Immune-Endocrine control of Leydig cell function

Dale Buck Hales, PhDUniversity of Illinois at Chicago

Department of Physiology and Biophysics

Cross section of rat testisSeminiferous tubules and Interstitium where Leydig cells reside.

Kent Christensen, Univ. Michigan

Interstitium of rat testis showing endothelium, Leydig cells (L), and macrophages (arrow). Note close association of macrophages and Leydig cells.

Scott Miller, Univ Utah

Close association of Leydig cell and macrophage, lower panel shows close up of “digitation” of Leydig cell process extending onto macrophage surface.

Scott Miller, Univ. Utah

Cytokines, ROS

Macrophage-Leydig cell interactions

?

cholesterol

Extracellularlipoprotein

Cholesterolpool

LH

ATP

cAMPPKA+

Pregnenolone

Progesterone

Androstenedione

TESTOSTERONE

m

3HSD

P450c17

17HSD

acetate

IN VIVO METHODSIN VIVO METHODSIN VIVO METHODSIN VIVO METHODS

• Inject mice ip with LPS• Sacrifice mice at various times• Collect blood for serum hormone analyses by RIA• Collect testes, adrenals, and other organs• Isolate Leydig cells and testicular macrophages

– RNA and Protein analyses

• Metabolically label Leydig cells ex vivo with 35S-methionine and immunoprecipitate StAR

• Aanalyze m by fluorescent microscopy

Effect of LPS on steroidogenic mRNA levelsEffect of LPS on steroidogenic mRNA levels Effect of LPS on steroidogenic mRNA levelsEffect of LPS on steroidogenic mRNA levels

P450scc

P450c17

3-HSD

actin

LPS - + - + - + - + - +

time 2h 4h 6h 8h 24h

0

2

4

6

8

10

12

14

LPS vs. serum testosterone: 2-24 hoursLPS vs. serum testosterone: 2-24 hoursLPS vs. serum testosterone: 2-24 hoursLPS vs. serum testosterone: 2-24 hours

Tes

tost

ero

ne

(ng

/ml)

control

LPS

Time post LPS

24 h2 h 4 h 8 h6 h

Steroidogenic Acute Steroidogenic Acute Regulatory Protein: StARRegulatory Protein: StAR

Steroidogenic Acute Steroidogenic Acute Regulatory Protein: StARRegulatory Protein: StAR

• Essential for steroid hormone biosynthesis• Cyclic-AMP dependent expression• Facilitates cholesterol transfer across inner-

mitochondrial (aqueous) space• Translated as a 37 kDa precursor protein that

is processed to the 30 kDa mature form as it translocates into the mitochondria

• Cholesterol transport activity depends on intact m

StAR facilitates cholesterol transfer

Domains and phosphorylation sites ofSteroidogenic Acute Regulatory protein (StAR)

signal peptides critical region

cholesterol transfer

Transfer across outer mitochondrialmembrane and cleavage of first peptide

Transfer across inner membrane, formation of contact sites for cholesteroltransfer, and cleavage of second peptide

Mature 30 kDa protein associated withinner mitochondrial membrane post

cholesterol transfer

N'-mutant protein associates only withouter mitochondrial membrane and still facilitates cholesterol transfer

C'-mutant protein neither associates with outer mitochondrial membrane nor

facilitates cholesterol transfer

Current working model of StAR mechanism of action via association of C´

with outer mitochondrial membrane

LPS vs. StAR protein LPS vs. StAR protein expression: 2 hr after injectionexpression: 2 hr after injection

LPS vs. StAR protein LPS vs. StAR protein expression: 2 hr after injectionexpression: 2 hr after injection

30 kDa

37 kDa

conLPS

LPS vs. StAR mRNA expressionLPS vs. StAR mRNA expressionLPS vs. StAR mRNA expressionLPS vs. StAR mRNA expression

LPS vs. StAR Synthesis: 2 hrLPS vs. StAR Synthesis: 2 hrLPS vs. StAR Synthesis: 2 hrLPS vs. StAR Synthesis: 2 hr

LPS vs. StAR synthesis: 2 hrLPS vs. StAR synthesis: 2 hrLPS vs. StAR synthesis: 2 hrLPS vs. StAR synthesis: 2 hr

0

20000

40000

60000

80000

100000

120000

140000

con-30 con-37 lps-30 lps-37

Effect of LPS on Steroidogenic ProteinsEffect of LPS on Steroidogenic ProteinsEffect of LPS on Steroidogenic ProteinsEffect of LPS on Steroidogenic Proteins

What mediates the acute LPS What mediates the acute LPS inhibition?inhibition?

What mediates the acute LPS What mediates the acute LPS inhibition?inhibition?

• Tested numerous inflammatory mediators in Leydig cells in vitro-- none mimicked the LPS “effect”– cytokines (TNF, IL-1, IL-6, IFN, TGF)– prostaglandins (PGF2, PGE) – catecholamines (norepi, isoproteranol) – ceramide (C2, C8)– nitric oxide donors (Sin-1, SNAP, SNP, Nor-3)– Calcium inophore (A23187)

Carbonyl cyanide Carbonyl cyanide mm--chlorophenylhydrazone (cccp)chlorophenylhydrazone (cccp)

Carbonyl cyanide Carbonyl cyanide mm--chlorophenylhydrazone (cccp)chlorophenylhydrazone (cccp)

• Carbonyl cyanide m-chlorophenyl-hydrazone (cccp): potent uncoupler of oxidative phosphorylation; protonophore, mitochondrial disrupter.

