Mitochondrial Control of Leydig Cell Steroidogenesis Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics.
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Mitochondrial Control of Leydig Cell Steroidogenesis Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics Cross section of rat testis Showing Seminiferous 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 Macrophage-Leydig cell interactions Cytokines, ROS ? Extracellular lipoprotein Cholesterol pool cholesterol PKA+ ATP cAMP Pregnenolone DYm 3bHSD ATP transcription acetate LH Progesterone P450c17 Androstenedione 17bHSD TESTOSTERONE Mitochondrial vs. Nuclear control of steroidogenesis cAMP ROS/mitochondrial disruptors - Acute regulation at the level of substrate availability + PKA Cytokines PKC agonists + + testosterone mitochondrial - Chronic regulation at the level of gene transcription nuclear Effect of LPS on steroidogenic mRNA levels P450scc P450c17 3b-HSD actin LPS - + - + - + - + time 2h 4h 6h - + 8h 24h Effect of LPS on P450c17 protein levels 2 and 24 h post injection control LPS contro LPS l 2 hours 24 hours LPS vs. serum testosterone: 2-24 hours control LPS 14 Testosterone (ng/ml) 12 10 8 6 4 2 0 2h 4h 6h 8 h 24 h Time post LPS LPS vs. StAR protein expression: 2 hr after injection 37 kDa 30 kDa LPS vs. StAR mRNA expression Steroidogenic Acute Regulatory Protein: StAR • Essential for steroid hormone biosynthesis • Cyclic-AMP dependent expression • Facilitates cholesterol transfer across innermitochondrial (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 DYm StAR facilitates cholesterol transfer 37 StAR Processing 32 30 Inner-mitochondrial forms Cytosol 37 kDa N' signal peptides cholesterol transfer critical region Outer mitochondrial membrane Inner- mitochondrial membrane N' 32 kDa matrix N' 30 kDa Time course of StAR decay density Time course of StAR decay 100000 80000 30 kDa 60000 40000 32 kDa 37 kDa 20000 0 0 15 30 45 60 minutes 75 90 105 120 StAR chol adx Mitochondrial matrix Adx-red scc chol Cytosol PBR chol 3bHSD N’ StAR C’ ? TOM TIM PBR Cytosol VDAC ANT Mitochondrial matrix CphD CK HK StAR N-terminal localization expression clones MTS 1-37 ITS 38-47 pCMV-StAR TAA StAR-stop MTS 1-37 Tom20 OMTS StAR D-ITS StAR D-N47 StAR/Tom20 CCHL IMSS StAR/CCHL What mediates the acute LPS inhibition? • Tested numerous inflammatory mediators in Leydig cells in vitro-- none mimicked the acute LPS “effect” – cytokines (TNFa, IL-1, IL-6, IFNg, TGFb) – prostaglandins (PGF2a, PGE) – catecholamines (norepi, isoproteranol) LPS vs. StAR protein expression: 2 hr after injection 37 kDa 30 kDa Carbonyl cyanide mchlorophenylhydrazone (cccp) • Carbonyl cyanide m-chlorophenylhydrazone (cccp): potent uncoupler of oxidative phosphorylation; protonophore, mitochondrial disrupter. • Causes transient disruption of DYm Mitochondrial respiration, OX-PHOS and DYm H+ DYm e- Effect of CCCP on StAR protein 37 kDa 30 kDa Control cAMP cAMP + cccp cccp Effect of CCCP on StAR mRNA 3.4 kB 2.9 kB StAR 1.6 kB cyclophilin con cA cA+cccp Effect of CCCP on StAR synthesis 37kDa 30kDa Control cAMP cccp cAMP + cccp Tetramethylrhodamine Ethyl Ester (TMRE) • Tetramethylrhodamine Ethyl Ester (TMRE): Uptake is dependent on DYm. Rapidly and reversibly taken up by allowing dynamic measurement of membrane potential by fluorescent microscopy and flow cytometry. CCCP disrupts DYm in MA10s control CCCP-treated Effect of mitochondrial agents on progesterone production 1000 900 800 ng/ml 700 600 500 400 300 200 100 0 con cAMP +Oligom +arsen +CCCP Effect of mitochondrial agents on StAR protein expression 37 kDa 30 kDa Effect of mitochondrial agents on StAR mRNA expression 3.2 kB StAR 1.6 kB cyclophilin Effect of H2O2 on StAR protein 4000 3000 2000 1000 0 Effect of H2O2 on StAR