
IL-6 vs. testosterone production
Calphostin C vs. AVP inhibition of P450c17 mRNA
- + - + + +- - + - + +- - - + - +
cAMPAVP
Calphostin C
P450c17
18S
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
Effect of AVP on MARKS phosphorylation
concAM
PAVP
4PDDIL-1
PMA
MARKS94
66
45
31
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
Cyclic-AMP responsive regions of the Cyp17 promoter
RE
LA
TIV
E C
AT
AC
TIV
ITY
20
40
60
80
100CONTROLcAMP
-2500 -1021 -346 -245
TNF and PMA stimulate translocation of PKC from cytoplasm to membrane
control
PMA TNF
No antibody
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.
Percent of WT induction by cAMP
0%20%40%60%80%
100%120%140%
WT mut1
mut2
mut3
mut4
mut5
mut6
mut7
mut8
mut9
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
-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
?
Effect of ATF2 on expression
02468
101214
pro 491pro
ATF2
ATF2+cA
JUN
JUN+cA
ATF2+JUN
ATF2+JUN+cA
mtv
mtv+cA
cAMP vs.ATF2 protein expression
807366
99
68
43
29
0 1h 3h 6h 9h 9h+TNF
+ cAMP
Summary and plans
• ATF2 may be the involved but is likely pairing with an as yet unidentified and cAMP-induced factor
• C/EBPis a likely candidate-- cAMP induces its expression
• More detailed mutagenesis, expression cloning, in vivo foot printing
Immune-Endocrine control of Leydig cell function
Return….
Overview and significance of Immune-endocrine interactions
in the regulation of Leydig cell function
Cytokine signaling