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σ-1 receptor at the mitochondrial-associated endoplasmic reticulum membrane is responsible for mitochondrial metabolic regulation - PubMed

σ-1 receptor at the mitochondrial-associated endoplasmic reticulum membrane is responsible for mitochondrial metabolic regulation

Karla-Sue C Marriott et al. J Pharmacol Exp Ther. 2012 Dec.

Abstract

The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a small section of the outer mitochondrial membrane tethered to the ER by lipid and protein filaments. One such MAM protein is the σ-1 receptor, which contributes to multiple signaling pathways. We found that short interfering RNA-mediated knockdown of σ-1 reduced pregnenolone synthesis by 95% without affecting expression of the inner mitochondrial membrane resident enzyme, 3-β-hydroxysteroid dehydrogenase 2. To explore the underlying mechanism of this effect, we generated a series of σ-receptor ligands: 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide (KSCM-1), 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide (KSCM-5), and 6-methoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl) propyl)benzofuran-2-carboxamide (KSCM-11) specifically bound to σ-1 in the nanomolar range, whereas KSCM-5 and KSCM-11 also bound to σ-2. Treatment of cells with the KSCM ligands led to decreased cell viability, with KSCM-5 having the most potent effect followed by KSCM-11. KSCM-1 increased σ-1 expression by 4-fold and progesterone synthesis, whereas the other compounds decreased progesterone synthesis. These differences probably are caused by ligand molecular structure. For example, KSCM-1 has two methoxy substituents at C-5 and C-6 of the benzofuran ring, whereas KSCM-11 has one at C-6. KSCM ligands or σ-1 knockdown did not alter the expression of ER resident enzymes that synthesize steroids. However, coimmunoprecipitation of the σ-1 receptor pulled down voltage-dependent anion channel 2 (VDAC2), whose expression was enhanced by KSCM-1. VDAC2 plays a key role in cholesterol transport into the mitochondria, suggesting that the σ-1 receptor at the MAM coordinates with steroidogenic acute regulatory protein for cholesterol trafficking into the mitochondria for metabolic regulation.

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Figures

Scheme 1.
Scheme 1.

Synthesis of different KSCM ligands from carboxamides via N-alkylation. The substitution of side chains is indicated above, generating KSCM-1, KSCM 5, and KSCM-11 ligands.

Fig. 1.
Fig. 1.

Expression and activity of the σ-1 receptor in σ-1 receptor-knockdown MA-10 cells. A, schematic presentation of a simplified version of the biosynthesis pathway and its relationship to the MAM with σ-1 receptor. B, left, all four siRNA oligonucleotides at 60 pmol reduced expression of the σ-1 receptor in MA-10 cells, but only siRNA4 reduced expression at 30 pmol. Western blot shows unchanged expression of IMM resident 3βHSD2 and OMM-associated Tom20 in MA-10 cells. Right, loading control of probing the corresponding left with β-actin. C, isolated mitochondria from MA-10 control and siRNA knockdown cells were incubated with [14C]cholesterol and NADPH in a metabolic conversion assay, and metabolites were separated by thin-layer chromatography. D, quantitative estimation of the metabolites from C. E, Western blot of the cellular fractions with the indicated antibodies, showing the purity of our mitochondrial fraction. Data presented are the mean ± S.E.M. of three independent experiments. F, proposed model of interaction between ER and mitochondria through the MAM resident σ-1 receptor.

Fig. 2.
Fig. 2.

A to C, pharmacokinetics of KSCM ligands. Ligand binding of KSCM-1 (A), KSCM-5 (B), and KSCM-11 (C) to σ-1 receptors. Affinities Ki (nM) were determined in rat brain homogenate in σ-binding buffer. σ-1 Receptors were labeled with [3H](+)- pentazocine, and haloperidol served as the reference compound. KSCM-5 exhibited the strongest binding followed by KSCM-1 and KSCM-11. D to F, ligand binding of KSCM-1 (D), KSCM-5 (E), and KSCM-11 (F) to σ-2 receptors. Affinities Ki (nM) were determined in PC12 cells in σ-binding buffer. σ-2 Receptors were labeled with [3H]1,3-di(2-tolyl) guanidine and haloperidol as the reference compound. KSCM-5 exhibited the strongest binding followed by KSCM-11 and then very weak binding by KSCM-1. Data represent Ki (nM) values obtained from nonlinear regression of radioligand competition binding isotherms. Ki values are calculated from best-fit IC50 values using the Cheng-Prusoff equation. Data presented are the mean ± S.E.M. of three independent experiments. PDSP compound 19613 (control), compound 19603 (control).