mRNA Northern Blot StAR mRNA Contr. cAMP. Cyclophilin mRNA 100 200 250 500 Effect of H2O2 on P450scc protein 5000 4000 3000 2000 1000 0 Effect of xanthine/xanthine oxidase on StAR protein Effect of xanthine/xanthine oxidase on StAR forms 2500 2000 1500 30+32 kDa 1000 37 kDa b 500 a a b a b b 0 con. cAMP +10 +50 +100 37/30+30 kDa StAR cAMP + Xanthine Ox. (mU) IOD Ratio IOD StAR cAMP + Xanthine Ox. (mU) a a 12 a 10 8 6 a b bb a cAMP +10 4 2 0 con. a b +50 +100 cAMP + Xanthine Ox. (mU) TMRE staining of MA-10 cells exposed to H2O2—time lapse Do reactive oxygen species (ROS) mediated the acute inhbitory effects of LPS? • Testicular Macrophages are known to produce ROS when activated • ROS are produced rapidly after exposure to LPS • Many potential sources of ROS in testicular interstitium LPS inhibits Leydig cells in vivo via ROS MDA + HNE (uM/10e6 LC) Increased lipid peroxidation and depolarization of Leydig cell mitochondria support involvement of ROS in LPS action in vivo Lipid peroxides 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 control LPS What is the Dym-sensitive component of steroidogenesis? • Protein import into matrix is Dymdependent– but likely not responsible for inhibition of StAR • PBR? • Perturbation of intra-mitochondrial Ca2+ and/or ATP levels? Ca2+ transport systems in mitochondria Ruthinium Red H+ e- Ca2+ uniporter (U) facilitates the transport of Ca2+ inward down the electrochemical gradient. Ca2+ activated permeability transition pore (PTP) also is shown Potential role for mitochondrial Ca2+ IOD Ru360 is a cell permeable derivative Con cAMP +H202 +5 on StAR +10 Proteinof Ruthinium Effects of Ru360 Red-- a specific uM Ru360 Mitochondrial 150 Ca2+ uptake blocker 100 50 0 Con cAMP +H202 +5 +10 uM Ru360 CCCP disrupts DYm in MA10s control CCCP-treated Excitation/Emission Spectra: Control vs. CCCP 350000 548 300000 250000 200000 576 150000 100000 50000 0 500 520 540 nm 560 580 Excitation/Emission Difference Spectra difference 548-573 fluoresence intensity 60000 40000 20000 0 -20000 500 550 -40000 -60000 -80000 -100000 nm Time-based dual emission spectra Fluorescence intensity 450000 400000 350000 300000 250000 200000 150000 100000 0 100 200 300 400 seconds 500 600 700 800 Ratiometric Fluorometry: Estimation of DYm Ratio 575/549 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0 100 200 300 400 seconds 500 600 700 800 Sites in the electron transport chain that inhibitors act Determination of NADH/NAD+ ratio fluorescence 150000 130000 5 uMCCCP 110000 90000 70000 5mM CN- 15mM CN1mM CN- 50000 30000 0 100 200 300 400 seconds 500 600 Effect of cAMP and Antimycin A on DYm Dym ratio 2 ratio 1.5 1 0.5 0 Control cAMP cAMP + Antimycin Effect of cAMP and Antimycin A on NADH/NAD+ NADH/NAD ratio ratio 1.2 1.19 1.18 1.17 1.16 1.15 1.14 1.13 Control cAMP cAMP + Antimycin StAR 37 kDa Star c l +a AM nt P im yc +C in C C +H P +o 2 lig O2 om +c ycin ya ni de 30 kDa Star co nt ro IOD Effect of mito compounds on StAR 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 StAR P450scc Con cAMP +AA +CCCP +H202 +oligo +CN M P +a nt im yc in +C C C P +H 2O +o 2 lig om yc in +c ya ni de co 50 cA P450scc 1600 1400 1200 1000 800 600 400 200 0 nt ro l 30 IOD 37 Steroidogenic machinery Sites of immune inhibition ROS Hales Lab Fred Lepore Neil Iyengar Tristan Shankara Marika Wrzosek John Allen Thorsten Diemer Paul Janus Steinunn Thorardottir Karen Held Hales NIH: HD25271 HD35544 Collaborators Judy Bolton—UIC Colin Jefcoate—UW Madison Jean-Guy Lehoux—Sherbrooke Yossi Orly—Hebrew Univ Anita Payne—Stanford Mariann Piano—UIC Catherine Rivier—Salk Inst Douglas Stocco—Texas Tech Gregory Thatcher—UIC StAR oxidative stress alcohol steroidogenesis “It takes balls to work on Leydig cells” Anita Payne circa 1984