Transcript Cytokines

Cytokines and the regulation of
steroidogenesis
Buck Hales
Department of Physiology & Biophysics
UIC
Immune factors vs. Cyp17 mRNA
I(RatiofP450ncte1g7rtaotecdyOlopptihciallnD)ensity
Immune factors vs. Cyp17 mRNA
0
.
6
0
.
5
0
.
4
0
.
3
0
.
2
0
.
1
0
.
0
C8
cAMP+TNF- 
cAMP+ 
P+IL-1
IcAML-1 
cAMP+IL-6
cAMP+
cAMP
Control
Immune factors vs. testosterone
4
0
0
)
3
5
0
3
0
0
2
5
0
T
e
s
t
o
e
r
o
n
e
6
(ng/10 Leydigcels/24h
2
0
0
1
5
0
1
0
0
5
0
0
TNF- 
cAMP+
IL-1 
cAMP+
-1 
cAMP+IL
-6
cAMP+IL
cAMP
Control
IL-6 vs. testosterone production
1
8
0
0
1
6
0
0
1
4
0
0
1
2
0
0
T
e
s
t
o
e
r
o
n
e
6
(ng/10 Leydigcels/24h)
1
0
0
0
8
0
0
6
0
0
4
0
0
2
0
0
0
0
cAMP+1
0
cAMP+1
0
cAMP+1
cAMP+1
cAMP
Control
I
L
6
(
n
g
/
m
l
)
Calphostin C vs. AVP inhibition
of P450c17 mRNA
18S
P450c17
cAMP
AVP
Calphostin C
- + - + + +
- - + - + +
- - - + - +
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
94
MARKS
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
CONTROL
cAMP
100
80
60
40
20
-2500 -1021 -346
-245
TNF and PMA stimulate translocation of
PKC from cytoplasm to membrane
control
No antibody
PMA
TNF
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
140%
120%
100%
80%
60%
40%
20%
0%
WT mut mut mut mut mut mut mut mut mut
1
2
3
4
5
6
7
8
9
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
ATF2/cjun
AhR/Arnt (core sequence)
SF-1
mut 9
mut 10
Putative regulatory motifs revealed by mutagenesis
?
-250
-440
ATF2/c-jun mutants 2,5,9
C/EBP
AhR/ARNT mutant 6
SF-1 mutant 7
ARE
Effect of ATF2 on expression
ATF2
14
12
10
8
6
4
2
0
ATF2+cA
JUN
JUN+cA
ATF2+JUN
ATF2+JUN+cA
mtv
pro
491pro
mtv+cA
cAMP vs.ATF2 protein expression
99
80
73
66
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/EBP is a likely candidate-- cAMP
induces its expression
• More detailed mutagenesis, expression
cloning, in vivo foot printing
Immune-Endocrine control
of Leydig cell function
Return….
Cytokine signaling
Overview and significance
of Immune-endocrine interactions
in the regulation of Leydig cell function