cytokines and the regulation of steroidogenesis buck hales department of physiology & biophysics...

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