Fig. 3.
Fig. 3.

Cell viability assay of the KSCM ligands after incubation for 24 h at varying concentrations in MA-10 cells. KSCM-5 (B) exhibited the greatest toxicity followed by KSCM-1 (A) and KSCM-11 (C). Data are the mean ± S.E.M. of at least three independent experiments.

Fig. 4.
Fig. 4.

Effect of different KSCM ligands on the σ-1 receptor expression. KSCM ligands were added to MA10 cells at concentrations ranging from 1 to 100 nM for 24 h. A, left, Western blots showed that expression of the σ-1 receptor increased with increasing the concentrations of KSCM-1 (top), moderately increased with increasing concentrations of KSCM-11 (bottom), but remained unchanged with KSCM-5 (middle). Right, Western blot of the same membranes probing with β-actin corresponding to each ligand. B, densitometric estimation of the Western blots in A illustrates the difference in expression with increasing concentrations of KSCM ligands. C, effect of incubating cells with KSCM-1, KSCM-5, and KSCM-11 on the expression of the inner mitochondrial steroidogenic enzyme 3-βHSD2. D, mitochondrial metabolic conversion of [3H]pregnenolone to progesterone after the addition of NAD in the presence of various concentrations of KSCM-1, KSCM-5, and KSCM-11. E, quantitative estimation of the metabolic conversion from D, which shows 27% enhanced increase in activity in the presence of KSCM-1 at 10 nM. Data presented in B and E are the mean ± S.E.M. of three independent experiments.

Fig. 5.
Fig. 5.

Effect of σ-1 receptor on the outer mitochondrial proteins. A, effect of increasing concentrations of KSCM-1, KSCM-5, and KSCM-11 on VDAC1 expression as determined by Western blotting. B, left, effect of increasing concentrations of KSCM-1 (top), KSCM-5 (middle), and KSCM-11(bottom) on VDAC2 expression as determined by Western blotting. Right, the expression of β-actin when probed the same membrane presented at left under identical conditions. C, densitometric estimation of the effect of KSCM ligands on VDAC2 from B, left expression compared with β-actin in B, right. VDAC2 expression was increased with KSCM-1 up to 50 nM, and then partially decreased. KSCM-5 reduced expression at 100 nM, and KSCM-11 reduced expression at 5 nM. D, effect of KSCM-1 on σ-1 receptor expression in VDAC1 knockdown cells. Western blot with σ-1 antibody showed increased expression after addition of KSCM-1 at any concentration, except 100 nM. E and F, σ-1 expression in VDAC1 knockdown cells incubated with KSCM-5 and KSCM-11 ranging from 1 to 100 nM. Western blots indicated that σ-1 expression remained unchanged in VDAC1 knockdown cells. Unaffected expression of β-actin is presented (bottom).

Fig. 6.
Fig. 6.

Expression of StAR and its interaction with proteins involved in steroidogenesis. A, expression showing the unchanged expression of mitochondrial unimported 37-kDa and imported 30-kDa StAR after incubation with KSCM-5 and KSCM-11. KSCM-1, at or above 10 nM, stabilizes the expression of unimported StAR, but imported 30-kDa StAR expression was unaffected. B, coimmunoprecipitation of the MA-10 mitochondria with the indicated antibodies followed by Western blotting with σ-1 antibody (top) and StAR antibody (middle). The experiment shows the interaction of StAR with σ-1. Western blotting of the same coimmunoprecipitation samples with IMM resident protein Tim23 (bottom) antibody showed that Tim23 was present in MA-10 cells and pig adrenals; it did not interact with the other proteins.

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