• Causes transient disruption of m

Mitochondrial respiration, OX-PHOS and Mitochondrial respiration, OX-PHOS and mmMitochondrial respiration, OX-PHOS and Mitochondrial respiration, OX-PHOS and mm

cytosol

mitochondria

CCCP vs. Progesterone in MA10sCCCP vs. Progesterone in MA10sCCCP vs. Progesterone in MA10sCCCP vs. Progesterone in MA10s

Effect of CCCP on StAR proteinEffect of CCCP on StAR proteinEffect of CCCP on StAR proteinEffect of CCCP on StAR protein

Control cAMP cAMP + cccp cccp

37 kDa

30 kDa

Effect of CCCP on StAR synthesisEffect of CCCP on StAR synthesisEffect of CCCP on StAR synthesisEffect of CCCP on StAR synthesis

Control cAMP cccp cAMP + cccp

37kDa

30kDa

0

500000

1000000

1500000

2000000

2500000D

PM

/ug

pro

tein

Effect of CCCP on protein Effect of CCCP on protein synthesissynthesis

Effect of CCCP on protein Effect of CCCP on protein synthesissynthesis

Effect of CCCP on StAR Effect of CCCP on StAR synthesissynthesis

Effect of CCCP on StAR Effect of CCCP on StAR synthesissynthesis

0

10

20

30

40

50

60

70

80C

orr

ecte

d d

ensi

ty

37 kDa30 kDa

con cA cA+cccp

StAR

cyclophilin

3.4 kB

1.6 kB

2.9 kB

Effect of CCCP on StAR mRNAEffect of CCCP on StAR mRNAEffect of CCCP on StAR mRNAEffect of CCCP on StAR mRNA

Tetramethylrhodamine Tetramethylrhodamine Ethyl Ester (TMRE)Ethyl Ester (TMRE)

Tetramethylrhodamine Tetramethylrhodamine Ethyl Ester (TMRE)Ethyl Ester (TMRE)

• Tetramethylrhodamine

Ethyl Ester (TMRE): Uptake is dependent on m. Rapidly and reversibly taken up by allowing dynamic measurement of membrane potential by fluorescent microscopy and flow cytometry.

CCCP disruptsCCCP disrupts mm in MA10sin MA10sCCCP disruptsCCCP disrupts mm in MA10sin MA10s

controlcontrolcontrolcontrol CCCP-treatedCCCP-treated CCCP-treatedCCCP-treated

HH22OO22 vs. Progesterone in MA10s vs. Progesterone in MA10sHH22OO22 vs. Progesterone in MA10s vs. Progesterone in MA10s

0

50

100

150

200

250

control cAMP cAMP+ 100

cAMP+ 200

cAMP+ 250

cAMP+ 500

ng/m

l

HH220022 vs. StAR and 3 vs. StAR and 3-HSD -HSD protein in MA10sprotein in MA10s

HH220022 vs. StAR and 3 vs. StAR and 3-HSD -HSD protein in MA10sprotein in MA10s

Effect of HEffect of H22OO22 on StAR mRNA on StAR mRNAEffect of HEffect of H22OO22 on StAR mRNA on StAR mRNA

Northern Blot

StAR mRNA

Contr. cAMP. 100 200 250 500

Cyclophilin mRNA

TMRE staining of MA-10 cells TMRE staining of MA-10 cells exposed to H2O2exposed to H2O2

TMRE staining of MA-10 cells TMRE staining of MA-10 cells exposed to H2O2exposed to H2O2

Control 100M H2O2

Summary: StAR and 3Summary: StAR and 3-HSD -HSD studies in MA10sstudies in MA10s

Summary: StAR and 3Summary: StAR and 3-HSD -HSD studies in MA10sstudies in MA10s

• CCCP transiently disrupts m

• CCCP inhibits StAR processing and progesterone production

• Disruption of m alone does not block 3-HSD protein-- activity??

• H2O2 inhibits StAR and 3-HSD protein and progesterone production

• H2O2 disrupts m

Hypothesis-oneHypothesis-oneHypothesis-oneHypothesis-one

• LPS causes an abrupt inhibition of steroidogenesis by disrupting Leydig cell mitochondria– LPS activates generation of reactive oxygen

species (ROS) from testicular interstitial macrophages

– ROS from adjacent macrophages disrupts Leydig cell mitochondria

Hypothesis-twoHypothesis-twoHypothesis-twoHypothesis-two

• LPS causes Leydig cell apoptosis– Long term depolarization precedes initiation of

apoptosis– Apoptosis of Leydig cells results in long-term

depression of serum testosterone– Perturbation of 3-HSD and StAR is an early

indictor of apoptosis

Summary: in vivo studiesSummary: in vivo studiesSummary: in vivo studiesSummary: in vivo studies

• LPS causes an abrupt and prolonged decrease in serum testosterone levels

• Abrupt decreases in testosterone are correlated to inhibition of Leydig cell StAR and 3-HSD protein

• LPS causes disruption of Leydig cell m,

• Preliminary data supports the role of ROS in mediating LPS effects in vivo

NIH: HD25271 HD35544NIH: HD25271 HD35544NIH: HD25271 HD35544NIH: HD25271 HD35544

Thorsten DiemerBeth NardulliSalil GindeJohn AllenJohn Choi

Thorsten DiemerBeth NardulliSalil GindeJohn AllenJohn Choi

Hales Lab

Bruce Bosmann Barbara ClarkJim FergusonLester Lau Jean-Guy LeHoux Mark McLean Yossi Orly Keith Parker Anita Payne Richard PestellCatherine Rivier Focko RommertsDouglas Stocco

Bruce Bosmann Barbara ClarkJim FergusonLester Lau Jean-Guy LeHoux Mark McLean Yossi Orly Keith Parker Anita Payne Richard PestellCatherine Rivier Focko RommertsDouglas Stocco

collaborators

Karen Held HalesKaren Held HalesKaren Held HalesKaren Held Hales