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.
Footnotes
K.-S.C.M. and M.P. contributed equally to this work.
H.S.B. was supported by the National Institutes of Health [Grant HD057876] and the Anderson Cancer Institute. K.-S.C.M. was supported by the National Institutes of Health National Institute on Drug Abuse [Grant DA027086] and the National Institutes of Health National Center on Minority Health and Health Disparities [Grant 5P20MD003941] (to K.-S.C.M. and H.S.B.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
ABBREVIATIONS:
- ER
- endoplasmic reticulum
- MAM
- mitochondria-associated ER membrane
- Ab
- antibody
- IMM
- inner mitochondrial membrane
- IP3
- inositol 1,4,5-triphosphate
- KSCM-1
- 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl) benzofuran-2-carboxamide
- KSCM-5
- 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide
- KSCM-11
- 6-methoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl) benzofuran-2-carboxamide
- OMM
- outer mitochondrial membrane
- SCC
- side-chain cleavage
- siRNA
- short interfering RNA
- SKF-10047
- 2S-[2α,6α,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol
- StAR
- steroidogenic acute regulatory protein
- TLC
- thin-layer chromatography
- VDAC
- voltage-dependent anion channel
- 3βHSD2
- 3-β-hydroxysteroid dehydrogenase.
- Received July 6, 2012.
- Accepted August 23, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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